Isolation and characterization of major histocompatibility complex class IIB genes from the nurse shark.

OpenAIRE

Bartl, S; Weissman, I L

1994-01-01

The major histocompatibility complex (MHC) contains a set of linked genes which encode cell surface proteins involved in the binding of small peptide antigens for their subsequent recognition by T lymphocytes. MHC proteins share structural features and the presence and location of polymorphic residues which play a role in the binding of antigens. In order to compare the structure of these molecules and gain insights into their evolution, we have isolated two MHC class IIB genes from the nurse...

Genetic Divergence of the Rhesus Macaque Major Histocompatibility Complex

Science.gov (United States)

Daza-Vamenta, Riza; Glusman, Gustavo; Rowen, Lee; Guthrie, Brandon; Geraghty, Daniel E.

2004-01-01

The major histocompatibility complex (MHC) is comprised of the class I, class II, and class III regions, including the MHC class I and class II genes that play a primary role in the immune response and serve as an important model in studies of primate evolution. Although nonhuman primates contribute significantly to comparative human studies, relatively little is known about the genetic diversity and genomics underlying nonhuman primate immunity. To address this issue, we sequenced a complete rhesus macaque MHC spanning over 5.3 Mb, and obtained an additional 2.3 Mb from a second haplotype, including class II and portions of class I and class III. A major expansion of from six class I genes in humans to as many as 22 active MHC class I genes in rhesus and levels of sequence divergence some 10-fold higher than a similar human comparison were found, averaging from 2% to 6% throughout extended portions of class I and class II. These data pose new interpretations of the evolutionary constraints operating between MHC diversity and T-cell selection by contrasting with models predicting an optimal number of antigen presenting genes. For the clinical model, these data and derivative genetic tools can be implemented in ongoing genetic and disease studies that involve the rhesus macaque. PMID:15289473

Molecular Genotype Identification of Different Chickens: Major Histocompatibility Complex

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Hongzhi Wang

2014-09-01

Full Text Available Chicken is a main poultry in China. Molecular breeding for disease resistance plays an important role in the control of diseases, especially infectious diseases. Choice of genes for disease resistance is the key technology of molecular breeding. The major histocompatibility complex (MHC is of great interest to poultry breeding scientists for its extraordinary polymorphism and close relation with traits of resistance against infectious diseases. The MHC-B haplotype plays an important role in the study of disease resistance in chicken. The traditional chicken MHC-B haplotype is commonly defined by serologic reactions of erythrocytes and the majority of studies have been conducted in Leghorn and broiler but study about other chicken breeds is little. In this study, firstly, the microsatellite marker LEI0258 which is located within the MHC was sequenced by using target sequence capture assay in different chicken breeds, and then according to the number of repeated structures and polymorphic sequences in microsatellite, sequence information for the region defined by LEI0258 was obtained for different haplotypes. Afterwards, we identified the relation between MHC-B haplotypes and disease resistance. Collectively, these observed results provided the reference data for disease-resistant breeding association with blood type and for further study of MHC gene function in poultry.

Overall major histocompatibility complex class I expression is not downregulated in cervix cancer, as detected by immunoelectron microscopy

NARCIS (Netherlands)

van Eijkeren, MA; Roovers, JP; Oorschot, [No Value; Geuze, HJ

2004-01-01

Downregulation of major histocompatibility complex (MHC) class I molecules in cervix cancer has been proposed as a mechanism for cancer cells to escape immunodetection. By means of light microscopic immunohistochemistry, it has been shown that in 20-70% of cervix cancers MHC class I is

Mechanisms of mono- and poly-ubiquitination: Ubiquitination specificity depends on compatibility between the E2 catalytic core and amino acid residues proximal to the lysine

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Sadowski Martin

2010-08-01

Full Text Available Abstract Ubiquitination involves the attachment of ubiquitin to lysine residues on substrate proteins or itself, which can result in protein monoubiquitination or polyubiquitination. Ubiquitin attachment to different lysine residues can generate diverse substrate-ubiquitin structures, targeting proteins to different fates. The mechanisms of lysine selection are not well understood. Ubiquitination by the largest group of E3 ligases, the RING-family E3 s, is catalyzed through co-operation between the non-catalytic ubiquitin-ligase (E3 and the ubiquitin-conjugating enzyme (E2, where the RING E3 binds the substrate and the E2 catalyzes ubiquitin transfer. Previous studies suggest that ubiquitination sites are selected by E3-mediated positioning of the lysine toward the E2 active site. Ultimately, at a catalytic level, ubiquitination of lysine residues within the substrate or ubiquitin occurs by nucleophilic attack of the lysine residue on the thioester bond linking the E2 catalytic cysteine to ubiquitin. One of the best studied RING E3/E2 complexes is the Skp1/Cul1/F box protein complex, SCFCdc4, and its cognate E2, Cdc34, which target the CDK inhibitor Sic1 for K48-linked polyubiquitination, leading to its proteasomal degradation. Our recent studies of this model system demonstrated that residues surrounding Sic1 lysines or lysine 48 in ubiquitin are critical for ubiquitination. This sequence-dependence is linked to evolutionarily conserved key residues in the catalytic region of Cdc34 and can determine if Sic1 is mono- or poly-ubiquitinated. Our studies indicate that amino acid determinants in the Cdc34 catalytic region and their compatibility to those surrounding acceptor lysine residues play important roles in lysine selection. This may represent a general mechanism in directing the mode of ubiquitination in E2 s.

Expression of the major histocompatibility antigens HLA-A2 and HLA-B7 by DNA-mediated gene transfer

NARCIS (Netherlands)

Bernabeu, C.; Finlay, D.; van de Rijn, M.; Maziarz, R. T.; Biro, P. A.; Spits, H.; de Vries, J.; Terhorst, C. P.

1983-01-01

Genes coding for the heavy chain of the class I antigens HLA-A2 or HLA-B7 of the human major histocompatibility complex have been introduced into mouse LtK- cells by cotransfection with the herpes simplex virus thymidine kinase gene. HAT-resistant colonies were isolated expressing either HLA-A2 or

Functional isotypes are not encoded by the constant region genes of the beta subunit of the T cell receptor for antigen/major histocompatibility complex

OpenAIRE

1984-01-01

Human T cell clones and a cDNA probe specific for constant regions of the beta subunit of the antigen/major histocompatibility complex (MHC) receptor, TiC beta 1 and TiC beta 2, were employed to determine whether these genes were differentially used by functional classes of T lymphocytes. DNA from 10 interleukin-2-dependent T cell clones including class I and class II MHC-specific cytotoxic T lymphocytes (n = 6), T4+ inducer T lymphocytes (n = 2), and T8+ suppressor T lymphocytes (n = 2) show...

Clinical, immunological and genetic features in eleven Algerian patients with major histocompatibility complex class II expression deficiency

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Djidjik Réda

2012-08-01

Full Text Available Abstract Presenting processed antigens to CD4+ lymphocytes during the immune response involves major histocompatibility complex class II molecules. MHC class II genes transcription is regulated by four transcription factors: CIITA, RFXANK, RFX5 and RFXAP. Defects in these factors result in major histocompatibility complex class II expression deficiency, a primary combined immunodeficiency frequent in North Africa. Autosomal recessive mutations in the RFXANK gene have been reported as being the principal defect found in North African patients with this disorder. In this paper, we describe clinical, immunological and genetic features of 11 unrelated Algerian patients whose monocytes display a total absence of MHC class II molecules. They shared mainly the same clinical picture which included protracted diarrhoea and respiratory tract recurrent infections. Genetic analysis revealed that 9 of the 11 patients had the same RFXANK founder mutation, a 26 bp deletion (named I5E6-25_I5E6+1, also known as 752delG26. Immunological and genetic findings in our series may facilitate genetic counselling implementation for Algerian consanguineous families. Further studies need to be conducted to determine 752delG26 heterozygous mutation frequency in Algerian population.

Chemotherapy inhibits skeletal muscle ubiquitin-proteasome-dependent proteolysis.

Science.gov (United States)

Tilignac, Thomas; Temparis, Sandrine; Combaret, Lydie; Taillandier, Daniel; Pouch, Marie-Noëlle; Cervek, Matjaz; Cardenas, Diana M; Le Bricon, Thierry; Debiton, Eric; Samuels, Susan E; Madelmont, Jean-Claude; Attaix, Didier

2002-05-15

Chemotherapy has cachectic effects, but it is unknown whether cytostatic agents alter skeletal muscle proteolysis. We hypothesized that chemotherapy-induced alterations in protein synthesis should result in the increased incidence of abnormal proteins, which in turn should stimulate ubiquitin-proteasome-dependent proteolysis. The effects of the nitrosourea cystemustine were investigated in skeletal muscles from both healthy and colon 26 adenocarcinoma-bearing mice, an appropriate model for testing the impact of cytostatic agents. Muscle wasting was seen in both groups of mice 4 days after a single cystemustine injection, and the drug further increased the loss of muscle proteins already apparent in tumor-bearing animals. Cystemustine cured the tumor-bearing mice with 100% efficacy. Surprisingly, within 11 days of treatment, rates of muscle proteolysis progressively decreased below basal levels observed in healthy control mice and contributed to the cessation of muscle wasting. Proteasome-dependent proteolysis was inhibited by mechanisms that include reduced mRNA levels for 20S and 26S proteasome subunits, decreased protein levels of 20S proteasome subunits and the S14 non-ATPase subunit of the 26S proteasome, and impaired chymotrypsin- and trypsin-like activities of the enzyme. A combination of cisplatin and ifosfamide, two drugs that are widely used in the treatment of cancer patients, also depressed the expression of proteasomal subunits in muscles from rats bearing the MatB adenocarcinoma below basal levels. Thus, a down-regulation of ubiquitin-proteasome-dependent proteolysis is observed with various cytostatic agents and contributes to reverse the chemotherapy-induced muscle wasting.

Olfactory fingerprints for major histocompatibility complex-determined body odors.

Science.gov (United States)

Schaefer, M L; Young, D A; Restrepo, D

2001-04-01

Recognition of individual body odors is analogous to human face recognition in that it provides information about identity. Individual body odors determined by differences at the major histocompatibility complex (MHC or H-2) have been shown to influence mate choice, pregnancy block, and maternal behavior in mice. Unfortunately, the mechanism and extent of the main olfactory bulb (MOB) and accessory olfactory bulb (AOB) involvement in the discrimination of animals according to H-2-type has remained ambiguous. Here we study the neuronal activation patterns evoked in the MOB in different individuals on exposure to these complex, biologically meaningful sensory stimuli. We demonstrate that body odors from H-2 disparate mice evoke overlapping but distinct maps of neuronal activation in the MOB. The spatial patterns of odor-evoked activity are sufficient to be used like fingerprints to predict H-2 identity using a novel computer algorithm. These results provide functional evidence for discrimination of H-2-determined body odors in the MOB, but do not preclude a role for the AOB. These data further our understanding of the neural strategies used to decode socially relevant odors.

Cycle Inhibiting Factors (Cifs: Cyclomodulins That Usurp the Ubiquitin-Dependent Degradation Pathway of Host Cells

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Eric Oswald

2011-03-01

Full Text Available Cycle inhibiting factors (Cifs are type III secreted effectors produced by diverse pathogenic bacteria. Cifs are “cyclomodulins” that inhibit the eukaryotic host cell cycle and also hijack other key cellular processes such as those controlling the actin network and apoptosis. This review summarizes current knowledge on Cif since its first characterization in enteropathogenic Escherichia coli, the identification of several xenologues in distant pathogenic bacteria, to its structure elucidation and the recent deciphering of its mode of action. Cif impairs the host ubiquitin proteasome system through deamidation of ubiquitin or the ubiquitin-like protein NEDD8 that regulates Cullin-Ring-ubiquitin Ligase (CRL complexes. The hijacking of the ubiquitin-dependent degradation pathway of host cells results in the modulation of various cellular functions such as epithelium renewal, apoptosis and immune response. Cif is therefore a powerful weapon in the continuous arm race that characterizes host-bacteria interactions.

Molecular chaperones in targeting misfolded proteins for ubiquitin-dependent degradation

DEFF Research Database (Denmark)

Kriegenburg, Franziska; Ellgaard, Lars; Hartmann-Petersen, Rasmus

2012-01-01

The accumulation of misfolded proteins presents a considerable threat to the health of individual cells and has been linked to severe diseases, including neurodegenerative disorders. Considering that, in nature, cells often are exposed to stress conditions that may lead to aberrant protein...... conformational changes, it becomes clear that they must have an efficient quality control apparatus to refold or destroy misfolded proteins. In general, cells rely on molecular chaperones to seize and refold misfolded proteins. If the native state is unattainable, misfolded proteins are targeted for degradation...... via the ubiquitin-proteasome system. The specificity of this proteolysis is generally provided by E3 ubiquitin-protein ligases, hundreds of which are encoded in the human genome. However, rather than binding the misfolded proteins directly, most E3s depend on molecular chaperones to recognize...

Mate choice for major histocompatibility complex genetic divergence as a bet-hedging strategy in the Atlantic salmon (Salmo salar)

Science.gov (United States)

Evans, Melissa L.; Dionne, Mélanie; Miller, Kristina M.; Bernatchez, Louis

2012-01-01

Major histocompatibility complex (MHC)-dependent mating preferences have been observed across vertebrate taxa and these preferences are expected to promote offspring disease resistance and ultimately, viability. However, little empirical evidence linking MHC-dependent mate choice and fitness is available, particularly in wild populations. Here, we explore the adaptive potential of previously observed patterns of MHC-dependent mate choice in a wild population of Atlantic salmon (Salmo salar) in Québec, Canada, by examining the relationship between MHC genetic variation and adult reproductive success and offspring survival over 3 years of study. While Atlantic salmon choose their mates in order to increase MHC diversity in offspring, adult reproductive success was in fact maximized between pairs exhibiting an intermediate level of MHC dissimilarity. Moreover, patterns of offspring survival between years 0+ and 1+, and 1+ and 2+ and population genetic structure at the MHC locus relative to microsatellite loci indicate that strong temporal variation in selection is likely to be operating on the MHC. We interpret MHC-dependent mate choice for diversity as a likely bet-hedging strategy that maximizes parental fitness in the face of temporally variable and unpredictable natural selection pressures. PMID:21697172

A Recombinant Antibody with the Antigen-Specific, Major Histocompatibility Complex-Restricted Specificity of T Cells

Science.gov (United States)

Andersen, Peter S.; Stryhn, Anette; Hansen, Bjarke E.; Fugger, Lars; Engberg, Jan; Buus, Soren

1996-03-01

Specific recognition of peptide/major histocompatibility complex (MHC) molecule complexes by the T-cell receptor is a key reaction in the specific immune response. Antibodies against peptide/MHC complexes would therefore be valuable tools in studying MHC function and T-cell recognition and might lead to novel approaches in immunotherapy. However, it has proven difficult to generate antibodies with the specificity of T cells by conventional hybridoma techniques. Here we report that the phage display technology is a feasible alternative to generate antibodies recognizing specific, predetermined peptide/MHC complexes.

Dynamic survey of mitochondria by ubiquitin

Science.gov (United States)

Escobar-Henriques, Mafalda; Langer, Thomas

2014-01-01

Ubiquitin is a post-translational modifier with proteolytic and non-proteolytic roles in many biological processes. At mitochondria, it performs regulatory homeostatic functions and contributes to mitochondrial quality control. Ubiquitin is essential for mitochondrial fusion, regulates mitochondria-ER contacts, and participates in maternal mtDNA inheritance. Under stress, mitochondrial dysfunction induces ubiquitin-dependent responses that involve mitochondrial proteome remodeling and culminate in organelle removal by mitophagy. In addition, many ubiquitin-dependent mechanisms have been shown to regulate innate immune responses and xenophagy. Here, we review the emerging roles of ubiquitin at mitochondria. PMID:24569520

Chicken major histocompatibility complex-encoded B-G antigens are found on many cell types that are important for the immune system

DEFF Research Database (Denmark)

Salomonsen, J; Dunon, D; Skjødt, K

1991-01-01

B-G antigens are a polymorphic multigene family of cell surface molecules encoded by the chicken major histocompatibility complex (MHC). They have previously been described only on cells of the erythroid lineage. By using flow cytometry, section staining, and immunoprecipitation with monoclonal a...

Spatial variation and low diversity in the major histocompatibility complex in walrus (Odobenus rosmarus)

Science.gov (United States)

Sonsthagen, Sarah A.; Fales, Krystal; Jay, Chadwick V.; Sage, George K.; Talbot, Sandra L.

2014-01-01

Increased global temperature and associated changes to Arctic habitats will likely result in the northward advance of species, including an influx of pathogens novel to the Arctic. How species respond to these immunological challenges will depend in part on the adaptive potential of their immune response system. We compared levels of genetic diversity at a gene associated with adaptive immune response [Class II major histocompatibility complex (MHC), DQB exon 2] between populations of walrus (Odobenus rosmarus), a sea ice-dependent Arctic species. Walrus was represented by only five MHC DQB alleles, with frequency differences observed between Pacific and Atlantic populations. MHC DQB alleles appear to be under balancing selection, and most (80 %; n = 4/5) of the alleles were observed in walruses from both oceans, suggesting broad scale differences in the frequency of exposure and diversity of pathogens may be influencing levels of heterozygosity at DQB in walruses. Limited genetic diversity at MHC, however, suggests that walrus may have a reduced capacity to respond to novel immunological challenges associated with shifts in ecological communities and environmental stressors predicted for changing climates. This is particularly pertinent for walrus, since reductions in summer sea ice may facilitate both northward expansion of marine species and associated pathogens from more temperate regions, and exchange of marine mammals and associated pathogens through the recently opened Northwest Passage between the Atlantic and Pacific Oceans in the Canadian high Arctic.

Evolutionary Analysis of Minor Histocompatibility Genes In Hydra

KAUST Repository

Aalismail, Nojood

2016-01-01

In the present study we took initiative to study the self/nonself recognition in hydra and its relation to the immune response. Moreover, performing phylogenetic analysis to look for annotated immune genes in hydra gave us a potential to analyze the expression of minor histocompatibility genes that have been shown to play a major role in grafting and transplantation in mammals. Here we obtained the cDNA library that shows expression of minor histocompatibility genes and confirmed that the annotated sequences in databases are actually present. In addition, grafting experiments suggested, although still preliminary, that homograft showed less rejection response than in heterograft. Involvement of possible minor histocompatibility gene orthologous in immune response was examined by qPCR.

The Major Histocompatibility Complex and Perfumers' Descriptions of Human Body Odors

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Claus Wedekind

2007-04-01

Full Text Available The MHC (major histocompatibility complex is a group of genes that play a crucial role in immune recognition and in tolerance of tissue grafting. The MHC has also been found to influence body odors, body odor preferences, and mate choice in mice and humans. Here we test whether verbal descriptions of human body odors can be linked to the MHC. We asked 45 male students to live as odor neutral as possible for two consecutive days and to wear a T-shirt during the nights. The odors of these T-shirts were then described by five evaluators: two professional perfumers and three laymen. One of the perfumers was able to describe the T-shirt odors in such a way that some of the allelic specificity of the MHC was significantly revealed (after Bonferroni correction for multiple testing. This shows that, although difficult, some people are able to describe MHC-correlated body odor components.

Constitutive endocytosis and turnover of the neuronal glycine transporter GlyT2 is dependent on ubiquitination of a C-terminal lysine cluster.

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Jaime de Juan-Sanz

Full Text Available Inhibitory glycinergic neurotransmission is terminated by sodium and chloride-dependent plasma membrane glycine transporters (GlyTs. The mainly glial glycine transporter GlyT1 is primarily responsible for the completion of inhibitory neurotransmission and the neuronal glycine transporter GlyT2 mediates the reuptake of the neurotransmitter that is used to refill synaptic vesicles in the terminal, a fundamental role in the physiology and pathology of glycinergic neurotransmission. Indeed, inhibitory glycinergic neurotransmission is modulated by the exocytosis and endocytosis of GlyT2. We previously reported that constitutive and Protein Kinase C (PKC-regulated endocytosis of GlyT2 is mediated by clathrin and that PKC accelerates GlyT2 endocytosis by increasing its ubiquitination. However, the role of ubiquitination in the constitutive endocytosis and turnover of this protein remains unexplored. Here, we show that ubiquitination of a C-terminus four lysine cluster of GlyT2 is required for constitutive endocytosis, sorting into the slow recycling pathway and turnover of the transporter. Ubiquitination negatively modulates the turnover of GlyT2, such that increased ubiquitination driven by PKC activation accelerates transporter degradation rate shortening its half-life while decreased ubiquitination increases transporter stability. Finally, ubiquitination of GlyT2 in neurons is highly responsive to the free pool of ubiquitin, suggesting that the deubiquitinating enzyme (DUB ubiquitin C-terminal hydrolase-L1 (UCHL1, as the major regulator of neuronal ubiquitin homeostasis, indirectly modulates the turnover of GlyT2. Our results contribute to the elucidation of the mechanisms underlying the dynamic trafficking of this important neuronal protein which has pathological relevance since mutations in the GlyT2 gene (SLC6A5 are the second most common cause of human hyperekplexia.

Allogeneic major histocompatibility complex-mismatched equine bone marrow-derived mesenchymal stem cells are targeted for death by cytotoxic anti-major histocompatibility complex antibodies.

Science.gov (United States)

Berglund, A K; Schnabel, L V

2017-07-01

Allogeneic mesenchymal stem cells (MSCs) are a promising cell source for treating musculoskeletal injuries in horses. Controversy exists, however, over whether major histocompatibility complex (MHC)-mismatched MSCs are recognised by the recipient immune system and targeted for death by a cytotoxic antibody response. To determine if cytotoxic anti-MHC antibodies generated in vivo following MHC-mismatched MSC injections are capable of initiating complement-dependent cytotoxicity of MSCs. Experimental controlled study. Antisera previously collected at Days 0, 7, 14 and 21 post-injection from 4 horses injected with donor MHC-mismatched equine leucocyte antigen (ELA)-A2 haplotype MSCs and one control horse injected with donor MHC-matched ELA-A2 MSCs were utilised in this study. Antisera were incubated with ELA-A2 MSCs before adding complement in microcytotoxicity assays and cell death was analysed via eosin dye exclusion. ELA-A2 peripheral blood leucocytes (PBLs) were used in the assays as a positive control. Antisera from all 4 horses injected with MHC-mismatched MSCs contained antibodies that caused the death of ELA-A2 haplotype MSCs in the microcytotoxicity assays. In 2 of the 4 horses, antibodies were present as early as Day 7 post-injection. MSC death was consistently equivalent to that of ELA-A2 haplotype PBL death at all time points and antisera dilutions. Antisera from the control horse that was injected with MHC-matched MSCs did not contain cytotoxic ELA-A2 antibodies at any of the time points examined. This study examined MSC death in vitro only and utilized antisera from a small number of horses. The cytotoxic antibody response induced in recipient horses following injection with donor MHC-mismatched MSCs is capable of killing donor MSCs in vitro. These results suggest that the use of allogeneic MHC-mismatched MSCs must be cautioned against, not only for potential adverse events, but also for reduced therapeutic efficacy due to targeted MSC death. © 2016 The

T-cell activation. VI. Inhibitory and stimulatory effects of anti-major histocompatibility complex class I antibodies in allogeneic mixed lymphocyte culture

DEFF Research Database (Denmark)

Röpke, M; Röpke, C; Claesson, Mogens Helweg

1993-01-01

Murine T splenocytes stimulated in primary allogeneic mixed lymphocyte culture (MLC) were incubated with soluble anti-major histocompatibility complex (MHC) class I monoclonal antibodies. These antibodies induced inhibition in the cytotoxicity of the responding population and this inhibition...... was not dependent on the domain on class I molecules recognized by the antibodies. Cross-reactivity of the antibodies between the responder and stimulating cell population caused a marked reduction in the inhibitory effect compared to systems where no such cross-reactivity was present. Saturating levels...... of the antibodies caused a reduction in generation of T-cell cytotoxicity, whereas low concentrations stimulated the same response. These results demonstrate that the MHC class I molecules of T cells are of significant importance in antigen-induced signal transduction....

The functional interplay between the HIF pathway and the ubiquitin system - more than a one-way road.

Science.gov (United States)

Günter, Julia; Ruiz-Serrano, Amalia; Pickel, Christina; Wenger, Roland H; Scholz, Carsten C

2017-07-15

The hypoxia inducible factor (HIF) pathway and the ubiquitin system represent major cellular processes that are involved in the regulation of a plethora of cellular signaling pathways and tissue functions. The ubiquitin system controls the ubiquitination of proteins, which is the covalent linkage of one or several ubiquitin molecules to specific targets. This ubiquitination is catalyzed by approximately 1000 different E3 ubiquitin ligases and can lead to different effects, depending on the type of internal ubiquitin chain linkage. The best-studied function is the targeting of proteins for proteasomal degradation. The activity of E3 ligases is antagonized by proteins called deubiquitinases (or deubiquitinating enzymes), which negatively regulate ubiquitin chains. This is performed in most cases by the catalytic removal of these chains from the targeted protein. The HIF pathway is regulated in an oxygen-dependent manner by oxygen-sensing hydroxylases. Covalent modification of HIFα subunits leads to the recruitment of an E3 ligase complex via the von Hippel-Lindau (VHL) protein and the subsequent polyubiquitination and proteasomal degradation of HIFα subunits, demonstrating the regulation of the HIF pathway by the ubiquitin system. This unidirectional effect of an E3 ligase on the HIF pathway is the best-studied example for the interplay between these two important cellular processes. However, additional regulatory mechanisms of the HIF pathway through the ubiquitin system are emerging and, more recently, also the reciprocal regulation of the ubiquitin system through components of the HIF pathway. Understanding these mechanisms and their relevance for the activity of each other is of major importance for the comprehensive elucidation of the oxygen-dependent regulation of cellular processes. This review describes the current knowledge of the functional bidirectional interplay between the HIF pathway and the ubiquitin system on the protein level. Copyright © 2017

Isolation and characterization of major histocompatibility complex class II B genes in cranes.

Science.gov (United States)

Kohyama, Tetsuo I; Akiyama, Takuya; Nishida, Chizuko; Takami, Kazutoshi; Onuma, Manabu; Momose, Kunikazu; Masuda, Ryuichi

2015-11-01

In this study, we isolated and characterized the major histocompatibility complex (MHC) class II B genes in cranes. Genomic sequences spanning exons 1 to 4 were amplified and determined in 13 crane species and three other species closely related to cranes. In all, 55 unique sequences were identified, and at least two polymorphic MHC class II B loci were found in most species. An analysis of sequence polymorphisms showed the signature of positive selection and recombination. A phylogenetic reconstruction based on exon 2 sequences indicated that trans-species polymorphism has persisted for at least 10 million years, whereas phylogenetic analyses of the sequences flanking exon 2 revealed a pattern of concerted evolution. These results suggest that both balancing selection and recombination play important roles in the crane MHC evolution.

Ubiquitin fusion expression and tissue-dependent targeting of hG-CSF in transgenic tobacco

Science.gov (United States)

2011-01-01

Background Human granulocyte colony-stimulating factor (hG-CSF) is an important human cytokine which has been widely used in oncology and infection protection. To satisfy clinical needs, expression of recombinant hG-CSF has been studied in several organisms, including rice cell suspension culture and transient expression in tobacco leaves, but there was no published report on its expression in stably transformed plants which can serve as a more economical expression platform with potential industrial application. Results In this study, hG-CSF expression was investigated in transgenic tobacco leaves and seeds in which the accumulation of hG-CSF could be enhanced through fusion with ubiquitin by up to 7 fold in leaves and 2 fold in seeds, leading to an accumulation level of 2.5 mg/g total soluble protein (TSP) in leaves and 1.3 mg/g TSP in seeds, relative to hG-CSF expressed without a fusion partner. Immunoblot analysis showed that ubiquitin was processed from the final protein product, and ubiquitination was up-regulated in all transgenic plants analyzed. Driven by CaMV 35S promoter and phaseolin signal peptide, hG-CSF was observed to be secreted into apoplast in leaves but deposited in protein storage vacuole (PSV) in seeds, indicating that targeting of the hG-CSF was tissue-dependent in transgenic tobacco. Bioactivity assay showed that hG-CSF expressed in both seeds and leaves was bioactive to support the proliferation of NFS-60 cells. Conclusions In this study, the expression of bioactive hG-CSF in transgenic plants was improved through ubiquitin fusion strategy, demonstrating that protein expression can be enhanced in both plant leaves and seeds through fusion with ubiquitin and providing a typical case of tissue-dependent expression of recombinant protein in transgenic plants. PMID:21985646

New horizons in mouse immunoinformatics: reliable in silico prediction of mouse class I histocompatibility major complex peptide binding affinity.

Science.gov (United States)

Hattotuwagama, Channa K; Guan, Pingping; Doytchinova, Irini A; Flower, Darren R

2004-11-21

Quantitative structure-activity relationship (QSAR) analysis is a main cornerstone of modern informatic disciplines. Predictive computational models, based on QSAR technology, of peptide-major histocompatibility complex (MHC) binding affinity have now become a vital component of modern day computational immunovaccinology. Historically, such approaches have been built around semi-qualitative, classification methods, but these are now giving way to quantitative regression methods. The additive method, an established immunoinformatics technique for the quantitative prediction of peptide-protein affinity, was used here to identify the sequence dependence of peptide binding specificity for three mouse class I MHC alleles: H2-D(b), H2-K(b) and H2-K(k). As we show, in terms of reliability the resulting models represent a significant advance on existing methods. They can be used for the accurate prediction of T-cell epitopes and are freely available online ( http://www.jenner.ac.uk/MHCPred).

Evaluation of two approaches to genotyping major histocompatibility complex class I in a passerine—CE-SSCP and 454 pyrosequencing

Czech Academy of Sciences Publication Activity Database

Promerová, Marta; Babik, W.; Bryja, Josef; Albrecht, Tomáš; Stuglik, M.; Radwan, J.

2012-01-01

Roč. 12, č. 2 (2012), s. 285-292 ISSN 1755-098X R&D Projects: GA AV ČR IAA600930608; GA ČR GA206/06/0851; GA ČR GAP505/10/1871 Institutional research plan: CEZ:AV0Z60930519 Keywords : avian * Carpodacus erythrinus * major histocompatibility complex * next-generation sequencing * scarlet rosefinch Subject RIV: EG - Zoology Impact factor: 7.432, year: 2012

Structural requirements and biological significance of interactions between peptides and the major histocompatibility complex

DEFF Research Database (Denmark)

Grey, H M; Buus, S; Colon, S

1989-01-01

Previous studies indicate that T cells recognize a complex between the major histocompatibility complex (MHC) restriction-element and peptide-antigen fragments. Two aspects of this complex formation are considered in this paper: (1) what is the nature of the specificity of the interactions that a...... of binding to Ia (i.e. determinant selection was operative), we found that about 40% of Ia-binding peptides were not immunogenic (i.e. there were also 'holes in the T-cell repertoire')....... responsiveness, we present data that suggest both mechanisms operate in concert with one another. Thus only about 30% of a collection of peptides that in sum represent the sequence of a protein molecule were found to bind to Ia. Although immunogenicity was restricted to those peptides that were capable...

Human major histocompatibility complex contains a minimum of 19 genes between the complement cluster and HLA-B

International Nuclear Information System (INIS)

Spies, T.; Bresnahan, M.; Strominger, J.L.

1989-01-01

A 600-kilobase (kb) DNA segment from the human major histocompatibility complex (MHC) class III region was isolated by extension of a previous 435-kb chromosome walk. The contiguous series of cloned overlapping cosmids contains the entire 555-kb interval between C2 in the complement gene cluster and HLA-B. This region is known to encode the tumor necrosis factors (TNFs) α and β, B144, and the major heat shock protein HSP70. Moreover, a cluster of genes, BAT1-BAT5 (HLA-B-associated transcripts) have been localized in the vicinity of the genes for TNFα and TNFβ. An additional four genes were identified by isolation of corresponding cDNA clones with cosmid DNA probes. These genes for BAT6-BAT9 were mapped near the gene for C2 within a 120-kb region that includes a HSP70 gene pair. These results, together with complementary data from a similar recent study, indicated the presence of a minimum of 19 genes within the C2-HLA-B interval of the MHC class III region. Although the functional properties of most of these genes are yet unknown, they may be involved in some aspects of immunity. This idea is supported by the genetic mapping of the hematopoietic histocompatibility locus-1 (Hh-1) in recombinant mice between TNFα and H-2S, which is homologous to the complement gene cluster in humans

The major histocompatibility complex genes impact pain response in DA and DA.1U rats.

Science.gov (United States)

Guo, Yuan; Yao, Fan-Rong; Cao, Dong-Yuan; Li, Li; Wang, Hui-Sheng; Xie, Wen; Zhao, Yan

2015-08-01

Our recent studies have shown that the difference in basal pain sensitivity to mechanical and thermal stimulation between Dark-Agouti (DA) rats and a novel congenic DA.1U rats is major histocompatibility complex (MHC) genes dependent. In the present study, we further used DA and DA.1U rats to investigate the role of MHC genes in formalin-induced pain model by behavioral, electrophysiological and immunohistochemical methods. Behavioral results showed biphasic nociceptive behaviors increased significantly following the intraplantar injection of formalin in the hindpaw of DA and DA.1U rats. The main nociceptive behaviors were lifting and licking, especially in DA rats (PDA rats were significantly higher than those in DA.1U rats in both phases of the formalin test (PDA rats was significantly higher than that of DA.1U rats (PDA was greater than that in DA.1U rats (PDA rats was significantly higher than that in DA.1U rats in the respective experimental group (PDA and DA.1U rats exhibited nociceptive responses in formalin-induced pain model and DA rats were more sensitive to noxious chemical stimulus than DA.1U rats, indicating that MHC genes might contribute to the difference in pain sensitivity. Copyright © 2015 Elsevier Inc. All rights reserved.

Cuz1/Ynl155w, a Zinc-dependent Ubiquitin-binding Protein, Protects Cells from Metalloid-induced Proteotoxicity*

Science.gov (United States)

Hanna, John; Waterman, David; Isasa, Marta; Elsasser, Suzanne; Shi, Yuan; Gygi, Steven; Finley, Daniel

2014-01-01

Protein misfolding is a universal threat to cells. The ubiquitin-proteasome system mediates a cellular stress response capable of eliminating misfolded proteins. Here we identify Cuz1/Ynl155w as a component of the ubiquitin system, capable of interacting with both the proteasome and Cdc48. Cuz1/Ynl155w is regulated by the transcription factor Rpn4, and is required for cells to survive exposure to the trivalent metalloids arsenic and antimony. A related protein, Yor052c, shows similar phenotypes, suggesting a multicomponent stress response pathway. Cuz1/Ynl155w functions as a zinc-dependent ubiquitin-binding protein. Thus, Cuz1/Ynl155w is proposed to protect cells from metalloid-induced proteotoxicity by delivering ubiquitinated substrates to Cdc48 and the proteasome for destruction. PMID:24297164

Opposing roles of RNF8/RNF168 and deubiquitinating enzymes in ubiquitination-dependent DNA double-strand break response signaling and DNA-repair pathway choice

International Nuclear Information System (INIS)

Nakada, Shinichiro

2016-01-01

The E3 ubiquitin ligases ring finger protein (RNF) 8 and RNF168 transduce the DNA double-strand break (DSB) response (DDR) signal by ubiquitinating DSB sites. The depletion of RNF8 or RNF168 suppresses the accumulation of DNA-repair regulating factors such as 53BP1 and RAP80 at DSB sites, suggesting roles for RNF8- and RNF168-mediated ubiquitination in DSB repair. This mini-review provides a brief overview of the RNF8- and RNF168-dependent DDR-signaling and DNA-repair pathways. The choice of DNA-repair pathway when RNF8- and RNF168-mediated ubiquitination-dependent DDR signaling is negatively regulated by deubiquitinating enzymes (DUBs) is reviewed to clarify how the opposing roles of RNF8/RNF168 and DUBs regulate ubiquitination-dependent DDR signaling and the choice of DNA-repair pathway

Multiple major histocompatibility complex class I genes in Asian anurans: Ontogeny and phylogeny.

Science.gov (United States)

Didinger, Chelsea; Eimes, John A; Lillie, Mette; Waldman, Bruce

2017-05-01

Amphibians, as the first terrestrial vertebrates, offer a window into early major histocompatibility complex (MHC) evolution. We characterized the MHC class I of two Korean amphibians, the Asiatic toad (Bufo gargarizans) and the Japanese tree frog (Hyla japonica). We found at least four transcribed MHC class I (MHC I) loci, the highest number confirmed in any anuran to date. Furthermore, we identified MHC I transcripts in terrestrial adults, and possibly in aquatic larvae, of both species. We conducted a phylogenetic analysis based on MHC I sequence data and found that B. gargarizans and H. japonica cluster together in the superfamily Nobleobatrachia. We further identified three supertypes shared by the two species. Our results reveal substantial variation in the number of MHC I loci in anurans and suggest that certain supertypes have particular physiochemical properties that may confer pathogen resistance. Copyright © 2016 Elsevier Ltd. All rights reserved.

Characterisation of major histocompatibility complex class I transcripts in an Australian dragon lizard.

Science.gov (United States)

Hacking, Jessica; Bertozzi, Terry; Moussalli, Adnan; Bradford, Tessa; Gardner, Michael

2018-07-01

Characterisation of squamate major histocompatibility complex (MHC) genes has lagged behind other taxonomic groups. MHC genes encode cell-surface glycoproteins that present self- and pathogen-derived peptides to T cells and play a critical role in pathogen recognition. Here we characterise MHC class I transcripts for an agamid lizard (Ctenophorus decresii) and investigate the evolution of MHC class I in Iguanian lizards. An iterative assembly strategy was used to identify six full-length C. decresii MHC class I transcripts, which were validated as likely to encode classical class I MHC molecules. Evidence for exon shuffling recombination was uncovered for C. decresii transcripts and Bayesian phylogenetic analysis of Iguanian MHC class I sequences revealed a pattern expected under a birth-and-death mode of evolution. This work provides a stepping stone towards further research on the agamid MHC class I region. Copyright © 2018 Elsevier Ltd. All rights reserved.

Reconstructing an ancestral mammalian immune supercomplex from a marsupial major histocompatibility complex.

Directory of Open Access Journals (Sweden)

Katherine Belov

2006-03-01

Full Text Available The first sequenced marsupial genome promises to reveal unparalleled insights into mammalian evolution. We have used the Monodelphis domestica (gray short-tailed opossum sequence to construct the first map of a marsupial major histocompatibility complex (MHC. The MHC is the most gene-dense region of the mammalian genome and is critical to immunity and reproductive success. The marsupial MHC bridges the phylogenetic gap between the complex MHC of eutherian mammals and the minimal essential MHC of birds. Here we show that the opossum MHC is gene dense and complex, as in humans, but shares more organizational features with non-mammals. The Class I genes have amplified within the Class II region, resulting in a unique Class I/II region. We present a model of the organization of the MHC in ancestral mammals and its elaboration during mammalian evolution. The opossum genome, together with other extant genomes, reveals the existence of an ancestral "immune supercomplex" that contained genes of both types of natural killer receptors together with antigen processing genes and MHC genes.

Detection of autoreactive CD4 T cells using major histocompatibility complex class II dextramers

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Kuszynski Charles

2011-07-01

Full Text Available Abstract Background Tetramers are useful tools to enumerate the frequencies of antigen-specific T cells. However, unlike CD8 T cells, CD4 T cells - especially self-reactive cells - are challenging to detect with major histocompatibility complex (MHC class II tetramers because of low frequencies and low affinities of their T cell receptors to MHC-peptide complexes. Here, we report the use of fluorescent multimers, designated MHC dextramers that contain a large number of peptide-MHC complexes per reagent. Results The utility of MHC dextramers was evaluated in three autoimmune disease models: 1 proteolipid protein (PLP 139-151-induced experimental autoimmune encephalomyelitis in SJL/J (H-2s mice; 2 myelin oligodendrocyte glycoprotein (MOG 35-55-induced experimental autoimmune encephalomyelitis in C57Bl/6 (H-2b mice; and 3 cardiac myosin heavy chain (Myhc-α 334-352-induced experimental autoimmune myocarditis in A/J (H-2a mice. Flow cytometrically, we demonstrate that IAs/PLP 139-151, IAb/MOG 35-55 and IAk/Myhc-α 334-352 dextramers detect the antigen-sensitized cells with specificity, and with a detection sensitivity significantly higher than that achieved with conventional tetramers. Furthermore, we show that binding of dextramers, but not tetramers, is less dependent on the activation status of cells, permitting enumeration of antigen-specific cells ex vivo. Conclusions The data suggest that MHC dextramers are useful tools to track the generation and functionalities of self-reactive CD4 cells in various experimental systems.

Terminating protein ubiquitination: Hasta la vista, ubiquitin.

Science.gov (United States)

Stringer, Daniel K; Piper, Robert C

2011-09-15

Ubiquitination is a post-translational modification that generally directs proteins for degradation by the proteasome or by lysosomes. However, ubiquitination has been implicated in many other cellular processes, including transcriptional regulation, DNA repair, regulation of protein-protein interactions and association with ubiquitin-binding scaffolds. Ubiquitination is a dynamic process. Ubiquitin is added to proteins by E3 ubiquitin ligases as a covalent modification to one or multiple lysine residues as well as non-lysine amino acids. Ubiquitin itself contains seven lysines, each of which can also be ubiquitinated, leading to polyubiquitin chains that are best characterized for linkages occurring through K48 and K63. Ubiquitination can also be reversed by the action of deubiquitination enzymes (DUbs). Like E3 ligases, DUbs play diverse and critical roles in cells. ( 1) Ubiquitin is expressed as a fusion protein, as a linear repeat or as a fusion to ribosomal subunits, and DUbs are necessary to liberate free ubiquitin, making them the first enzyme of the ubiquitin cascade. Proteins destined for degradation by the proteasome or by lysosomes are deubiquitinated prior to their degradation, which allows ubiquitin to be recycled by the cell, contributing to the steady-state pool of free ubiquitin. Proteins destined for degradation by lysosomes are also acted upon by both ligases and DUbs. Deubiquitination can also act as a means to prevent protein degradation, and many proteins are thought to undergo rounds of ubiquitination and deubiquitination, ultimately resulting in either the degradation or stabilization of those proteins. Despite years of study, examining the effects of the ubiquitination of proteins remains quite challenging. This is because the methods that are currently being employed to study ubiquitination are limiting. Here, we briefly examine current strategies to study the effects of ubiquitination and describe an additional novel approach that we have

SUMO and ubiquitin-dependent XPC exchange drives nucleotide excision repair

DEFF Research Database (Denmark)

Van Cuijk, Loes; Van Belle, Gijsbert J.; Turkyilmaz, Yasemin

2015-01-01

XPC recognizes UV-induced DNA lesions and initiates their removal by nucleotide excision repair (NER). Damage recognition in NER is tightly controlled by ubiquitin and SUMO modifications. Recent studies have shown that the SUMO-targeted ubiquitin ligase RNF111 promotes K63-linked ubiquitylation o...

The Roles of VHL-Dependent Ubiquitination in Signaling and Cancer

Energy Technology Data Exchange (ETDEWEB)

Zhang, Qing [Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA (United States); Yang, Haifeng, E-mail: qing_zhang@dfci.harvard.edu [Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH (United States); Haifeng Yang, E-mail: yangh2@ccf.org [Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, NB-40, 9500 Euclid Avenue, Cleveland, OH 44195 (United States)

2012-04-12

The function of tumor suppressor VHL is compromised in the vast majority of clear cell renal cell carcinoma, and its mutations or loss of expression was causal for this disease. pVHL was found to be a substrate recognition subunit of an E3 ubiquitin ligase, and most of the tumor-derived mutations disrupt this function. pVHL was found to bind to the alpha subunits of hypoxia-inducible factor (HIF) and promote their ubiquitination and proteasomal degradation. Proline hydroxylation on key sites of HIFα provides the binding signal for pVHL E3 ligase complex. Beside HIFα, several other VHL targets have been identified, including activated epidermal growth factor receptor (EGFR), RNA polymerase II subunits RPB1 and hsRPB7, atypical protein kinase C (PKC), Sprouty2, β-adrenergic receptor II, and Myb-binding protein p160. HIFα is the most well studied substrate and has been proven to be critical for pVHL’s tumor suppressor function, but the activated EGFR and PKC and other pVHL substrates might also be important for tumor growth and drug response. Their regulations by pVHL and their relevance to signaling and cancer are discussed.

RFWD3-Dependent Ubiquitination of RPA Regulates Repair at Stalled Replication Forks.

Science.gov (United States)

Elia, Andrew E H; Wang, David C; Willis, Nicholas A; Boardman, Alexander P; Hajdu, Ildiko; Adeyemi, Richard O; Lowry, Elizabeth; Gygi, Steven P; Scully, Ralph; Elledge, Stephen J

2015-10-15

We have used quantitative proteomics to profile ubiquitination in the DNA damage response (DDR). We demonstrate that RPA, which functions as a protein scaffold in the replication stress response, is multiply ubiquitinated upon replication fork stalling. Ubiquitination of RPA occurs on chromatin, involves sites outside its DNA binding channel, does not cause proteasomal degradation, and increases under conditions of fork collapse, suggesting a role in repair at stalled forks. We demonstrate that the E3 ligase RFWD3 mediates RPA ubiquitination. RFWD3 is necessary for replication fork restart, normal repair kinetics during replication stress, and homologous recombination (HR) at stalled replication forks. Mutational analysis suggests that multisite ubiquitination of the entire RPA complex is responsible for repair at stalled forks. Multisite protein group sumoylation is known to promote HR in yeast. Our findings reveal a similar requirement for multisite protein group ubiquitination during HR at stalled forks in mammalian cells. Copyright © 2015 Elsevier Inc. All rights reserved.

Ubiquitin Proteasome System in Parkinson Disease: a keeper or a witness?

Science.gov (United States)

Martins-Branco, Diogo; Esteves, Ana R.; Santos, Daniel; Arduino, Daniela M.; Swerdlow, Russell H.; Oliveira, Catarina R.; Januario, Cristina; Cardoso, Sandra M.

2014-01-01

Objective The aim of this work was to evaluate the role of Ubiquitin-Proteasome System (UPS) on mitochondrial-driven alpha-synuclein (aSN) clearance in in vitro, ex vivo and in vivo Parkinson disease (PD) cellular models. Method We used SH-SY5Y ndufa2 knock-down (KD) cells, PD cybrids and peripheral blood mononuclear cells (PBMC) from patients meeting the diagnostic criteria for PD. We quantified aSN aggregation, proteasome activity and protein ubiquitination levels. In PBMC of PD patients population we evaluated aSN levels in plasma and the influence of several demographic characteristics in the above mentioned determinations. Results We found that ubiquitin-independent proteasome activity was up-regulated in SH-SY5Y ndufa2 KD cells while a down regulation was observed in PD cybrids and PBMC. Moreover, we observed an increase in protein ubiquitination that correlates with a decrease in ubiquitin-dependent proteasome activity. Accordingly, proteasome inhibition prevented ubiquitin-dependent aSN clearance. Ubiquitin-independent proteasome activity was positively correlated with ubiquitination in PBMC. We also report a negative correlation of chymotrypsin-like activity with age in control and late-onset PD groups. Total ubiquitin content is positively correlated with aSN oligomers levels, which leads to an age-dependent increase of aSN ubiquitination in LOPD. Moreover, aSN levels are increased in the plasma of PD patients. Interpretation aSN oligomers are ubiquitinated and we identified an ubiquitin-dependent clearance insufficiency with accumulation of both aSN and ubiquitin. However, SH-SY5Y ndufa2 KD cells showed a significant up-regulation of ubiquitin-independent proteasomal enzymatic activity that could mean a cell rescue attempt. Moreover, we identified that UPS function is age-dependent in PBMC. PMID:22921536

The human otubain2-ubiquitin structure provides insights into the cleavage specificity of poly-ubiquitin-linkages.

Directory of Open Access Journals (Sweden)

Mikael Altun

Full Text Available Ovarian tumor domain containing proteases cleave ubiquitin (Ub and ubiquitin-like polypeptides from proteins. Here we report the crystal structure of human otubain 2 (OTUB2 in complex with a ubiquitin-based covalent inhibitor, Ub-Br2. The ubiquitin binding mode is oriented differently to how viral otubains (vOTUs bind ubiquitin/ISG15, and more similar to yeast and mammalian OTUs. In contrast to OTUB1 which has exclusive specificity towards Lys48 poly-ubiquitin chains, OTUB2 cleaves different poly-Ub linked chains. N-terminal tail swapping experiments between OTUB1 and OTUB2 revealed how the N-terminal structural motifs in OTUB1 contribute to modulating enzyme activity and Ub-chain selectivity, a trait not observed in OTUB2, supporting the notion that OTUB2 may affect a different spectrum of substrates in Ub-dependent pathways.

Oriented coupling of major histocompatibility complex (MHC) to sensor surfaces using light assisted immobilisation technology

DEFF Research Database (Denmark)

Snabe, Torben; Røder, Gustav Andreas; Neves-Petersen, Maria Teresa

2005-01-01

Controlled and oriented immobilisation of proteins for biosensor purposes is of extreme interest since this provides more efficient sensors with a larger density of active binding sites per area compared to sensors produced by conventional immobilisation. In this paper oriented coupling of a major...... histocompatibility complex (MHC class I) to a sensor surface is presented. The coupling was performed using light assisted immobilisation--a novel immobilisation technology which allows specific opening of particular disulphide bridges in proteins which then is used for covalent bonding to thiol-derivatised surfaces...... via a new disulphide bond. Light assisted immobilisation specifically targets the disulphide bridge in the MHC-I molecule alpha(3)-domain which ensures oriented linking of the complex with the peptide binding site exposed away from the sensor surface. Structural analysis reveals that a similar...

Ubiquitination of specific mitochondrial matrix proteins

International Nuclear Information System (INIS)

Lehmann, Gilad; Ziv, Tamar; Braten, Ori; Admon, Arie; Udasin, Ronald G.; Ciechanover, Aaron

2016-01-01

Several protein quality control systems in bacteria and/or mitochondrial matrix from lower eukaryotes are absent in higher eukaryotes. These are transfer-messenger RNA (tmRNA), The N-end rule ATP-dependent protease ClpAP, and two more ATP-dependent proteases, HslUV and ClpXP (in yeast). The lost proteases resemble the 26S proteasome and the role of tmRNA and the N-end rule in eukaryotic cytosol is performed by the ubiquitin proteasome system (UPS). Therefore, we hypothesized that the UPS might have substituted these systems – at least partially – in the mitochondrial matrix of higher eukaryotes. Using three independent experimental approaches, we demonstrated the presence of ubiquitinated proteins in the matrix of isolated yeast mitochondria. First, we show that isolated mitochondria contain ubiquitin (Ub) conjugates, which remained intact after trypsin digestion. Second, we demonstrate that the mitochondrial soluble fraction contains Ub-conjugates, several of which were identified by mass spectrometry and are localized to the matrix. Third, using immunoaffinity enrichment by specific antibodies recognizing digested ubiquitinated peptides, we identified a group of Ub-modified matrix proteins. The modification was further substantiated by separation on SDS-PAGE and immunoblots. Last, we attempted to identify the ubiquitin ligase(s) involved, and identified Dma1p as a trypsin-resistant protein in our mitochondrial preparations. Taken together, these data suggest a yet undefined role for the UPS in regulation of the mitochondrial matrix proteins. -- Highlights: •Mitochondrial matrix contains ubiquitinated proteins. •Ubiquitination occurs most probably in the matrix. •Dma1p is a ubiquitin ligase present in mitochondrial preparations.

Ubiquitination of specific mitochondrial matrix proteins

Energy Technology Data Exchange (ETDEWEB)

Lehmann, Gilad [The Janet and David Polak Tumor and Vascular Biology Research Center and the Technion Integrated Cancer Center (TICC), The Rappaport Faculty of Medicine and Research Institute, Haifa, 31096 (Israel); Ziv, Tamar [The Smoler Proteomics Center, Faculty of Biology – Technion-Israel Institute of Technology, Haifa, 32000 (Israel); Braten, Ori [The Janet and David Polak Tumor and Vascular Biology Research Center and the Technion Integrated Cancer Center (TICC), The Rappaport Faculty of Medicine and Research Institute, Haifa, 31096 (Israel); Admon, Arie [The Smoler Proteomics Center, Faculty of Biology – Technion-Israel Institute of Technology, Haifa, 32000 (Israel); Udasin, Ronald G. [The Janet and David Polak Tumor and Vascular Biology Research Center and the Technion Integrated Cancer Center (TICC), The Rappaport Faculty of Medicine and Research Institute, Haifa, 31096 (Israel); Ciechanover, Aaron, E-mail: aaroncie@tx.technion.ac.il [The Janet and David Polak Tumor and Vascular Biology Research Center and the Technion Integrated Cancer Center (TICC), The Rappaport Faculty of Medicine and Research Institute, Haifa, 31096 (Israel)

2016-06-17

Several protein quality control systems in bacteria and/or mitochondrial matrix from lower eukaryotes are absent in higher eukaryotes. These are transfer-messenger RNA (tmRNA), The N-end rule ATP-dependent protease ClpAP, and two more ATP-dependent proteases, HslUV and ClpXP (in yeast). The lost proteases resemble the 26S proteasome and the role of tmRNA and the N-end rule in eukaryotic cytosol is performed by the ubiquitin proteasome system (UPS). Therefore, we hypothesized that the UPS might have substituted these systems – at least partially – in the mitochondrial matrix of higher eukaryotes. Using three independent experimental approaches, we demonstrated the presence of ubiquitinated proteins in the matrix of isolated yeast mitochondria. First, we show that isolated mitochondria contain ubiquitin (Ub) conjugates, which remained intact after trypsin digestion. Second, we demonstrate that the mitochondrial soluble fraction contains Ub-conjugates, several of which were identified by mass spectrometry and are localized to the matrix. Third, using immunoaffinity enrichment by specific antibodies recognizing digested ubiquitinated peptides, we identified a group of Ub-modified matrix proteins. The modification was further substantiated by separation on SDS-PAGE and immunoblots. Last, we attempted to identify the ubiquitin ligase(s) involved, and identified Dma1p as a trypsin-resistant protein in our mitochondrial preparations. Taken together, these data suggest a yet undefined role for the UPS in regulation of the mitochondrial matrix proteins. -- Highlights: •Mitochondrial matrix contains ubiquitinated proteins. •Ubiquitination occurs most probably in the matrix. •Dma1p is a ubiquitin ligase present in mitochondrial preparations.

Evolutionary Analysis of Minor Histocompatibility Genes In Hydra

KAUST Repository

Aalismail, Nojood

2016-05-01

Hydra is a simple freshwater solitary polyp used as a model system to study evolutionary aspects. The immune response of this organism has not been studied extensively and the immune response genes have not been identified and characterized. On the other hand, immune response has been investigated and genetic analysis has been initiated in other lower invertebrates. In the present study we took initiative to study the self/nonself recognition in hydra and its relation to the immune response. Moreover, performing phylogenetic analysis to look for annotated immune genes in hydra gave us a potential to analyze the expression of minor histocompatibility genes that have been shown to play a major role in grafting and transplantation in mammals. Here we obtained the cDNA library that shows expression of minor histocompatibility genes and confirmed that the annotated sequences in databases are actually present. In addition, grafting experiments suggested, although still preliminary, that homograft showed less rejection response than in heterograft. Involvement of possible minor histocompatibility gene orthologous in immune response was examined by qPCR.

High-Resolution Patterns of Meiotic Recombination across the Human Major Histocompatibility Complex

Science.gov (United States)

Cullen, Michael; Perfetto, Stephen P.; Klitz, William; Nelson, George; Carrington, Mary

2002-01-01

Definitive characteristics of meiotic recombination events over large (i.e., >1 Mb) segments of the human genome remain obscure, yet they are essential for establishing the haplotypic structure of the genome and for efficient mapping of complex traits. We present a high-resolution map of recombination at the kilobase level across a 3.3-Mb interval encompassing the major histocompatibility complex (MHC). Genotyping of 20,031 single sperm from 12 individuals resulted in the identification and fine mapping of 325 recombinant chromosomes within genomic intervals as small as 7 kb. Several principal characteristics of recombination in this region were observed: (1) rates of recombination can differ significantly between individuals; (2) intense hot spots of recombination occur at least every 0.8 Mb but are not necessarily evenly spaced; (3) distribution in the location of recombination events can differ significantly among individuals; (4) between hot spots, low levels of recombination occur fairly evenly across 100-kb segments, suggesting the presence of warm spots of recombination; and (5) specific sequence motifs associate significantly with recombination distribution. These data provide a plausible model for recombination patterns of the human genome overall. PMID:12297984

Inhibition of the HDAC/Suv39/G9a pathway restores the expression of DNA damage-dependent major histocompatibility complex class I-related chain A and B in cancer cells.

Science.gov (United States)

Nakajima, Nakako Izumi; Niimi, Atsuko; Isono, Mayu; Oike, Takahiro; Sato, Hiro; Nakano, Takashi; Shibata, Atsushi

2017-08-01

Immunotherapy is expected to be promising as a next generation cancer therapy. Immunoreceptors are often activated constitutively in cancer cells, however, such levels of ligand expression are not effectively recognized by the native immune system due to tumor microenvironmental adaptation. Studies have demonstrated that natural-killer group 2, member D (NKG2D), a major activating immunoreceptor, responds to DNA damage. The upregulation of major histocompatibility complex class I-related chain A and B (MICA/B) (members of NKG2D ligands) expression after DNA damage is associated with NK cell-mediated killing of cancer cells. However, the regulation of DNA damage-induced MICA/B expression has not been fully elucidated in the context of the types of cancer cell lines. In the present study, we found that MICA/B expression varied between cancer cell lines after DNA damage. Screening in terms of chromatin remodeling identified that inhibitors related to chromatin relaxation via post-translational modification on histone H3K9, i.e. HDAC, Suv39 or G9a inhibition, restored DNA damage-dependent MICA/B expression in insensitive cells. In addition, we revealed that the restored MICA/B expression was dependent on ATR as well as E2F1, a transcription factor. We further revealed that low‑dose treatment of an HDAC inhibitor was sufficient to restore MICA/B expression in insensitive cells. Finally, we demonstrated that HDAC inhibition restored DNA damage‑dependent cytotoxic NK activity against insensitive cells. Thus, the present study revealed that DNA damage‑dependent MICA/B expression in insensitive cancer cells can be restored by chromatin relaxation via the HDAC/Suv39/G9a pathway. Collectively, manipulation of chromatin status by therapeutic cancer drugs may potentiate the antitumor effect by enhancing immune activation following radiotherapy and DNA damage-associated chemotherapy.

Clinical, Immunological, and Molecular Findings in Five Patients with Major Histocompatibility Complex Class II Deficiency from India

Directory of Open Access Journals (Sweden)

Jahnavi Aluri

2018-02-01

Full Text Available Major histocompatibility complex (MHC class II deficiency is a rare autosomal recessive form of primary immunodeficiency disorder (PID characterized by the deficiency of MHC class II molecules. This deficiency affects the cellular and humoral immune response by impairing the development of CD4+ T helper (Th cells and Th cell-dependent antibody production by B cells. Affected children typically present with severe respiratory and gastrointestinal tract infections. Hematopoietic stem cell transplantation (HSCT is the only curative therapy available for treating these patients. This is the first report from India wherein we describe the clinical, immunological, and molecular findings in five patients with MHC class II deficiency. Our patients presented with recurrent lower respiratory tract infection as the most common clinical presentation within their first year of life and had a complete absence of human leukocyte antigen-antigen D-related (HLA-DR expression on B cells and monocytes. Molecular characterization revealed novel mutations in RFAXP, RFX5, and CIITA genes. Despite genetic heterogeneity, these patients were clinically indistinguishable. Two patients underwent HSCT but had a poor survival outcome. Detectable level of T cell receptor excision circles (TRECs were measured in our patients, highlighting that this form of PID may be missed by TREC-based newborn screening program for severe combined immunodeficiency.

ABA-dependent inhibition of the ubiquitin proteasome system during germination at high temperature in Arabidopsis.

Science.gov (United States)

Chiu, Rex Shun; Pan, Shiyue; Zhao, Rongmin; Gazzarrini, Sonia

2016-12-01

During germination, endogenous and environmental factors trigger changes in the transcriptome, translatome and proteome to break dormancy. In Arabidopsis thaliana, the ubiquitin proteasome system (UPS) degrades proteins that promote dormancy to allow germination. While research on the UPS has focused on the identification of proteasomal substrates, little information is known about the regulation of its activity. Here we characterized the activity of the UPS during dormancy release and maintenance by monitoring protein ubiquitination and degradation of two proteasomal substrates: Suc-LLVY-AMC, a well characterized synthetic substrate, and FUSCA3 (FUS3), a dormancy-promoting transcription factor degraded by the 26S proteasome. Our data indicate that proteasome activity and protein ubiquitination increase during imbibition at optimal temperature (21°C), and are required for seed germination. However, abscisic acid (ABA) and supraoptimal temperature (32°C) inhibit germination by dampening both protein ubiquitination and proteasome activity. Inhibition of UPS function by high temperature is reduced by the ABA biosynthesis inhibitor, fluridone, and in ABA biosynthetic mutants, suggesting that it is ABA dependent. Accordingly, inhibition of FUS3 degradation at 32°C is also dependent on ABA. Native gels show that inhibition of proteasome activity is caused by interference with the 26S/30S ratio as well as free 19S and 20S levels, impacting the proteasome degradation cycle. Transfer experiments show that ABA-mediated inhibition of proteasome activity at 21°C is restricted to the first 2 days of germination, a time window corresponding to seed sensitivity to environmental and ABA-mediated growth inhibition. Our data show that ABA and high temperature inhibit germination under unfavourable growth conditions by repressing the UPS. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

The canonical wnt signal restricts the glycogen synthase kinase 3/fbw7-dependent ubiquitination and degradation of eya1 phosphatase.

Science.gov (United States)

Sun, Ye; Li, Xue

2014-07-01

Haploinsufficiency of Eya1 causes the branchio-oto-renal (BOR) syndrome, and abnormally high levels of Eya1 are linked to breast cancer progression and poor prognosis. Therefore, regulation of Eya1 activity is key to its tissue-specific functions and oncogenic activities. Here, we show that Eya1 is posttranslationally modified by ubiquitin and that its ubiquitination level is self-limited to prevent premature degradation. Eya1 has an evolutionarily conserved CDC4 phosphodegron (CPD) signal, a target site of glycogen synthase kinase 3 (GSK3) kinase and Fbw7 ubiquitin ligase, which is required for Eya1 ubiquitination. Genetic deletion of Fbw7 and pharmacological inhibition of GSK3 significantly decrease Eya1 ubiquitination. Conversely, activation of the phosphatidylinositol 3-kinase (PI3K)/Akt and the canonical Wnt signal suppresses Eya1 ubiquitination. Compound Eya1(+/-); Wnt9b(+/-) mutants exhibit an increased penetrance of renal defect, indicating that they function in the same genetic pathway in vivo. Together, these findings reveal that the canonical Wnt and PI3K/Akt signal pathways restrain the GSK3/Fbw7-dependent Eya1 ubiquitination, and they further suggest that dysregulation of this novel axis contributes to tumorigenesis. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Porcine major histocompatibility complex (MHC) class I molecules and analysis of their peptide-binding specificities

DEFF Research Database (Denmark)

Pedersen, Lasse Eggers; Harndahl, Mikkel; Rasmussen, Michael

2011-01-01

a HLA-I molecule (HLA-A*11:01), thereby generating recombinant human/swine chimeric MHC-I molecules as well as the intact SLA-1*0401 molecule. Biochemical peptide-binding assays and positional scanning combinatorial peptide libraries were used to analyze the peptide-binding motifs of these molecules....... A pan-specific predictor of peptide–MHC-I binding, NetMHCpan, which was originally developed to cover the binding specificities of all known HLA-I molecules, was successfully used to predict the specificities of the SLA-1*0401 molecule as well as the porcine/human chimeric MHC-I molecules. These data......In all vertebrate animals, CD8+ cytotoxic T lymphocytes (CTLs) are controlled by major histocompatibility complex class I (MHC-I) molecules. These are highly polymorphic peptide receptors selecting and presenting endogenously derived epitopes to circulating CTLs. The polymorphism of the MHC...

Major Histocompatibility Complex, demographic, and environmental predictors of antibody presence in a free-ranging mammal.

Science.gov (United States)

Ruiz-López, María José; Monello, Ryan J; Schuttler, Stephanie G; Lance, Stacey L; Gompper, Matthew E; Eggert, Lori S

2014-12-01

Major Histocompatibility Complex (MHC) variability plays a key role in pathogen resistance, but its relative importance compared to environmental and demographic factors that also influence resistance is unknown. We analyzed the MHC II DRB exon 2 for 165 raccoons (Procyon lotor) in Missouri (USA). For each animal we also determined the presence of immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies to two highly virulent pathogens, canine distemper virus (CDV) and parvovirus. We investigated the role of MHC polymorphism and other demographic and environmental factors previously associated with predicting seroconversion. In addition, using an experimental approach, we studied the relative importance of resource availability and contact rates. We found important associations between IgG antibody presence and several MHC alleles and supertypes but not between IgM antibody presence and MHC. No effect of individual MHC diversity was found. For CDV, supertype S8, one allele within S8 (Prlo-DRB(∗)222), and a second allele (Prlo-DRB(∗)204) were positively associated with being IgG+, while supertype S4 and one allele within the supertype (Prlo-DRB(∗)210) were negatively associated with being IgG+. Age, year, and increased food availability were also positively associated with being IgG+, but allele Prlo-DRB(∗)222 was a stronger predictor. For parvovirus, only one MHC allele was negatively associated with being IgG+ and age and site were stronger predictors of seroconversion. Our results show that negative-frequency dependent selection is likely acting on the raccoon MHC and that while the role of MHC in relation to other factors depends on the pathogen of interest, it may be one of the most important factors predicting successful immune response. Copyright © 2014 Elsevier B.V. All rights reserved.

Characterisation of four major histocompatibility complex class II genes of the koala (Phascolarctos cinereus).

Science.gov (United States)

Lau, Quintin; Jobbins, Sarah E; Belov, Katherine; Higgins, Damien P

2013-01-01

Major histocompatibility complex (MHC) class II molecules have an integral role in the adaptive immune response, as they bind and present antigenic peptides to T helper lymphocytes. In this study of koalas, species-specific primers were designed to amplify exon 2 of the MHC class II DA and DB genes, which contain much of the peptide-binding regions of the α and β chains. A total of two DA α1 domain variants and eight DA β1 (DAB), three DB α1 and five DB β1 variants were amplified from 20 koalas from two free-living populations from South East Queensland and the Port Macquarie region in northern New South Wales. We detected greater variation in the β1 than in the α1 domains as well as evidence of positive selection in DAB. The present study provides a springboard to future investigation of the role of MHC in disease susceptibility in koalas.

Ligation of major histocompatibility complex class I antigens (MHC-I) prevents apoptosis induced by Fas or SAPK/JNK activation in T-lymphoma cells

DEFF Research Database (Denmark)

Lamberth, K; Claesson, M H

2001-01-01

Early apoptosis in Jurkat T-lymphoma cells was induced by agonistic anti-Fas Ab or by anisomycin which activates the stress kinases SAPK/JNK. Apoptosis was inhibited by ligation of major histocompatibility complex class I antigens (MHC-I). MHC-I ligation induced upregulation of the anti-apoptotic......Early apoptosis in Jurkat T-lymphoma cells was induced by agonistic anti-Fas Ab or by anisomycin which activates the stress kinases SAPK/JNK. Apoptosis was inhibited by ligation of major histocompatibility complex class I antigens (MHC-I). MHC-I ligation induced upregulation of the anti......-apoptotic Bcl-2 protein and stabilized the mitochondrial membrane potential (Deltapsim). MHC-I ligation also prevented downregulation of Bcl-2 and destabilization of Deltapsim induced by anti-Fas Ab treatment or anisomycin exposure. Studies on three different Jurkat cell mutants deficient for src p56(lck), ZAP......-70 kinase, or TCR/CD3 gamma-chain showed that the cells undergo apoptosis after Fas ligation. Anisomycin exposure induced apoptosis in the src p56(lck)-deficient cell line but not in the two other mutant cell lines. Simultaneous cross-linking of MHC-I and Fas ligation inhibited apoptosis in the ZAP...

Non-degradative Ubiquitination of Protein Kinases.

Directory of Open Access Journals (Sweden)

K Aurelia Ball

2016-06-01

Full Text Available Growing evidence supports other regulatory roles for protein ubiquitination in addition to serving as a tag for proteasomal degradation. In contrast to other common post-translational modifications, such as phosphorylation, little is known about how non-degradative ubiquitination modulates protein structure, dynamics, and function. Due to the wealth of knowledge concerning protein kinase structure and regulation, we examined kinase ubiquitination using ubiquitin remnant immunoaffinity enrichment and quantitative mass spectrometry to identify ubiquitinated kinases and the sites of ubiquitination in Jurkat and HEK293 cells. We find that, unlike phosphorylation, ubiquitination most commonly occurs in structured domains, and on the kinase domain, ubiquitination is concentrated in regions known to be important for regulating activity. We hypothesized that ubiquitination, like other post-translational modifications, may alter the conformational equilibrium of the modified protein. We chose one human kinase, ZAP-70, to simulate using molecular dynamics with and without a monoubiquitin modification. In Jurkat cells, ZAP-70 is ubiquitinated at several sites that are not sensitive to proteasome inhibition and thus may have other regulatory roles. Our simulations show that ubiquitination influences the conformational ensemble of ZAP-70 in a site-dependent manner. When monoubiquitinated at K377, near the C-helix, the active conformation of the ZAP-70 C-helix is disrupted. In contrast, when monoubiquitinated at K476, near the kinase hinge region, an active-like ZAP-70 C-helix conformation is stabilized. These results lead to testable hypotheses that ubiquitination directly modulates kinase activity, and that ubiquitination is likely to alter structure, dynamics, and function in other protein classes as well.

Regulation of nucleotide excision repair through ubiquitination

Institute of Scientific and Technical Information of China (English)

Jia Li; Audesh Bhat; Wei Xiao

2011-01-01

Nucleotide excision repair (NER) is the most versatile DNA-repair pathway in all organisms.While bacteria require only three proteins to complete the incision step of NER,eukaryotes employ about 30 proteins to complete the same step.Here we summarize recent studies demonstrating that ubiquitination,a post-translational modification,plays critical roles in regulating the NER activity either dependent on or independent of ubiquitin-proteolysis.Several NER components have been shown as targets of ubiquitination while others are actively involved in the ubiquitination process.We argue through this analysis that ubiquitination serves to coordinate various steps of NER and meanwhile connect NER with other related pathways to achieve the efficient global DNA-damage response.

Shared fine specificity between T-cell receptors and an antibody recognizing a peptide/major histocompatibility class I complex

DEFF Research Database (Denmark)

Stryhn, A; Andersen, P S; Pedersen, L O

1996-01-01

Cytotoxic T cells recognize mosaic structures consisting of target peptides embedded within self-major histocompatibility complex (MHC) class I molecules. This structure has been described in great detail for several peptide-MHC complexes. In contrast, how T-cell receptors recognize peptide...... each other showing that peptide residues 1, 3, 4, 6, and 7 were exposed on the MHC surface and recognized by the T cells. Thus, the majority, and perhaps all, of the side chains of the non-primary anchor residues may be available for T-cell recognition, and contribute to the stringent specificity of T...... cells. A striking similarity between the specificity of the T cells and that of the pSAN antibody was found and most of the peptide residues, which could be recognized by the T cells, could also be recognized by the antibody....

Expression of major histocompatibility complex class II and costimulatory molecules in oral carcinomas in vitro.

Science.gov (United States)

Villarroel-Dorrego, Mariana; Speight, Paul M; Barrett, A William

2005-01-01

Recognition in the 1980 s that keratinocytes can express class II molecules of the Major Histocompatibility Complex (MHC) first raised the possibility that these cells might have an immunological function, and may even act as antigen presenting cells (APC). For effective T lymphocyte activation, APC require, in addition to MHC II, appropriate costimulatory signals. The aim of this study was to determine the expression of MHC class II and the co-stimulatory molecules CD40, CD80 and CD86 in keratinocytes derived from healthy oral mucosa and oral carcinomas. Using flow cytometry, it was confirmed that oral keratinocytes, switch on, expression of MHC class II molecules after stimulation with IFNgamma in vitro. All keratinocyte lines expressed CD40 constitutively; by contrast, CD80 and CD86 were universally absent. Loss of CD80 and CD86 may be one means whereby tumours escape immunological surveillance.

Odour-based discrimination of similarity at the major histocompatibility complex in birds.

Science.gov (United States)

Leclaire, Sarah; Strandh, Maria; Mardon, Jérôme; Westerdahl, Helena; Bonadonna, Francesco

2017-01-11

Many animals are known to preferentially mate with partners that are dissimilar at the major histocompatibility complex (MHC) in order to maximize the antigen binding repertoire (or disease resistance) in their offspring. Although several mammals, fish or lizards use odour cues to assess MHC similarity with potential partners, the ability of birds to assess MHC similarity using olfactory cues has not yet been explored. Here we used a behavioural binary choice test and high-throughput-sequencing of MHC class IIB to determine whether blue petrels can discriminate MHC similarity based on odour cues alone. Blue petrels are seabirds with particularly good sense of smell, they have a reciprocal mate choice and are known to preferentially mate with MHC-dissimilar partners. Incubating males preferentially approached the odour of the more MHC-dissimilar female, whereas incubating females showed opposite preferences. Given their mating pattern, females were, however, expected to show preference for the odour of the more MHC-dissimilar male. Further studies are needed to determine whether, as in women and female mice, the preference varies with the reproductive cycle in blue petrel females. Our results provide the first evidence that birds can use odour cues only to assess MHC dissimilarity. © 2017 The Author(s).

Ubiquitin

DEFF Research Database (Denmark)

Vinther-Jensen, T.; Simonsen, A. H.; Budtz-Jorgensen, E.

2015-01-01

-expansion negative individuals using surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectrometry. Differences in peak intensity from SELDI-TOF spectra were evaluated. RESULTS: Levels of 10 peaks were statistically significantly different between manifest gene-expansion carriers...... and controls. One of them identified as ubiquitin was shown to be dependent on the Unified Huntington Disease Rating Scale Total Functional Capacity, a pseudo-measure of disease severity (P = 0.001), and the Symbol Digit Modalities Test (0.04) in manifest and CAG-age product score (P = 0.019) in all gene......-expansion carriers. CONCLUSIONS AND RELEVANCE: Multiple studies have shown that the ubiquitin-proteasome system is involved in Huntington's disease pathogenesis and understanding of this involvement may have therapeutic potential in humans. This is the first study on cerebrospinal fluid to confirm the involvement...

Ubiquitination of Cdc20 by the APC occurs through an intramolecular mechanism

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Foe, Ian T.; Foster, Scott A.; Cheung, Stephanie K.; DeLuca, Steven Z.; Morgan, David O.; Toczyski, David P.

2012-01-01

SUMMARY Background Cells control progression through late mitosis by regulating Cdc20 and Cdh1, the two mitotic activators of the Anaphase Promoting Complex (APC). The control of Cdc20 protein levels during the cell cycle is not well understood. Results Here, we demonstrate that Cdc20 is degraded in budding yeast by multiple APC-dependent mechanisms. We find that the majority of Cdc20 turnover does not involve a second activator molecule, but instead depends on in cis Cdc20 autoubiquitination while it is bound to its activator-binding site on the APC core. Unlike in trans ubiquitination of Cdc20 substrates, the APC ubiquitinates Cdc20 independent of APC activation by Cdc20’s C-box. Cdc20 turnover by this intramolecular mechanism is cell cycle-regulated, contributing to the decline in Cdc20 levels that occurs after anaphase. Interestingly, high substrate levels in vitro significantly reduce Cdc20 autoubiquitination. Conclusion We show here that Cdc20 fluctuates through the cell cycle via a distinct form of APC-mediated ubiquitination. This in cis autoubiquitination may preferentially occur in early anaphase, following depletion of Cdc20 substrates. This suggests that distinct mechanisms are able to target Cdc20 for ubiquitination at different points during the cell cycle. PMID:22079111

Nonperiodic activity of the human anaphase-promoting complex-Cdh1 ubiquitin ligase results in continuous DNA synthesis uncoupled from mitosis

DEFF Research Database (Denmark)

Lukas, C; Kramer, E R; Peters, J M

2000-01-01

Ubiquitin-proteasome-mediated destruction of rate-limiting proteins is required for timely progression through the main cell cycle transitions. The anaphase-promoting complex (APC), periodically activated by the Cdh1 subunit, represents one of the major cellular ubiquitin ligases which, in Saccha......Ubiquitin-proteasome-mediated destruction of rate-limiting proteins is required for timely progression through the main cell cycle transitions. The anaphase-promoting complex (APC), periodically activated by the Cdh1 subunit, represents one of the major cellular ubiquitin ligases which......, in Saccharomyces cerevisiae and Drosophila spp., triggers exit from mitosis and during G(1) prevents unscheduled DNA replication. In this study we investigated the importance of periodic oscillation of the APC-Cdh1 activity for the cell cycle progression in human cells. We show that conditional interference...... transition and lowered the rate of DNA synthesis during S phase, some of the activities essential for DNA replication became markedly amplified, mainly due to a progressive increase of E2F-dependent cyclin E transcription and a rapid turnover of the p27(Kip1) cyclin-dependent kinase inhibitor. Consequently...

Selective loss of mouse embryos due to the expression of transgenic major histocompatibility class I molecules early in embryogenesis.

Science.gov (United States)

Aït-Azzouzene, D; Langkopf, A; Cohen, J; Bleux, C; Gendron, M C; Kanellopoulos-Langevin, C

1998-05-01

Among the numerous hypotheses proposed to explain the absence of fetal rejection by the mother in mammals, it has been suggested that regulation of expression of the polymorphic major histocompatibility complex (MHC) at the fetal-maternal interface plays a major role. In addition to a lack of MHC gene expression in the placenta throughout gestation, the absence of polymorphic MHC molecules on the early embryo, as well as their low level of expression after midgestation, could contribute to this important biologic phenomenon. In order to test this hypothesis, we have produced transgenic mice able to express polymorphic MHC class I molecules early in embryogenesis. We have placed the MHC class la gene H-2Kb under the control of a housekeeping gene promoter, the hydroxy-methyl-glutaryl coenzyme A reductase (HMG) gene minimal promoter. This construct has been tested for functionality after transfection into mouse fibroblast L cells. The analysis of three founder transgenic mice and their progeny suggested that fetoplacental units that could express the H-2Kb heavy chains are unable to survive in utero beyond midgestation. We have shown further that a much higher resorption rate, on days 11 to 13 of embryonic development, is observed among transgenic embryos developing from eggs microinjected at the one-cell stage with the pHMG-Kb construct than in control embryos. This lethality is not due to immune phenomena, since it is observed in histocompatible combinations between mother and fetus. These results are discussed in the context of what is currently known about the regulation of MHC expression at the fetal-maternal interface and in various transgenic mouse models.

Heat shock induced change in protein ubiquitination in Chlamydomonas

International Nuclear Information System (INIS)

Shimogawara, K.; Muto, S.

1989-01-01

Ubiquitin was purified from pea (Pisum sativum L.) and its antibody was produced. Western blot analysis showed that the antibody cross-reacted with ubiquitins from a green alga Chlamydomonas reinhardtii, a brown alga Laminaria angustata and a red alga Porphyridium cruentum but not with ubiquitin from a blue-green alga Synechococcus sp. In Chlamydomonas, the antibody also reacted with some ubiquitinated proteins including 28- and 31-kDa polypeptides. The isoelectric points of Chlamydomonas ubiquitin and the 28- and 31-kDa ubiquitinated proteins were 8.0, 8.9 and 10.3, respectively. The ubiquitinated proteins, including the 28- and 31-kDa polypeptides were detected after in vitro ATP-dependent ubiquitination of Chlamydomonas cell extract with l25 I-labeled bovine ubiquitin. Heat treatment of Chlamydomonas cells (>40°C) caused drastic increase of ubiquitinated proteins with high mol wt (>60kDa), and coordinated redistribution or decrease of other ubiquitinated proteins and free ubiquitin. Quantitative analysis revealed that the 28- and 31-kDa ubiquitinated proteins showed different responses against heat stress, i.e. the former being more sensitive than the latter. (author)

Nucleosome acidic patch promotes RNF168- and RING1B/BMI1-dependent H2AX and H2A ubiquitination and DNA damage signaling.

Directory of Open Access Journals (Sweden)

Justin W Leung

2014-03-01

Full Text Available Histone ubiquitinations are critical for the activation of the DNA damage response (DDR. In particular, RNF168 and RING1B/BMI1 function in the DDR by ubiquitinating H2A/H2AX on Lys-13/15 and Lys-118/119, respectively. However, it remains to be defined how the ubiquitin pathway engages chromatin to provide regulation of ubiquitin targeting of specific histone residues. Here we identify the nucleosome acid patch as a critical chromatin mediator of H2A/H2AX ubiquitination (ub. The acidic patch is required for RNF168- and RING1B/BMI1-dependent H2A/H2AXub in vivo. The acidic patch functions within the nucleosome as nucleosomes containing a mutated acidic patch exhibit defective H2A/H2AXub by RNF168 and RING1B/BMI1 in vitro. Furthermore, direct perturbation of the nucleosome acidic patch in vivo by the expression of an engineered acidic patch interacting viral peptide, LANA, results in defective H2AXub and RNF168-dependent DNA damage responses including 53BP1 and BRCA1 recruitment to DNA damage. The acidic patch therefore is a critical nucleosome feature that may serve as a scaffold to integrate multiple ubiquitin signals on chromatin to compose selective ubiquitinations on histones for DNA damage signaling.

Conditional analysis identifies three novel major histocompatibility complex loci associated with psoriasis.

Science.gov (United States)

Knight, Jo; Spain, Sarah L; Capon, Francesca; Hayday, Adrian; Nestle, Frank O; Clop, Alex; Barker, Jonathan N; Weale, Michael E; Trembath, Richard C

2012-12-01

Psoriasis is a common, chronic, inflammatory skin disorder. A number of genetic loci have been shown to confer risk for psoriasis. Collectively, these offer an integrated model for the inherited basis for susceptibility to psoriasis that combines altered skin barrier function together with the dysregulation of innate immune pathogen sensing and adap-tive immunity. The major histocompatibility complex (MHC) harbours the psoriasis susceptibility region which exhibits the largest effect size, driven in part by variation contained on the HLA-Cw*0602 allele. However, the resolution of the number and genomic location of potential independent risk loci are hampered by extensive linkage disequilibrium across the region. We leveraged the power of large psoriasis case and control data sets and the statistical approach of conditional analysis to identify potential further association signals distributed across the MHC. In addition to the major loci at HLA-C (P = 2.20 × 10(-236)), we observed and replicated four additional independent signals for disease association, three of which are novel. We detected evidence for association at SNPs rs2507971 (P = 6.73 × 10(-14)), rs9260313 (P = 7.93 × 10(-09)), rs66609536 (P = 3.54 × 10(-07)) and rs380924 (P = 6.24 × 10(-06)), located within the class I region of the MHC, with each observation replicated in an independent sample (P ≤ 0.01). The previously identified locus is close to MICA, the other three lie near MICB, HLA-A and HCG9 (a non-coding RNA gene). The identification of disease associations with both MICA and MICB is particularly intriguing, since each encodes an MHC class I-related protein with potent immunological function.

DNA methylation requires a DNMT1 ubiquitin interacting motif (UIM) and histone ubiquitination.

Science.gov (United States)

Qin, Weihua; Wolf, Patricia; Liu, Nan; Link, Stephanie; Smets, Martha; La Mastra, Federica; Forné, Ignasi; Pichler, Garwin; Hörl, David; Fellinger, Karin; Spada, Fabio; Bonapace, Ian Marc; Imhof, Axel; Harz, Hartmann; Leonhardt, Heinrich

2015-08-01

DNMT1 is recruited by PCNA and UHRF1 to maintain DNA methylation after replication. UHRF1 recognizes hemimethylated DNA substrates via the SRA domain, but also repressive H3K9me3 histone marks with its TTD. With systematic mutagenesis and functional assays, we could show that chromatin binding further involved UHRF1 PHD binding to unmodified H3R2. These complementation assays clearly demonstrated that the ubiquitin ligase activity of the UHRF1 RING domain is required for maintenance DNA methylation. Mass spectrometry of UHRF1-deficient cells revealed H3K18 as a novel ubiquitination target of UHRF1 in mammalian cells. With bioinformatics and mutational analyses, we identified a ubiquitin interacting motif (UIM) in the N-terminal regulatory domain of DNMT1 that binds to ubiquitinated H3 tails and is essential for DNA methylation in vivo. H3 ubiquitination and subsequent DNA methylation required UHRF1 PHD binding to H3R2. These results show the manifold regulatory mechanisms controlling DNMT1 activity that require the reading and writing of epigenetic marks by UHRF1 and illustrate the multifaceted interplay between DNA and histone modifications. The identification and functional characterization of the DNMT1 UIM suggests a novel regulatory principle and we speculate that histone H2AK119 ubiquitination might also lead to UIM-dependent recruitment of DNMT1 and DNA methylation beyond classic maintenance.

Recombinational hotspot specific to female meiosis in the mouse major histocompatibility complex.

Science.gov (United States)

Shiroishi, T; Hanzawa, N; Sagai, T; Ishiura, M; Gojobori, T; Steinmetz, M; Moriwaki, K

1990-01-01

The wm7 haplotype of the major histocompatibility complex (MHC), derived from the Japanese wild mouse Mus musculus molossinus, enhances recombination specific to female meiosis in the K/A beta interval of the MHC. We have mapped crossover points of fifteen independent recombinants from genetic crosses of the wm7 and laboratory haplotypes. Most of them were confined to a short segment of approximately 1 kilobase (kb) of DNA between the A beta 3 and A beta 2 genes, indicating the presence of a female-specific recombinational hotspot. Its location overlaps with a sex-independent hotspot previously identified in the Mus musculus castaneus CAS3 haplotype. We have cloned and sequenced DNA fragments surrounding the hotspot from the wm7 haplotype and the corresponding regions from the hotspot-negative B10.A and C57BL/10 strains. There is no significant difference between the sequences of these three strains, or between these and the published sequences of the CAS3 and C57BL/6 strains. However, a comparison of this A beta 3/A beta 2 hotspot with a previously characterized hotspot in the E beta gene revealed that they have a very similar molecular organization. Each hotspot consists of two elements, the consensus sequence of the mouse middle repetitive MT family and the tetrameric repeated sequences, which are separated by 1 kb of DNA.

Polarisation of Major Histocompatibility Complex II Host Genotype with Pathogenesis of European Brown Hare Syndrome Virus

DEFF Research Database (Denmark)

Iacovakis, Christos; Mamuris, Zissis; Moutou, Katerina A

2013-01-01

A study was conducted in order to determine the occurrence of European Brown Hare Syndrome virus (EBHSV) in Denmark and possible relation between disease pathogenesis and Major Histocompatibility Complex (MHC) host genotype. Liver samples were examined from 170 brown hares (hunted, found sick...... were found to be EBHSV-positive (RT-PCR, VP60 gene). In order to investigate associations between viral pathogenesis and host genotype, variation within the exon 2 DQA gene of MHC was assessed. DQA exon 2 analysis revealed the occurrence of seven different alleles in Denmark. Consistent with other...... populations examined so far in Europe, observed heterozygosity of DQA (H o = 0.1180) was lower than expected (H e = 0.5835). The overall variation for both nucleotide and amino acid differences (2.9% and 14.9%, respectively) were lower in Denmark than those assessed in other European countries (8.3% and 16...

Novel Phosphorylation and Ubiquitination Sites Regulate Reactive Oxygen Species-dependent Degradation of Anti-apoptotic c-FLIP Protein*

Science.gov (United States)

Wilkie-Grantham, Rachel P.; Matsuzawa, Shu-Ichi; Reed, John C.

2013-01-01

The cytosolic protein c-FLIP (cellular Fas-associated death domain-like interleukin 1β-converting enzyme inhibitory protein) is an inhibitor of death receptor-mediated apoptosis that is up-regulated in a variety of cancers, contributing to apoptosis resistance. Several compounds found to restore sensitivity of cancer cells to TRAIL, a TNF family death ligand with promising therapeutic potential, act by targeting c-FLIP ubiquitination and degradation by the proteasome. The generation of reactive oxygen species (ROS) has been implicated in c-FLIP protein degradation. However, the mechanism by which ROS post-transcriptionally regulate c-FLIP protein levels is not well understood. We show here that treatment of prostate cancer PPC-1 cells with the superoxide generators menadione, paraquat, or buthionine sulfoximine down-regulates c-FLIP long (c-FLIPL) protein levels, which is prevented by the proteasome inhibitor MG132. Furthermore, pretreatment of PPC-1 cells with a ROS scavenger prevented ubiquitination and loss of c-FLIPL protein induced by menadione or paraquat. We identified lysine 167 as a novel ubiquitination site of c-FLIPL important for ROS-dependent degradation. We also identified threonine 166 as a novel phosphorylation site and demonstrate that Thr-166 phosphorylation is required for ROS-induced Lys-167 ubiquitination. The mutation of either Thr-166 or Lys-167 was sufficient to stabilize c-FLIP protein levels in PPC-1, HEK293T, and HeLa cancer cells treated with menadione or paraquat. Accordingly, expression of c-FLIP T166A or K167R mutants protected cells from ROS-mediated sensitization to TRAIL-induced cell death. Our findings reveal novel ROS-dependent post-translational modifications of the c-FLIP protein that regulate its stability, thus impacting sensitivity of cancer cells to TRAIL. PMID:23519470

Development of a rapid in vitro protein refolding assay which discriminates between peptide-bound and peptide-free forms of recombinant porcine major histocompatibility class I complex (SLA-I)

DEFF Research Database (Denmark)

Oleksiewicz, M.B.; Kristensen, B.; Ladekjaer-Mikkelsen, A.S.

2002-01-01

The extracellular domains of swine leukocyte antigen class I (SLA-I, major histocompatibility complex protein class 1) were cloned and sequenced for two haplotypes (114 and H7) which do not share any alleles based on serological typing, and which are the most important in Danish farmed pigs...

Tau protein degradation is catalyzed by the ATP/ubiquitin-independent 20S proteasome under normal cell conditions

OpenAIRE

Grune, Tilman; Botzen, Diana; Engels, Martina; Voss, Peter; Kaiser, Barbara; Jung, Tobias; Grimm, Stefanie; Ermak, Gennady; Davies, Kelvin J. A.

2010-01-01

Tau is the major protein exhibiting intracellular accumulation in Alzheimer disease. The mechanisms leading to its accumulation are not fully understood. It has been proposed that the proteasome is responsible for degrading tau but, since proteasomal inhibitors block both the ubiquitin-dependent 26S proteasome and the ubiqutin-independent 20S proteasome pathways, it is not clear which of these pathways is involved in tau degradation. Some involvement of the ubiquitin ligase, CHIP in tau degra...

Auto-ubiquitination of Mdm2 Enhances Its Substrate Ubiquitin Ligase Activity*

Science.gov (United States)

Ranaweera, Ruchira S.; Yang, Xiaolu

2013-01-01

The RING domain E3 ubiquitin ligase Mdm2 is the master regulator of the tumor suppressor p53. It targets p53 for proteasomal degradation, restraining the potent activity of p53 and enabling cell survival and proliferation. Like most E3 ligases, Mdm2 can also ubiquitinate itself. How Mdm2 auto-ubiquitination may influence its substrate ubiquitin ligase activity is undefined. Here we show that auto-ubiquitination of Mdm2 is an activating event. Mdm2 that has been conjugated to polyubiquitin chains, but not to single ubiquitins, exhibits substantially enhanced activity to polyubiquitinate p53. Mechanistically, auto-ubiquitination of Mdm2 facilitates the recruitment of the E2 ubiquitin-conjugating enzyme. This occurs through noncovalent interactions between the ubiquitin chains on Mdm2 and the ubiquitin binding domain on E2s. Mutations that diminish the noncovalent interactions render auto-ubiquitination unable to stimulate Mdm2 substrate E3 activity. These results suggest a model in which polyubiquitin chains on an E3 increase the local concentration of E2 enzymes and permit the processivity of substrate ubiquitination. They also support the notion that autocatalysis may be a prevalent mode for turning on the activity of latent enzymes. PMID:23671280

Isolation and characterization of major histocompatibility complex class IIB genes from the nurse shark.

Science.gov (United States)

Bartl, S; Weissman, I L

1994-01-04

The major histocompatibility complex (MHC) contains a set of linked genes which encode cell surface proteins involved in the binding of small peptide antigens for their subsequent recognition by T lymphocytes. MHC proteins share structural features and the presence and location of polymorphic residues which play a role in the binding of antigens. In order to compare the structure of these molecules and gain insights into their evolution, we have isolated two MHC class IIB genes from the nurse shark, Ginglymostoma cirratum. Two clones, most probably alleles, encode proteins which differ by 13 amino acids located in the putative antigen-binding cleft. The protein structure and the location of polymorphic residues are similar to their mammalian counterparts. Although these genes appear to encode a typical MHC protein, no T-cell-mediated responses have been demonstrated in cartilaginous fish. The nurse shark represents the most phylogenetically primitive organism in which both class IIA [Kasahara, M., Vazquez, M., Sato, K., McKinney, E.C. & Flajnik, M.F. (1992) Proc. Natl. Acad. Sci USA 89, 6688-6692] and class IIB genes, presumably encoding the alpha/beta heterodimer, have been isolated.

Size polymorphism of chicken major histocompatibility complex-encoded B-G molecules is due to length variation in the cytoplasmic heptad repeat region

DEFF Research Database (Denmark)

Kaufman, J; Salomonsen, J; Skjødt, K

1990-01-01

B-G antigens are cell-surface molecules encoded by a highly polymorphic multigene family located in the chicken major histocompatibility complex (MHC). Rabbit antisera to B-G molecules immunoprecipitate 3-6 bands from iodinated erythrocytes by sodium dodecyl sulfate (SDS) gels under reducing......, which bear intrachain disulfide bonds. All 3-6 bands have different mobilities in SDS gels between different haplotypes, ranging from 30 to 55 kDa. This size polymorphism is not affected by glycosidase treatment or addition of protease inhibitors. Partial proteolysis of cell surface-iodinated B...

Genomic polymorphism, recombination, and linkage disequilibrium in human major histocompatibility complex-encoded antigen-processing genes.

Science.gov (United States)

van Endert, P M; Lopez, M T; Patel, S D; Monaco, J J; McDevitt, H O

1992-01-01

Recently, two subunits of a large cytosolic protease and two putative peptide transporter proteins were found to be encoded by genes within the class II region of the major histocompatibility complex (MHC). These genes have been suggested to be involved in the processing of antigenic proteins for presentation by MHC class I molecules. Because of the high degree of polymorphism in MHC genes, and previous evidence for both functional and polypeptide sequence polymorphism in the proteins encoded by the antigen-processing genes, we tested DNA from 27 consanguineous human cell lines for genomic polymorphism by restriction fragment length polymorphism (RFLP) analysis. These studies demonstrate a strong linkage disequilibrium between TAP1 and LMP2 RFLPs. Moreover, RFLPs, as well as a polymorphic stop codon in the telomeric TAP2 gene, appear to be in linkage disequilibrium with HLA-DR alleles and RFLPs in the HLA-DO gene. A high rate of recombination, however, seems to occur in the center of the complex, between the TAP1 and TAP2 genes. Images PMID:1360671

Lipofection indirectly increases expression of endogenous major histocompatibility complex class I molecules on tumor cells.

Science.gov (United States)

Fox, B A; Drury, M; Hu, H M; Cao, Z; Huntzicker, E G; Qie, W; Urba, W J

1998-01-01

Direct intratumoral injection of a lipid/DNA complex encoding an allogeneic major histocompatibility complex (MHC) class I molecule leads to regression of both an immunogenic murine tumor and also melanoma lesions in some patients. We have sought to understand the mechanism(s) for this augmentation of antitumor activity. While optimizing parameters for in vitro gene transfer into the D5 subclone of B16BL6, it was noted that lipofected tumors not only expressed the new alloantigen but also exhibited increased expression of endogenous MHC class I, both H-2 Kb and H-2 Db. This increase in expression was not restricted to the small percentage of cells that expressed the transfected gene, but appeared to affect the majority of cells in culture. Class I expression was not increased by lipopolysaccharide, DNA alone, lipid, or lipid/lipopolysaccharide mixtures. Enhanced class I expression required a DNA/lipid complex and was greatest when parameters optimized for gene transfer of the alloantigen were used. All DNA plasmids tested had this effect, including one plasmid whose DNA was not transcribed because it lacked an expression cassette. Because of the critical role that MHC class I antigens play in immune recognition, we propose that lipid complex-mediated gene transfer may provide immunological advantages beyond those that are attributable to expression of the specific gene transferred.

Major histocompatibility complex class II compatibility, but not class I, predicts mate choice in a bird with highly developed olfaction.

Science.gov (United States)

Strandh, Maria; Westerdahl, Helena; Pontarp, Mikael; Canbäck, Björn; Dubois, Marie-Pierre; Miquel, Christian; Taberlet, Pierre; Bonadonna, Francesco

2012-11-07

Mate choice for major histocompatibility complex (MHC) compatibility has been found in several taxa, although rarely in birds. MHC is a crucial component in adaptive immunity and by choosing an MHC-dissimilar partner, heterozygosity and potentially broad pathogen resistance is maximized in the offspring. The MHC genotype influences odour cues and preferences in mammals and fish and hence olfactory-based mate choice can occur. We tested whether blue petrels, Halobaena caerulea, choose partners based on MHC compatibility. This bird is long-lived, monogamous and can discriminate between individual odours using olfaction, which makes it exceptionally well suited for this analysis. We screened MHC class I and II B alleles in blue petrels using 454-pyrosequencing and quantified the phylogenetic, functional and allele-sharing similarity between individuals. Partners were functionally more dissimilar at the MHC class II B loci than expected from random mating (p = 0.033), whereas there was no such difference at the MHC class I loci. Phylogenetic and non-sequence-based MHC allele-sharing measures detected no MHC dissimilarity between partners for either MHC class I or II B. Our study provides evidence of mate choice for MHC compatibility in a bird with a high dependency on odour cues, suggesting that MHC odour-mediated mate choice occurs in birds.

Histone deacetylase inhibitor, Trichostatin A induces ubiquitin-dependent cyclin D1 degradation in MCF-7 breast cancer cells

Directory of Open Access Journals (Sweden)

Charles Coombes R

2006-02-01

Full Text Available Abstract Background Cyclin D1 is an important regulator of G1-S phase cell cycle transition and has been shown to be important for breast cancer development. GSK3β phosphorylates cyclin D1 on Thr-286, resulting in enhanced ubiquitylation, nuclear export and degradation of the cyclin in the cytoplasm. Recent findings suggest that the development of small-molecule cyclin D1 ablative agents is of clinical relevance. We have previously shown that the histone deacetylase inhibitor trichostatin A (TSA induces the rapid ubiquitin-dependent degradation of cyclin D1 in MCF-7 breast cancer cells prior to repression of cyclin D1 gene (CCND1 transcription. TSA treatment also resulted in accumulation of polyubiquitylated GFP-cyclin D1 species and reduced levels of the recombinant protein within the nucleus. Results Here we provide further evidence for TSA-induced ubiquitin-dependent degradation of cyclin D1 and demonstrate that GSK3β-mediated nuclear export facilitates this activity. Our observations suggest that TSA treatment results in enhanced cyclin D1 degradation via the GSK3β/CRM1-dependent nuclear export/26S proteasomal degradation pathway in MCF-7 cells. Conclusion We have demonstrated that rapid TSA-induced cyclin D1 degradation in MCF-7 cells requires GSK3β-mediated Thr-286 phosphorylation and the ubiquitin-dependent 26S proteasome pathway. Drug induced cyclin D1 repression contributes to the inhibition of breast cancer cell proliferation and can sensitize cells to CDK and Akt inhibitors. In addition, anti-cyclin D1 therapy may be highly specific for treating human breast cancer. The development of potent and effective cyclin D1 ablative agents is therefore of clinical relevance. Our findings suggest that HDAC inhibitors may have therapeutic potential as small-molecule cyclin D1 ablative agents.

Quantitative disease resistance: to better understand parasite-mediated selection on major histocompatibility complex.

Science.gov (United States)

Westerdahl, Helena; Asghar, Muhammad; Hasselquist, Dennis; Bensch, Staffan

2012-02-07

We outline a descriptive framework of how candidate alleles of the immune system associate with infectious diseases in natural populations of animals. Three kinds of alleles can be separated when both prevalence of infection and infection intensity are measured--qualitative disease resistance, quantitative disease resistance and susceptibility alleles. Our descriptive framework demonstrates why alleles for quantitative resistance and susceptibility cannot be separated based on prevalence data alone, but are distinguishable on infection intensity. We then present a case study to evaluate a previous finding of a positive association between prevalence of a severe avian malaria infection (GRW2, Plasmodium ashfordi) and a major histocompatibility complex (MHC) class I allele (B4b) in great reed warblers Acrocephalus arundinaceus. Using the same dataset, we find that individuals with allele B4b have lower GRW2 infection intensities than individuals without this allele. Therefore, allele B4b provides quantitative resistance rather than increasing susceptibility to infection. This implies that birds carrying B4b can mount an immune response that suppresses the acute-phase GRW2 infection, while birds without this allele cannot and may die. We argue that it is important to determine whether MHC alleles related to infections are advantageous (quantitative and qualitative resistance) or disadvantageous (susceptibility) to obtain a more complete picture of pathogen-mediated balancing selection.

The ubiquitin ligase SEVEN IN ABSENTIA (SINA) ubiquitinates a defense-related NAC transcription factor and is involved in defense signaling.

Science.gov (United States)

Miao, Min; Niu, Xiangli; Kud, Joanna; Du, Xinran; Avila, Julian; Devarenne, Timothy P; Kuhl, Joseph C; Liu, Yongsheng; Xiao, Fangming

2016-07-01

We recently identified a defense-related tomato (Solanum lycopersicum) NAC (NAM, ATAF1,2, CUC2) transcription factor, NAC1, that is subjected to ubiquitin-proteasome system-dependent degradation in plant cells. In this study, we identified a tomato ubiquitin ligase (termed SEVEN IN ABSENTIA3; SINA3) that ubiquitinates NAC1, promoting its degradation. We conducted coimmunoprecipitation and bimolecular fluorescence complementation to determine that SINA3 specifically interacts with the NAC1 transcription factor in the nucleus. Moreover, we found that SINA3 ubiquitinates NAC1 in vitro and promotes NAC1 degradation via polyubiquitination in vivo, indicating that SINA3 is a ubiquitin ligase that ubiquitinates NAC1, promoting its degradation. Our real-time PCR analysis indicated that, in contrast to our previous finding that NAC1 mRNA abundance increases upon Pseudomonas infection, the SINA3 mRNA abundance decreases in response to Pseudomonas infection. Moreover, using Agrobacterium-mediated transient expression, we found that overexpression of SINA3 interferes with the hypersensitive response cell death triggered by multiple plant resistance proteins. These results suggest that SINA3 ubiquitinates a defense-related NAC transcription factor for degradation and plays a negative role in defense signaling. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

K63-Linked Ubiquitination in Kinase Activation and Cancer

Energy Technology Data Exchange (ETDEWEB)

Wang, Guocan [Department of Cancer Biology, The University of Texas M. D. Anderson Cancer Center, Houston, TX (United States); Gao, Yuan [Department of Molecular and Cellular Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, TX (United States); The University of Texas Graduate School of Biomedical Sciences at Houston, Houston, TX (United States); Li, Liren [Department of Genomic Medicine, The University of Texas M. D. Anderson Cancer Center, Houston, TX (United States); Jin, Guoxiang; Cai, Zhen [Department of Molecular and Cellular Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, TX (United States); The University of Texas Graduate School of Biomedical Sciences at Houston, Houston, TX (United States); Chao, Jui-I [Department of Molecular and Cellular Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, TX (United States); Department of Biological Science and Technology, National Chiao Tung University, Hsinchu, Taiwan (China); Lin, Hui-Kuan, E-mail: hklin@mdanderson.org [Department of Molecular and Cellular Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, TX (United States); The University of Texas Graduate School of Biomedical Sciences at Houston, Houston, TX (United States)

2012-01-31

Ubiquitination has been demonstrated to play a pivotal role in multiple biological functions, which include cell growth, proliferation, apoptosis, DNA damage response, innate immune response, and neuronal degeneration. Although the role of ubiquitination in targeting proteins for proteasome-dependent degradation have been extensively studied and well-characterized, the critical non-proteolytic functions of ubiquitination, such as protein trafficking and kinase activation, involved in cell survival and cancer development, just start to emerge, In this review, we will summarize recent progresses in elucidating the non-proteolytic function of ubiquitination signaling in protein kinase activation and its implications in human cancers. The advancement in the understanding of the novel functions of ubiquitination in signal transduction pathways downstream of growth factor receptors may provide novel paradigms for the treatment of human cancers.

K63-Linked Ubiquitination in Kinase Activation and Cancer

International Nuclear Information System (INIS)

Wang, Guocan; Gao, Yuan; Li, Liren; Jin, Guoxiang; Cai, Zhen; Chao, Jui-I; Lin, Hui-Kuan

2012-01-01

Ubiquitination has been demonstrated to play a pivotal role in multiple biological functions, which include cell growth, proliferation, apoptosis, DNA damage response, innate immune response, and neuronal degeneration. Although the role of ubiquitination in targeting proteins for proteasome-dependent degradation have been extensively studied and well-characterized, the critical non-proteolytic functions of ubiquitination, such as protein trafficking and kinase activation, involved in cell survival and cancer development, just start to emerge, In this review, we will summarize recent progresses in elucidating the non-proteolytic function of ubiquitination signaling in protein kinase activation and its implications in human cancers. The advancement in the understanding of the novel functions of ubiquitination in signal transduction pathways downstream of growth factor receptors may provide novel paradigms for the treatment of human cancers.

DNA variation of the mammalian major histocompatibility complex reflects genomic diversity and population history

Energy Technology Data Exchange (ETDEWEB)

Yuhki, Naoya; O' Brien, S.J. (National Cancer Institute, Frederick, MD (USA))

1990-01-01

The major histocompatibility complex (MHC) is a multigene complex of tightly linked homologous genes that encode cell surface antigens that play a key role in immune regulation and response to foreign antigens. In most species, MHC gene products display extreme antigenic polymorphism, and their variability has been interpreted to reflect an adaptive strategy for accommodating rapidly evolving infectious agents that periodically afflict natural populations. Determination of the extent of MHC variation has been limited to populations in which skin grafting is feasible or for which serological reagents have been developed. The authors present here a quantitative analysis of restriction fragment length polymorphism of MHC class I genes in several mammalian species (cats, rodents, humans) known to have very different levels of genetic diversity based on functional MHC assays and on allozyme surveys. When homologous class I probes were employed, a notable concordance was observed between the extent of MHC restriction fragment variation and functional MHC variation detected by skin grafts or genome-wide diversity estimated by allozyme screens. These results confirm the genetically depauperate character of the African cheetah, Acinonyx jubatus, and the Asiatic lion, Panthera leo persica; further, they support the use of class I MHC molecular reagents in estimating the extent and character of genetic diversity in natural populations.

DNA variation of the mammalian major histocompatibility complex reflects genomic diversity and population history

International Nuclear Information System (INIS)

Yuhki, Naoya; O'Brien, S.J.

1990-01-01

The major histocompatibility complex (MHC) is a multigene complex of tightly linked homologous genes that encode cell surface antigens that play a key role in immune regulation and response to foreign antigens. In most species, MHC gene products display extreme antigenic polymorphism, and their variability has been interpreted to reflect an adaptive strategy for accommodating rapidly evolving infectious agents that periodically afflict natural populations. Determination of the extent of MHC variation has been limited to populations in which skin grafting is feasible or for which serological reagents have been developed. The authors present here a quantitative analysis of restriction fragment length polymorphism of MHC class I genes in several mammalian species (cats, rodents, humans) known to have very different levels of genetic diversity based on functional MHC assays and on allozyme surveys. When homologous class I probes were employed, a notable concordance was observed between the extent of MHC restriction fragment variation and functional MHC variation detected by skin grafts or genome-wide diversity estimated by allozyme screens. These results confirm the genetically depauperate character of the African cheetah, Acinonyx jubatus, and the Asiatic lion, Panthera leo persica; further, they support the use of class I MHC molecular reagents in estimating the extent and character of genetic diversity in natural populations

Restriction fragment length polymorphism of the major histocompatibility complex of the dog.

Science.gov (United States)

Sarmiento, U M; Storb, R F

1988-01-01

Human major histocompatibility complex (HLA) cDNA probes were used to analyze the restriction fragment length polymorphism (RFLP) of the DLA-D region in dogs. Genomic DNA from peripheral blood leucocytes of 23 unrelated DLA-D-homozygous dogs representing nine DLA-D types (defined by mixed leucocyte reaction) was digested with restriction enzymes (Bam HI, Eco RI, Hind III, Pvu II, Taq I, Rsa I, Msp I, Pst I, and Bgl II), separated by agarose gel electrophoresis, and transferred onto Biotrace membrane. The Southern blots were successively hybridized with radiolabeled HLA cDNA probes corresponding to DR, DQ, DP, and DO beta genes. The autoradiograms for all nine enzyme digests displayed multiple bands with the DRb, DQb, and DPb probes while the DOb probe hybridized with one to two bands. The RFLP patterns were highly polymorphic but consistent within each DLA-D type. Standard RFLP patterns were established for nine DLA-D types which could be discriminated from each other by using two enzymes (Rsa I and Pst I) and the HLA-DPb probe. Cluster analysis of the polymorphic restriction fragments detected by the DRb probe revealed four closely related supertypic groups or DLA-DR families: Dw3 + Dw4 + D1, Dw8 + D10, D7 + D16 + D9, and Dw1. This study provides the basis for DLA-D genotyping at a population level by RFLP analysis. These results also suggest that the genetic organization of the DLA-D region may closely resemble that of the HLA complex.

Blood parasites shape extreme major histocompatibility complex diversity in a migratory passerine.

Science.gov (United States)

Biedrzycka, Aleksandra; Bielański, Wojciech; Ćmiel, Adam; Solarz, Wojciech; Zając, Tadeusz; Migalska, Magdalena; Sebastian, Alvaro; Westerdahl, Helena; Radwan, Jacek

2018-06-01

Pathogens are one of the main forces driving the evolution and maintenance of the highly polymorphic genes of the vertebrate major histocompatibility complex (MHC). Although MHC proteins are crucial in pathogen recognition, it is still poorly understood how pathogen-mediated selection promotes and maintains MHC diversity, and especially so in host species with highly duplicated MHC genes. Sedge warblers (Acrocephalus schoenobaenus) have highly duplicated MHC genes, and using data from high-throughput MHC genotyping, we were able to investigate to what extent avian malaria parasites explain temporal MHC class I supertype fluctuations in a long-term study population. We investigated infection status and infection intensities of two different strains of Haemoproteus, that is avian malaria parasites that are known to have significant fitness consequences in sedge warblers. We found that prevalence of avian malaria in carriers of specific MHC class I supertypes was a significant predictor of their frequency changes between years. This finding suggests that avian malaria infections partly drive the temporal fluctuations of the MHC class I supertypes. Furthermore, we found that individuals with a large number of different supertypes had higher resistance to avian malaria, but there was no evidence for an optimal MHC class I diversity. Thus, the two studied malaria parasite strains appear to select for a high MHC class I supertype diversity. Such selection may explain the maintenance of the extremely high number of MHC class I gene copies in sedge warblers and possibly also in other passerines where avian malaria is a common disease. © 2018 John Wiley & Sons Ltd.

Red Queen Processes Drive Positive Selection on Major Histocompatibility Complex (MHC Genes.

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Maciej Jan Ejsmond

2015-11-01

Full Text Available Major Histocompatibility Complex (MHC genes code for proteins involved in the incitation of the adaptive immune response in vertebrates, which is achieved through binding oligopeptides (antigens of pathogenic origin. Across vertebrate species, substitutions of amino acids at sites responsible for the specificity of antigen binding (ABS are positively selected. This is attributed to pathogen-driven balancing selection, which is also thought to maintain the high polymorphism of MHC genes, and to cause the sharing of allelic lineages between species. However, the nature of this selection remains controversial. We used individual-based computer simulations to investigate the roles of two phenomena capable of maintaining MHC polymorphism: heterozygote advantage and host-pathogen arms race (Red Queen process. Our simulations revealed that levels of MHC polymorphism were high and driven mostly by the Red Queen process at a high pathogen mutation rate, but were low and driven mostly by heterozygote advantage when the pathogen mutation rate was low. We found that novel mutations at ABSs are strongly favored by the Red Queen process, but not by heterozygote advantage, regardless of the pathogen mutation rate. However, while the strong advantage of novel alleles increased the allele turnover rate, under a high pathogen mutation rate, allelic lineages persisted for a comparable length of time under Red Queen and under heterozygote advantage. Thus, when pathogens evolve quickly, the Red Queen is capable of explaining both positive selection and long coalescence times, but the tension between the novel allele advantage and persistence of alleles deserves further investigation.

Detecting Site-Specific Physicochemical Selective Pressures: Applications to the Class I HLA of the Human Major Histocompatibility Complex and the SRK of the Plant Sporophytic Self-Incompatibility System

DEFF Research Database (Denmark)

Sainudiin, Raazesh; Wong, Wendy Shuk Wan; Yogeeswaran, Krithika

2005-01-01

:transversion biases. Here, we apply this method to two positively selected receptors involved in ligand-recognition: the class I alleles of the human major histocompatibility complex (MHC) of known structure and the S-locus receptor kinase (SRK) of the sporophytic self-incompatibility system (SSI) in cruciferous...... Bayes approach is used to identify sites that may be important for ligand recognition in these proteins....

CD54/intercellular adhesion molecule 1 and major histocompatibility complex II signaling induces B cells to express interleukin 2 receptors and complements help provided through CD40 ligation

DEFF Research Database (Denmark)

Poudrier, J; Owens, T

1994-01-01

We have examined signaling roles for CD54 intercellular adhesion molecule 1 and major histocompatibility complex (MHC) II as contact ligands during T help for B cell activation. We used a T helper 1 (Th1)-dependent helper system that was previously shown to be contact as well as interleukin 2 (IL-2......) dependent to demonstrate the relative roles of CD54, MHC II, and CD40 signaling in the events leading to the induction of B cell proliferation and responsiveness to IL-2. Paraformaldehyde-fixed activated Th1-induced expression of IL-2R alpha, IL-2R beta, and B7, and upregulated MHC II and CD54 on B cells...... resulted in the upregulated expression of MHC II and of CD54 and B7, respectively, analogous to the effect of fixed activated Th1 cells. B7 expression was further enhanced by co-cross-linking CD54 and MHC II. Cross-linking of CD40 achieved comparable effects. Strikingly, cross-linking ligation of CD54...

The combination of major histocompatibility complex (MHC) and non-MHC genes influences murine lymphocytic choriomeningitis virus pathogenesis

DEFF Research Database (Denmark)

Eyler, Y L; Pfau, C J; Broomhall, K S

1989-01-01

with the recessive disease phenotype. In all cases, susceptibility was dominant. In backcross progeny obtained from matings of parental strains differing in both major histocompatibility complex (MHC) and non-MHC (SWR; C3H), 90% of the challenged mice died, indicating that at least three loci controlled...... susceptibility to the disease. When the parental strains carried similar MHC haplotypes but dissimilar background genes (B10.BR; CBA), 78% of the backcross mice succumbed, indicating that at least two non-MHC loci influenced disease susceptibility. It is unlikely, however, that the same two non-MHC loci...... are critical in all genetic combinations, since F1 produced from two H-2 identical, resistant strains (B10.BR; C3H) were found to be fully susceptible. When congenic mice, differing only in the D-end of the MHC region, were analysed, 50% of the backcross animals died, indicating that one gene in the MHC region...

Effects of major histocompatibility complex class II knockout on mouse bone mechanical properties during development

Science.gov (United States)

Simske, Steven J.; Bateman, Ted A.; Smith, Erin E.; Ferguson, Virginia L.; Chapes, Stephen K.

2002-01-01

We investigated the effect of major histocompatibility complex class II (MHC II) knockout on the development of the mouse peripheral skeleton. These C2D mice had less skeletal development at 8, 12 and 16 weeks of age compared to wild-type C57BL/6J (B6) male mice. The C2D mice had decreased femur mechanical, geometric and compositional measurements compared to wild type mice at each of these ages. C2D femur stiffness (S), peak force in 3-pt bending (Pm), and mineral mass (Min-M) were 74%, 64% and 66%, respectively, of corresponding B6 values at 8 weeks of age. Similar differences were measured at 12 weeks (for which C2D femoral S, Pm and Min-M were 71%, 72% and 73%, respectively, of corresponding B6 values) and at 16 weeks (for which C2D femoral S, Pm and Min-M were 80%, 66% and 61%, respectively, of corresponding B6 values). MHC II knockout delays the development of adult bone properties and is accompanied by lower body mass compared to wild-type controls.

A Plastid Protein That Evolved from Ubiquitin and Is Required for Apicoplast Protein Import in Toxoplasma gondii

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Justin D. Fellows

2017-06-01

Full Text Available Apicomplexan parasites cause a variety of important infectious diseases, including malaria, toxoplasma encephalitis, and severe diarrhea due to Cryptosporidium. Most apicomplexans depend on an organelle called the apicoplast which is derived from a red algal endosymbiont. The apicoplast is essential for the parasite as the compartment of fatty acid, heme, and isoprenoid biosynthesis. The majority of the approximate 500 apicoplast proteins are nucleus encoded and have to be imported across the four membranes that surround the apicoplast. Import across the second outermost membrane of the apicoplast, the periplastid membrane, depends on an apicoplast-specific endoplasmic reticulum-associated protein degradation (ERAD complex and on enzymes of the associated ubiquitination cascade. However, identification of an apicoplast ubiquitin associated with this machinery has long been elusive. Here we identify a plastid ubiquitin-like protein (PUBL, an apicoplast protein that is derived from a ubiquitin ancestor but that has significantly changed in its primary sequence. PUBL is distinct from known ubiquitin-like proteins, and phylogenomic analyses suggest a clade specific to apicomplexans. We demonstrate that PUBL and the AAA ATPase CDC48AP both act to translocate apicoplast proteins across the periplastid membrane during protein import. Conditional null mutants and genetic complementation show that both proteins are critical for this process and for parasite survival. PUBL residues homologous to those that are required for ubiquitin conjugation onto target proteins are essential for this function, while those required for polyubiquitination and preprotein processing are dispensable. Our experiments provide a mechanistic understanding of the molecular machinery that drives protein import across the membranes of the apicoplast.

Hijacking of the Host Ubiquitin Network by Legionella pneumophila

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Jiazhang Qiu

2017-12-01

Full Text Available Protein ubiquitination is critical for regulation of numerous eukaryotic cellular processes such as protein homeostasis, cell cycle progression, immune response, DNA repair, and vesicular trafficking. Ubiquitination often leads to the alteration of protein stability, subcellular localization, or interaction with other proteins. Given the importance of ubiquitination in the regulation of host immunity, it is not surprising that many infectious agents have evolved strategies to interfere with the ubiquitination network with sophisticated mechanisms such as functional mimicry. The facultative intracellular pathogen Legionella pneumophila is the causative agent of Legionnaires' disease. L. pneumophila is phagocytosed by macrophages and is able to replicate within a niche called Legionella-containing vacuole (LCV. The biogenesis of LCV is dependent upon the Dot/Icm type IV secretion system which delivers more than 330 effector proteins into host cytosol. The optimal intracellular replication of L. pneumophila requires the host ubiquitin-proteasome system. Furthermore, membranes of the bacterial phagosome are enriched with ubiquitinated proteins in a way that requires its Dot/Icm type IV secretion system, suggesting the involvement of effectors in the manipulation of the host ubiquitination machinery. Here we summarize recent advances in our understanding of mechanisms exploited by L. pneumophila effector proteins to hijack the host ubiquitination pathway.

HUWE1 and TRIP12 Collaborate in Degradation of Ubiquitin-Fusion Proteins and Misframed Ubiquitin

DEFF Research Database (Denmark)

Poulsen, Esben G; Steinhauer, Cornelia; Lees, Michael

2012-01-01

In eukaryotic cells an uncleavable ubiquitin moiety conjugated to the N-terminus of a protein signals the degradation of the fusion protein via the proteasome-dependent ubiquitin fusion degradation (UFD) pathway. In yeast the molecular mechanism of the UFD pathway has been well characterized...... in degradation of the UFD substrate Ub(G76V)-YFP. The most significant hits from the screen were the E3 ubiquitin-protein ligase HUWE1, as well as PSMD7 and PSMD14 that encode proteasome subunits. Accordingly, knock down of HUWE1 led to an increase in the steady state level and a retarded degradation of the UFD...... substrate. Knock down of HUWE1 also led to a stabilization of the physiological UFD substrate UBB(+1). Precipitation experiments revealed that HUWE1 is associated with both the Ub(G76V)-YFP substrate and the 26S proteasome, indicating that it functions late in the UFD pathway. Double knock down of HUWE1...

Activity-Dependent Ubiquitination of GluA1 Mediates a Distinct AMPAR Endocytosis and Sorting Pathway

Science.gov (United States)

Schwarz, Lindsay A.; Hall, Benjamin J.; Patrick, Gentry N.

2010-01-01

The accurate trafficking of AMPA receptors (AMPARs) to and from the synapse is a critical component of learning and memory in the brain, while dysfunction of AMPAR trafficking is hypothesized to be an underlying mechanism of Alzheimer’s disease. Previous work has shown that ubiquitination of integral membrane proteins is a common post-translational modification used to mediate endocytosis and endocytic sorting of surface proteins in eukaryotic cells. Here we report that mammalian AMPARs become ubiquitinated in response to their activation. Using a mutant of GluA1 that is unable to be ubiquitinated at lysines on its carboxy-terminus, we demonstrate that ubiquitination is required for internalization of surface AMPARs and their trafficking to the lysosome in response to the AMPAR agonist AMPA, but not for internalization of AMPARs in response to the NMDA receptor (NMDAR) agonist NMDA. Through over-expression or RNAi-mediated knockdown, we identify that a specific E3 ligase, Nedd4-1, is necessary for this process. Finally, we show that ubiquitination of GluA1 by Nedd4-1 becomes more prevalent as neurons mature. Together, these data show that ubiquitination of GluA1-containing AMPARs by Nedd4-1 mediates their endocytosis and trafficking to the lysosome. Furthermore, these results provide insight into how hippocampal neurons regulate AMPAR trafficking and degradation with high specificity in response to differing neuronal signaling cues, and suggest that changes to this pathway may occur as neurons mature. PMID:21148011

A role for PCNA ubiquitination in immunoglobulin hypermutation.

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Hiroshi Arakawa

2006-11-01

Full Text Available Proliferating cell nuclear antigen (PCNA is a DNA polymerase cofactor and regulator of replication-linked functions. Upon DNA damage, yeast and vertebrate PCNA is modified at the conserved lysine K164 by ubiquitin, which mediates error-prone replication across lesions via translesion polymerases. We investigated the role of PCNA ubiquitination in variants of the DT40 B cell line that are mutant in K164 of PCNA or in Rad18, which is involved in PCNA ubiquitination. Remarkably, the PCNA(K164R mutation not only renders cells sensitive to DNA-damaging agents, but also strongly reduces activation induced deaminase-dependent single-nucleotide substitutions in the immunoglobulin light-chain locus. This is the first evidence, to our knowledge, that vertebrates exploit the PCNA-ubiquitin pathway for immunoglobulin hypermutation, most likely through the recruitment of error-prone DNA polymerases.

Linear ubiquitination in immunity.

Science.gov (United States)

Shimizu, Yutaka; Taraborrelli, Lucia; Walczak, Henning

2015-07-01

Linear ubiquitination is a post-translational protein modification recently discovered to be crucial for innate and adaptive immune signaling. The function of linear ubiquitin chains is regulated at multiple levels: generation, recognition, and removal. These chains are generated by the linear ubiquitin chain assembly complex (LUBAC), the only known ubiquitin E3 capable of forming the linear ubiquitin linkage de novo. LUBAC is not only relevant for activation of nuclear factor-κB (NF-κB) and mitogen-activated protein kinases (MAPKs) in various signaling pathways, but importantly, it also regulates cell death downstream of immune receptors capable of inducing this response. Recognition of the linear ubiquitin linkage is specifically mediated by certain ubiquitin receptors, which is crucial for translation into the intended signaling outputs. LUBAC deficiency results in attenuated gene activation and increased cell death, causing pathologic conditions in both, mice, and humans. Removal of ubiquitin chains is mediated by deubiquitinases (DUBs). Two of them, OTULIN and CYLD, are constitutively associated with LUBAC. Here, we review the current knowledge on linear ubiquitination in immune signaling pathways and the biochemical mechanisms as to how linear polyubiquitin exerts its functions distinctly from those of other ubiquitin linkage types. © 2015 The Authors. Immunological Reviews Published by John Wiley & Sons Ltd.

Antibodies against major histocompatibility complex class II antigens directly inhibit the growth of T cells infected with Theileria parva without affecting their state of activation

OpenAIRE

Eichhorn, M; Prospero, T D; Heussler, Volker; Dobbelaere, D A

1993-01-01

We have analyzed the effect of antibodies (Abs) directed against major histocompatibility complex (MHC) class II Abs on the proliferation of Theileria parva-infected (Tpi) T cells. Anti-MHC class II Abs exert a direct effect on Tpi T cells causing an acute block in their proliferation. The inhibition does not involve apoptosis and is also entirely reversible. The rapid arrest of DNA synthesis caused by anti- MHC class II Abs is not due to interference with the state of activation of the T cel...

Regulation of AMPA Receptor Trafficking by Protein Ubiquitination

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Jocelyn Widagdo

2017-10-01

Full Text Available The molecular mechanisms underlying plastic changes in the strength and connectivity of excitatory synapses have been studied extensively for the past few decades and remain the most attractive cellular models of learning and memory. One of the major mechanisms that regulate synaptic plasticity is the dynamic adjustment of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA-type glutamate receptor content on the neuronal plasma membrane. The expression of surface AMPA receptors (AMPARs is controlled by the delicate balance between the biosynthesis, dendritic transport, exocytosis, endocytosis, recycling and degradation of the receptors. These processes are dynamically regulated by AMPAR interacting proteins as well as by various post-translational modifications that occur on their cytoplasmic domains. In the last few years, protein ubiquitination has emerged as a major regulator of AMPAR intracellular trafficking. Dysregulation of AMPAR ubiquitination has also been implicated in the pathophysiology of Alzheimer’s disease. Here we review recent advances in the field and provide insights into the role of protein ubiquitination in regulating AMPAR membrane trafficking and function. We also discuss how aberrant ubiquitination of AMPARs contributes to the pathogenesis of various neurological disorders, including Alzheimer’s disease, chronic stress and epilepsy.

Quantification of HLA-DM-Dependent Major Histocompatibility Complex of Class II Immunopeptidomes by the Peptide Landscape Antigenic Epitope Alignment Utility

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Miguel Álvaro-Benito

2018-05-01

Full Text Available The major histocompatibility complex of class II (MHCII immunopeptidome represents the repertoire of antigenic peptides with the potential to activate CD4+ T cells. An understanding of how the relative abundance of specific antigenic epitopes affects the outcome of T cell responses is an important aspect of adaptive immunity and offers a venue to more rationally tailor T cell activation in the context of disease. Recent advances in mass spectrometric instrumentation, computational power, labeling strategies, and software analysis have enabled an increasing number of stratified studies on HLA ligandomes, in the context of both basic and translational research. A key challenge in the case of MHCII immunopeptidomes, often determined for different samples at distinct conditions, is to derive quantitative information on consensus epitopes from antigenic peptides of variable lengths. Here, we present the design and benchmarking of a new algorithm [peptide landscape antigenic epitope alignment utility (PLAtEAU] allowing the identification and label-free quantification (LFQ of shared consensus epitopes arising from series of nested peptides. The algorithm simplifies the complexity of the dataset while allowing the identification of nested peptides within relatively short segments of protein sequences. Moreover, we apply this algorithm to the comparison of the ligandomes of cell lines with two different expression levels of the peptide-exchange catalyst HLA-DM. Direct comparison of LFQ intensities determined at the peptide level is inconclusive, as most of the peptides are not significantly enriched due to poor sampling. Applying the PLAtEAU algorithm for grouping of the peptides into consensus epitopes shows that more than half of the total number of epitopes is preferentially and significantly enriched for each condition. This simplification and deconvolution of the complex and ambiguous peptide-level dataset highlights the value of the PLAt

Predicting binding affinities of protein ligands from three-dimensional models: application to peptide binding to class I major histocompatibility proteins

DEFF Research Database (Denmark)

Rognan, D; Lauemoller, S L; Holm, A

1999-01-01

A simple and fast free energy scoring function (Fresno) has been developed to predict the binding free energy of peptides to class I major histocompatibility (MHC) proteins. It differs from existing scoring functions mainly by the explicit treatment of ligand desolvation and of unfavorable protein...... coordinates of the MHC-bound peptide have first been determined with an accuracy of about 1-1.5 A. Furthermore, it may be easily recalibrated for any protein-ligand complex.......) and of a series of 16 peptides to H-2K(k). Predictions were more accurate for HLA-A2-binding peptides as the training set had been built from experimentally determined structures. The average error in predicting the binding free energy of the test peptides was 3.1 kJ/mol. For the homology model-derived equation...

High-throughput siRNA screening applied to the ubiquitin-proteasome system

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Poulsen, Esben Guldahl; Nielsen, Sofie V.; Pietras, Elin J.

2016-01-01

The ubiquitin-proteasome system is the major pathway for intracellular protein degradation in eukaryotic cells. Due to the large number of genes dedicated to the ubiquitin-proteasome system, mapping degradation pathways for short lived proteins is a daunting task, in particular in mammalian cells...

Structural basis for ubiquitin recognition by ubiquitin-binding zinc finger of FAAP20.

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Aya Toma

Full Text Available Several ubiquitin-binding zinc fingers (UBZs have been reported to preferentially bind K63-linked ubiquitin chains. In particular, the UBZ domain of FAAP20 (FAAP20-UBZ, a member of the Fanconi anemia core complex, seems to recognize K63-linked ubiquitin chains, in order to recruit the complex to DNA interstrand crosslinks and mediate DNA repair. By contrast, it is reported that the attachment of a single ubiquitin to Rev1, a translesion DNA polymerase, increases binding of Rev1 to FAAP20. To clarify the specificity of FAAP20-UBZ, we determined the crystal structure of FAAP20-UBZ in complex with K63-linked diubiquitin at 1.9 Å resolution. In this structure, FAAP20-UBZ interacts only with one of the two ubiquitin moieties. Consistently, binding assays using surface plasmon resonance spectrometry showed that FAAP20-UBZ binds ubiquitin and M1-, K48- and K63-linked diubiquitin chains with similar affinities. Residues in the vicinity of Ala168 within the α-helix and the C-terminal Trp180 interact with the canonical Ile44-centered hydrophobic patch of ubiquitin. Asp164 within the α-helix and the C-terminal loop mediate a hydrogen bond network, which reinforces ubiquitin-binding of FAAP20-UBZ. Mutations of the ubiquitin-interacting residues disrupted binding to ubiquitin in vitro and abolished the accumulation of FAAP20 to DNA damage sites in vivo. Finally, structural comparison among FAAP20-UBZ, WRNIP1-UBZ and RAD18-UBZ revealed distinct modes of ubiquitin binding. UBZ family proteins could be divided into at least three classes, according to their ubiquitin-binding modes.

Nitric oxide prodrug JS-K inhibits ubiquitin E1 and kills tumor cells retaining wild-type p53.

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Kitagaki, J; Yang, Y; Saavedra, J E; Colburn, N H; Keefer, L K; Perantoni, A O

2009-01-29

Nitric oxide (NO) is a major effector molecule in cancer prevention. A number of studies have shown that NO prodrug JS-K (O(2)-(2,4-dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate) induces apoptotic cell death in vitro and in vivo, indicating that it is a promising new therapeutic for cancer. However, the mechanism of its tumor-killing activity remains unclear. Ubiquitin plays an important role in the regulation of tumorigenesis and cell apoptosis. Our earlier report has shown that inactivation of the ubiquitin system through blocking E1 (ubiquitin-activating enzyme) activity preferentially induces apoptosis in p53-expressing transformed cells. As E1 has an active cysteine residue that could potentially interact with NO, we hypothesized that JS-K could inactivate E1 activity. E1 activity was evaluated by detecting ubiquitin-E1 conjugates through immunoblotting. JS-K strikingly inhibits the ubiquitin-E1 thioester formation in cells in a dose-dependent manner with an IC(50) of approximately 2 microM, whereas a JS-K analog that cannot release NO did not affect these levels in cells. Moreover, JS-K decreases total ubiquitylated proteins and increases p53 levels, which is mainly regulated by ubiquitin and proteasomal degradation. Furthermore, JS-K preferentially induces cell apoptosis in p53-expressing transformed cells. These findings indicate that JS-K inhibits E1 activity and kills transformed cells harboring wild-type p53.

Major histocompatibility complex harbors widespread genotypic variability of non-additive risk of rheumatoid arthritis including epistasis.

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Wei, Wen-Hua; Bowes, John; Plant, Darren; Viatte, Sebastien; Yarwood, Annie; Massey, Jonathan; Worthington, Jane; Eyre, Stephen

2016-04-25

Genotypic variability based genome-wide association studies (vGWASs) can identify potentially interacting loci without prior knowledge of the interacting factors. We report a two-stage approach to make vGWAS applicable to diseases: firstly using a mixed model approach to partition dichotomous phenotypes into additive risk and non-additive environmental residuals on the liability scale and secondly using the Levene's (Brown-Forsythe) test to assess equality of the residual variances across genotype groups per marker. We found widespread significant (P 5e-05) vGWAS signals within the major histocompatibility complex (MHC) across all three study cohorts of rheumatoid arthritis. We further identified 10 epistatic interactions between the vGWAS signals independent of the MHC additive effects, each with a weak effect but jointly explained 1.9% of phenotypic variance. PTPN22 was also identified in the discovery cohort but replicated in only one independent cohort. Combining the three cohorts boosted power of vGWAS and additionally identified TYK2 and ANKRD55. Both PTPN22 and TYK2 had evidence of interactions reported elsewhere. We conclude that vGWAS can help discover interacting loci for complex diseases but require large samples to find additional signals.

Gene duplication and fragmentation in the zebra finch major histocompatibility complex.

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Balakrishnan, Christopher N; Ekblom, Robert; Völker, Martin; Westerdahl, Helena; Godinez, Ricardo; Kotkiewicz, Holly; Burt, David W; Graves, Tina; Griffin, Darren K; Warren, Wesley C; Edwards, Scott V

2010-04-01

Due to its high polymorphism and importance for disease resistance, the major histocompatibility complex (MHC) has been an important focus of many vertebrate genome projects. Avian MHC organization is of particular interest because the chicken Gallus gallus, the avian species with the best characterized MHC, possesses a highly streamlined minimal essential MHC, which is linked to resistance against specific pathogens. It remains unclear the extent to which this organization describes the situation in other birds and whether it represents a derived or ancestral condition. The sequencing of the zebra finch Taeniopygia guttata genome, in combination with targeted bacterial artificial chromosome (BAC) sequencing, has allowed us to characterize an MHC from a highly divergent and diverse avian lineage, the passerines. The zebra finch MHC exhibits a complex structure and history involving gene duplication and fragmentation. The zebra finch MHC includes multiple Class I and Class II genes, some of which appear to be pseudogenes, and spans a much more extensive genomic region than the chicken MHC, as evidenced by the presence of MHC genes on each of seven BACs spanning 739 kb. Cytogenetic (FISH) evidence and the genome assembly itself place core MHC genes on as many as four chromosomes with TAP and Class I genes mapping to different chromosomes. MHC Class II regions are further characterized by high endogenous retroviral content. Lastly, we find strong evidence of selection acting on sites within passerine MHC Class I and Class II genes. The zebra finch MHC differs markedly from that of the chicken, the only other bird species with a complete genome sequence. The apparent lack of synteny between TAP and the expressed MHC Class I locus is in fact reminiscent of a pattern seen in some mammalian lineages and may represent convergent evolution. Our analyses of the zebra finch MHC suggest a complex history involving chromosomal fission, gene duplication and translocation in the

Gene duplication and fragmentation in the zebra finch major histocompatibility complex

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Burt David W

2010-04-01

Full Text Available Abstract Background Due to its high polymorphism and importance for disease resistance, the major histocompatibility complex (MHC has been an important focus of many vertebrate genome projects. Avian MHC organization is of particular interest because the chicken Gallus gallus, the avian species with the best characterized MHC, possesses a highly streamlined minimal essential MHC, which is linked to resistance against specific pathogens. It remains unclear the extent to which this organization describes the situation in other birds and whether it represents a derived or ancestral condition. The sequencing of the zebra finch Taeniopygia guttata genome, in combination with targeted bacterial artificial chromosome (BAC sequencing, has allowed us to characterize an MHC from a highly divergent and diverse avian lineage, the passerines. Results The zebra finch MHC exhibits a complex structure and history involving gene duplication and fragmentation. The zebra finch MHC includes multiple Class I and Class II genes, some of which appear to be pseudogenes, and spans a much more extensive genomic region than the chicken MHC, as evidenced by the presence of MHC genes on each of seven BACs spanning 739 kb. Cytogenetic (FISH evidence and the genome assembly itself place core MHC genes on as many as four chromosomes with TAP and Class I genes mapping to different chromosomes. MHC Class II regions are further characterized by high endogenous retroviral content. Lastly, we find strong evidence of selection acting on sites within passerine MHC Class I and Class II genes. Conclusion The zebra finch MHC differs markedly from that of the chicken, the only other bird species with a complete genome sequence. The apparent lack of synteny between TAP and the expressed MHC Class I locus is in fact reminiscent of a pattern seen in some mammalian lineages and may represent convergent evolution. Our analyses of the zebra finch MHC suggest a complex history involving

Stressing the ubiquitin-proteasome system without 20S proteolytic inhibition selectively kills cervical cancer cells.

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Ravi K Anchoori

Full Text Available Cervical cancer cells exhibit an increased requirement for ubiquitin-dependent protein degradation associated with an elevated metabolic turnover rate, and for specific signaling pathways, notably HPV E6-targeted degradation of p53 and PDZ proteins. Natural compounds with antioxidant properties including flavonoids and triterpenoids hold promise as anticancer agents by interfering with ubiquitin-dependent protein degradation. An increasing body of evidence indicates that their α-β unsaturated carbonyl system is the molecular determinant for inhibition of ubiquitin-mediated protein degradation up-stream of the catalytic sites of the 20S proteasome. Herein we report the identification and characterization of a new class of chalcone-based, potent and cell permeable chemical inhibitors of ubiquitin-dependent protein degradation, and a lead compound RAMB1. RAMB1 inhibits ubiquitin-dependent protein degradation without compromising the catalytic activities of the 20S proteasome, a mechanism distinct from that of Bortezomib. Treatment of cervical cancer cells with RAMB1 triggers unfolded protein responses, including aggresome formation and Hsp90 stabilization, and increases p53 steady state levels. RAMB1 treatment results in activation of lysosomal-dependent degradation pathways as a mechanism to compensate for increasing levels of poly-ubiquitin enriched toxic aggregates. Importantly, RAMB1 synergistically triggers cell death of cervical cancer cells when combined with the lysosome inhibitor Chloroquine.

HUWE1 and TRIP12 collaborate in degradation of ubiquitin-fusion proteins and misframed ubiquitin.

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Esben G Poulsen

Full Text Available In eukaryotic cells an uncleavable ubiquitin moiety conjugated to the N-terminus of a protein signals the degradation of the fusion protein via the proteasome-dependent ubiquitin fusion degradation (UFD pathway. In yeast the molecular mechanism of the UFD pathway has been well characterized. Recently the human E3 ubiquitin-protein ligase TRIP12 was connected with the UFD pathway, but little is otherwise known about this system in mammalian cells. In the present work, we utilized high-throughput imaging on cells transfected with a targeted siRNA library to identify components involved in degradation of the UFD substrate Ub(G76V-YFP. The most significant hits from the screen were the E3 ubiquitin-protein ligase HUWE1, as well as PSMD7 and PSMD14 that encode proteasome subunits. Accordingly, knock down of HUWE1 led to an increase in the steady state level and a retarded degradation of the UFD substrate. Knock down of HUWE1 also led to a stabilization of the physiological UFD substrate UBB(+1. Precipitation experiments revealed that HUWE1 is associated with both the Ub(G76V-YFP substrate and the 26S proteasome, indicating that it functions late in the UFD pathway. Double knock down of HUWE1 and TRIP12 resulted in an additive stabilization of the substrate, suggesting that HUWE1 and TRIP12 function in parallel during UFD. However, even when both HUWE1 and TRIP12 are downregulated, ubiquitylation of the UFD substrate was still apparent, revealing functional redundancy between HUWE1, TRIP12 and yet other ubiquitin-protein ligases.

Activity-dependent ubiquitination of GluA1 mediates a distinct AMPA receptor endocytosis and sorting pathway.

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Schwarz, Lindsay A; Hall, Benjamin J; Patrick, Gentry N

2010-12-08

The accurate trafficking of AMPA receptors (AMPARs) to and from the synapse is a critical component of learning and memory in the brain, whereas dysfunction of AMPAR trafficking is hypothesized to be an underlying mechanism of Alzheimer's disease. Previous work has shown that ubiquitination of integral membrane proteins is a common posttranslational modification used to mediate endocytosis and endocytic sorting of surface proteins in eukaryotic cells. Here we report that mammalian AMPARs become ubiquitinated in response to their activation. Using a mutant of GluA1 that is unable to be ubiquitinated at lysines on its C-terminus, we demonstrate that ubiquitination is required for internalization of surface AMPARs and their trafficking to the lysosome in response to the AMPAR agonist AMPA but not for internalization of AMPARs in response to the NMDA receptor agonist NMDA. Through overexpression or RNA interference-mediated knockdown, we identify that a specific E3 ligase, Nedd4-1 (neural-precursor cell-expressed developmentally downregulated gene 4-1), is necessary for this process. Finally, we show that ubiquitination of GluA1 by Nedd4-1 becomes more prevalent as neurons mature. Together, these data show that ubiquitination of GluA1-containing AMPARs by Nedd4-1 mediates their endocytosis and trafficking to the lysosome. Furthermore, these results provide insight into how hippocampal neurons regulate AMPAR trafficking and degradation with high specificity in response to differing neuronal signaling cues and suggest that changes to this pathway may occur as neurons mature.

Dynamics of major histocompatibility complex class I association with the human peptide-loading complex.

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Panter, Michaela S; Jain, Ankur; Leonhardt, Ralf M; Ha, Taekjip; Cresswell, Peter

2012-09-07

Although the human peptide-loading complex (PLC) is required for optimal major histocompatibility complex class I (MHC I) antigen presentation, its composition is still incompletely understood. The ratio of the transporter associated with antigen processing (TAP) and MHC I to tapasin, which is responsible for MHC I recruitment and peptide binding optimization, is particularly critical for modeling of the PLC. Here, we characterized the stoichiometry of the human PLC using both biophysical and biochemical approaches. By means of single-molecule pulldown (SiMPull), we determined a TAP/tapasin ratio of 1:2, consistent with previous studies of insect-cell microsomes, rat-human chimeric cells, and HeLa cells expressing truncated TAP subunits. We also report that the tapasin/MHC I ratio varies, with the PLC population comprising both 2:1 and 2:2 complexes, based on mutational and co-precipitation studies. The MHC I-saturated PLC may be particularly prevalent among peptide-selective alleles, such as HLA-C4. Additionally, MHC I association with the PLC increases when its peptide supply is reduced by inhibiting the proteasome or by blocking TAP-mediated peptide transport using viral inhibitors. Taken together, our results indicate that the composition of the human PLC varies under normal conditions and dynamically adapts to alterations in peptide supply that may arise during viral infection. These findings improve our understanding of the quality control of MHC I peptide loading and may aid the structural and functional modeling of the human PLC.

Identification of naturally processed hepatitis C virus-derived major histocompatibility complex class I ligands.

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Benno Wölk

Full Text Available Fine mapping of human cytotoxic T lymphocyte (CTL responses against hepatitis C virus (HCV is based on external loading of target cells with synthetic peptides which are either derived from prediction algorithms or from overlapping peptide libraries. These strategies do not address putative host and viral mechanisms which may alter processing as well as presentation of CTL epitopes. Therefore, the aim of this proof-of-concept study was to identify naturally processed HCV-derived major histocompatibility complex (MHC class I ligands. To this end, continuous human cell lines were engineered to inducibly express HCV proteins and to constitutively express high levels of functional HLA-A2. These cell lines were recognized in an HLA-A2-restricted manner by HCV-specific CTLs. Ligands eluted from HLA-A2 molecules isolated from large-scale cultures of these cell lines were separated by high performance liquid chromatography and further analyzed by electrospray ionization quadrupole time of flight mass spectrometry (MS/tandem MS. These analyses allowed the identification of two HLA-A2-restricted epitopes derived from HCV nonstructural proteins (NS 3 and 5B (NS3₁₄₀₆₋₁₄₁₅ and NS5B₂₅₉₄₋₂₆₀₂. In conclusion, we describe a general strategy that may be useful to investigate HCV pathogenesis and may contribute to the development of preventive and therapeutic vaccines in the future.

Promoters active in interphase are bookmarked during mitosis by ubiquitination

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Arora, Mansi; Zhang, Jie; Heine, George F.; Ozer, Gulcin; Liu, Hui-wen; Huang, Kun; Parvin, Jeffrey D.

2012-01-01

We analyzed modification of chromatin by ubiquitination in human cells and whether this mark changes through the cell cycle. HeLa cells were synchronized at different stages and regions of the genome with ubiquitinated chromatin were identified by affinity purification coupled with next-generation sequencing. During interphase, ubiquitin marked the chromatin on the transcribed regions of ∼70% of highly active genes and deposition of this mark was sensitive to transcriptional inhibition. Promoters of nearly half of the active genes were highly ubiquitinated specifically during mitosis. The ubiquitination at the coding regions in interphase but not at promoters during mitosis was enriched for ubH2B and dependent on the presence of RNF20. Ubiquitin labeling of both promoters during mitosis and transcribed regions during interphase, correlated with active histone marks H3K4me3 and H3K36me3 but not a repressive histone modification, H3K27me3. The high level of ubiquitination at the promoter chromatin during mitosis was transient and was removed within 2 h after the cells exited mitosis and entered the next cell cycle. These results reveal that the ubiquitination of promoter chromatin during mitosis is a bookmark identifying active genes during chromosomal condensation in mitosis, and we suggest that this process facilitates transcriptional reactivation post-mitosis. PMID:22941662

The mechanism of OTUB1-mediated inhibition of ubiquitination

Energy Technology Data Exchange (ETDEWEB)

Wiener, Reuven; Zhang, Xiangbin; Wang, Tao; Wolberger, Cynthia (JHU)

2013-04-08

Histones are ubiquitinated in response to DNA double-strand breaks (DSB), promoting recruitment of repair proteins to chromatin. UBC13 (also known as UBE2N) is a ubiquitin-conjugating enzyme (E2) that heterodimerizes with UEV1A (also known as UBE2V1) and synthesizes K63-linked polyubiquitin (K63Ub) chains at DSB sites in concert with the ubiquitin ligase (E3), RNF168 (ref. 3). K63Ub synthesis is regulated in a non-canonical manner by the deubiquitinating enzyme, OTUB1 (OTU domain-containing ubiquitin aldehyde-binding protein 1), which binds preferentially to the UBC13-Ub thiolester. Residues amino-terminal to the OTU domain, which had been implicated in ubiquitin binding, are required for binding to UBC13-Ub and inhibition of K63Ub synthesis. Here we describe structural and biochemical studies elucidating how OTUB1 inhibits UBC13 and other E2 enzymes. We unexpectedly find that OTUB1 binding to UBC13-Ub is allosterically regulated by free ubiquitin, which binds to a second site in OTUB1 and increases its affinity for UBC13-Ub, while at the same time disrupting interactions with UEV1A in a manner that depends on the OTUB1 N terminus. Crystal structures of an OTUB1-UBC13 complex and of OTUB1 bound to ubiquitin aldehyde and a chemical UBC13-Ub conjugate show that binding of free ubiquitin to OTUB1 triggers conformational changes in the OTU domain and formation of a ubiquitin-binding helix in the N terminus, thus promoting binding of the conjugated donor ubiquitin in UBC13-Ub to OTUB1. The donor ubiquitin thus cannot interact with the E2 enzyme, which has been shown to be important for ubiquitin transfer. The N-terminal helix of OTUB1 is positioned to interfere with UEV1A binding to UBC13, as well as with attack on the thiolester by an acceptor ubiquitin, thereby inhibiting K63Ub synthesis. OTUB1 binding also occludes the RING E3 binding site on UBC13, thus providing a further component of inhibition. The general features of the inhibition mechanism explain how OTUB1

Glucocorticoids activate the ATP-ubiquitin-dependent proteolytic system in skeletal muscle during fasting

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Wing, S. S.; Goldberg, A. L.; Goldberger, A. L. (Principal Investigator)

1993-01-01

Glucocorticoids are essential for the increase in protein breakdown in skeletal muscle normally seen during fasting. To determine which proteolytic pathway(s) are activated upon fasting, leg muscles from fed and fasted normal rats were incubated under conditions that block or activate different proteolytic systems. After food deprivation (1 day), the nonlysosomal ATP-dependent process increased by 250%, as shown in experiments involving depletion of muscle ATP. Also, the maximal capacity of the lysosomal process increased 60-100%, but no changes occurred in the Ca(2+)-dependent or the residual energy-independent proteolytic processes. In muscles from fasted normal and adrenalectomized (ADX) rats, the protein breakdown sensitive to inhibitors of the lysosomal or Ca(2+)-dependent pathways did not differ. However, the ATP-dependent process was 30% slower in muscles from fasted ADX rats. Administering dexamethasone to these animals or incubating their muscles with dexamethasone reversed this defect. During fasting, when the ATP-dependent process rises, muscles show a two- to threefold increase in levels of ubiquitin (Ub) mRNA. However, muscles of ADX animals failed to show this response. Injecting dexamethasone into the fasted ADX animals increased muscle Ub mRNA within 6 h. Thus glucocorticoids activate the ATP-Ub-dependent proteolytic pathway in fasting apparently by enhancing the expression of components of this system such as Ub.

Transplantation of islet cells across major histocompatibility barriers after total lymphoid irradiation and infusion of allogeneic bone marrow cells

International Nuclear Information System (INIS)

Britt, L.D.; Scharp, D.W.; Lacy, P.E.; Slavin, S.

1982-01-01

Diabetic Lewis rats (AgB1/L) were evaluated as recipients of allogeneic Wistar-Furth (AgB2/2) isolated adult islets without the use of standard recipient immunosuppression. One group was treated with fractionated total lymphoid irradiation (TLI) and Wistar-Furth bone marrow cell reconstitution to proven chimerism prior to islet transplantation. This group returned to a prediabetic state following Wistar-Furth islet transplantation without any evidence of rejection for 100 days posttransplant. A second group of Lewis rats received only TLI without bone marrow treatment. They gave a varying result following islet transplantation with one recipient showing evidence of prolonged islet survival. A third chimeric control group did not receive isolated islets and did not alter their diabetic state. A fourth group was not given TLI nor donor bone marrow cells and uniformly rejected their allogeneic islets by 7 days. Thus, allogeneic adult islets will survive across major rat histocompatibility barriers using TLI and donor bone marrow chimerism as the only form of immunosuppression

The Ubiquitin System and Jasmonate Signaling

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Astrid Nagels Durand

2016-01-01

Full Text Available The ubiquitin (Ub system is involved in most, if not all, biological processes in eukaryotes. The major specificity determinants of this system are the E3 ligases, which bind and ubiquitinate specific sets of proteins and are thereby responsible for target recruitment to the proteasome or other cellular processing machineries. The Ub system contributes to the regulation of the production, perception and signal transduction of plant hormones. Jasmonic acid (JA and its derivatives, known as jasmonates (JAs, act as signaling compounds regulating plant development and plant responses to various biotic and abiotic stress conditions. We provide here an overview of the current understanding of the Ub system involved in JA signaling.

Mcl-1 Ubiquitination: Unique Regulation of an Essential Survival Protein

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Barbara Mojsa

2014-05-01

Full Text Available Mcl-1 is an anti-apoptotic protein of the Bcl-2 family that is essential for the survival of multiple cell lineages and that is highly amplified in human cancer. Under physiological conditions, Mcl-1 expression is tightly regulated at multiple levels, involving transcriptional, post-transcriptional and post-translational processes. Ubiquitination of Mcl-1, that targets it for proteasomal degradation, allows for rapid elimination of the protein and triggering of cell death, in response to various cellular events. In the last decade, a number of studies have elucidated different pathways controlling Mcl-1 ubiquitination and degradation. Four different E3 ubiquitin-ligases (e.g., Mule, SCFβ-TrCP, SCFFbw7 and Trim17 and one deubiquitinase (e.g., USP9X, that respectively mediate and oppose Mcl-1 ubiquitination, have been formerly identified. The interaction between Mule and Mcl-1 can be modulated by other Bcl-2 family proteins, while recognition of Mcl-1 by the other E3 ubiquitin-ligases and deubiquitinase is influenced by phosphorylation of specific residues in Mcl-1. The protein kinases and E3 ubiquitin-ligases that are involved in the regulation of Mcl-1 stability vary depending on the cellular context, highlighting the complexity and pivotal role of Mcl-1 regulation. In this review, we attempt to recapitulate progress in understanding Mcl-1 regulation by the ubiquitin-proteasome system.

Availability of endogenous peptides limits expression of an M3a-Ld major histocompatibility complex class I chimera

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1994-01-01

Taking advantage of our understanding of the peptide specificity of the major histocompatibility complex class I-b molecule M3a, we sought to determine why these molecules are poorly represented on the cell surface. To this end we constructed a chimeric molecule with the alpha 1 and alpha 2 domains of M3a and alpha 3 of Ld thereby allowing use of available monoclonal antibodies to quantify surface expression. Transfected, but not control, B10.CAS2 (H-2M3b) cells were lysed readily by M3a-restricted monoclonal cytotoxic T lymphocytes. Thus, the chimera bound, trafficked, and presented endogenous mitochondrial peptides. However, despite high levels of M3a-Ld mRNA, transfectants were negative by surface staining. This finding was consistent with inefficient trafficking to the cell surface. Incubation at 26 degrees C, thought to permit trafficking of unoccupied heavy (H) chains, resulted in detectable cell surface expression of chimeric molecules. Incubation with exogenous peptide at 26 degrees C (but not at 37 degrees C) greatly enhanced expression of M3a-Ld molecules in a dose- dependent manner, suggesting stabilization of unoccupied molecules. Stable association of beta 2-microglobulin with the chimeric H chain was observed in labeled cell lysates only in the presence of exogenous specific peptide, indicating that peptide is required for the formation of a ternary complex. These results indicate that surface expression of M3a-Ld is limited largely by the steady-state availability of endogenous peptides. Since most known M3a-binding peptides are N- formylated, native M3a may normally be expressed at high levels only during infection by intracellular bacteria. PMID:8270862

The BAH domain of BAF180 is required for PCNA ubiquitination

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Niimi, Atsuko [Department of Genome Dynamics, Research Institute of Environmental Medicine, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601 (Japan); Hopkins, Suzanna R; Downs, Jessica A [Genome Damage and Stability Centre, University of Sussex, Falmer, Brighton BN1 9RQ (United Kingdom); Masutani, Chikahide, E-mail: masutani@riem.nagoya-u.ac.jp [Department of Genome Dynamics, Research Institute of Environmental Medicine, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601 (Japan)

2015-09-15

Highlights: • The expression of BAF180 promotes UV-induced PCNA ubiquitination during S phase. • The BAH domains of BAF180 alone are sufficient to promote PCNA ubiquitination. • The BAH domains are not assembled into the PBAF in the absence of the C-terminus. - Abstract: Monoubiquitination of proliferating cell nuclear antigen (PCNA) is a critical regulator of post replication repair (PRR). The depletion of BAF180, a unique subunit of the PBAF chromatin remodeling complex in human cells results in reduced PCNA ubiquitination leading to less efficient fork progression following DNA damage, but little is known about the mechanism. Here, we report that the expression of exogenous BAF180 in cells promotes PCNA ubiquitination during S-phase after UV irradiation and it persists for many hours. No correlation was observed between the protein level of ubiquitin-specific protease 1 (USP1) and ubiquitinated PCNA in BAF180 expressing cells. Analysis of cells expressing BAF180 deletion mutants showed that the bromo-adjacent homology (BAH) domains are responsible for this effect. Surprisingly, a deletion construct encoding only the BAH domain region is able to increase the level of ubiquitinated PCNA, even though it is unable to be assembled into the PBAF complex. These results suggest that the ATPase-dependent chromatin remodeling activity of PBAF is not necessary, but instead the BAH domains are sufficient to promote PCNA ubiquitination.

Automated Analysis of Flow Cytometry Data to Reduce Inter-Lab Variation in the Detection of Major Histocompatibility Complex Multimer-Binding T Cells

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Pedersen, Natasja Wulff; Chandran, P. Anoop; Qian, Yu

2017-01-01

Manual analysis of flow cytometry data and subjective gate-border decisions taken by individuals continue to be a source of variation in the assessment of antigen-specific T cells when comparing data across laboratories, and also over time in individual labs. Therefore, strategies to provide...... automated analysis of major histocompatibility complex (MHC) multimer-binding T cells represent an attractive solution to decrease subjectivity and technical variation. The challenge of using an automated analysis approach is that MHC multimer-binding T cell populations are often rare and therefore...... laboratories. We used three different methods, FLOw Clustering without K (FLOCK), Scalable Weighted Iterative Flow-clustering Technique (SWIFT), and ReFlow to analyze flow cytometry data files from 28 laboratories. Each laboratory screened for antigen-responsive T cell populations with frequency ranging from 0...

Regulation of synaptic structure by ubiquitin C-terminal hydrolase L1.

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Cartier, Anna E; Djakovic, Stevan N; Salehi, Afshin; Wilson, Scott M; Masliah, Eliezer; Patrick, Gentry N

2009-06-17

Ubiquitin C-terminal hydrolase L1 (UCH-L1) is a deubiquitinating enzyme that is selectively and abundantly expressed in the brain, and its activity is required for normal synaptic function. Here, we show that UCH-L1 functions in maintaining normal synaptic structure in hippocampal neurons. We found that UCH-L1 activity is rapidly upregulated by NMDA receptor activation, which leads to an increase in the levels of free monomeric ubiquitin. Conversely, pharmacological inhibition of UCH-L1 significantly reduces monomeric ubiquitin levels and causes dramatic alterations in synaptic protein distribution and spine morphology. Inhibition of UCH-L1 activity increases spine size while decreasing spine density. Furthermore, there is a concomitant increase in the size of presynaptic and postsynaptic protein clusters. Interestingly, however, ectopic expression of ubiquitin restores normal synaptic structure in UCH-L1-inhibited neurons. These findings point to a significant role of UCH-L1 in synaptic remodeling, most likely by modulating free monomeric ubiquitin levels in an activity-dependent manner.

Ubiquitination in Periodontal Disease: A Review.

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Tsuchida, Sachio; Satoh, Mamoru; Takiwaki, Masaki; Nomura, Fumio

2017-07-10

Periodontal disease (periodontitis) is a chronic inflammatory condition initiated by microbial infection that leads to gingival tissue destruction and alveolar bone resorption. The periodontal tissue's response to dental plaque is characterized by the accumulation of polymorphonuclear leukocytes, macrophages, and lymphocytes, all of which release inflammatory mediators and cytokines to orchestrate the immunopathogenesis of periodontal disease. Ubiquitination is achieved by a mechanism that involves a number of factors, including an ubiquitin-activating enzyme, ubiquitin-conjugating enzyme, and ubiquitin-protein ligase. Ubiquitination is a post-translational modification restricted to eukaryotes that are involved in essential host processes. The ubiquitin system has been implicated in the immune response, development, and programmed cell death. Increasing numbers of recent reports have provided evidence that many approaches are delivering promising reports for discovering the relationship between ubiquitination and periodontal disease. The scope of this review was to investigate recent progress in the discovery of ubiquitinated protein in diseased periodontium and to discuss the ubiquitination process in periodontal diseases.

Gammaherpesviral Tegument Proteins, PML-Nuclear Bodies and the Ubiquitin-Proteasome System

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Florian Full

2017-10-01

Full Text Available Gammaherpesviruses like Epstein-Barr virus (EBV and Kaposi’s sarcoma-associated herpesvirus (KSHV subvert the ubiquitin proteasome system for their own benefit in order to facilitate viral gene expression and replication. In particular, viral tegument proteins that share sequence homology to the formylglycineamide ribonucleotide amidotransferase (FGARAT, or PFAS, an enzyme in the cellular purine biosynthesis, are important for disrupting the intrinsic antiviral response associated with Promyelocytic Leukemia (PML protein-associated nuclear bodies (PML-NBs by proteasome-dependent and independent mechanisms. In addition, all herpesviruses encode for a potent ubiquitin protease that can efficiently remove ubiquitin chains from proteins and thereby interfere with several different cellular pathways. In this review, we discuss mechanisms and functional consequences of virus-induced ubiquitination and deubiquitination for early events in gammaherpesviral infection.

In vivo immunologic selection of class I major histocompatibility complex gene deletion variants from the B16-BL6 melanoma.

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Talmadge, J E; Talmadge, C B; Zbar, B; McEwen, R; Meeker, A K; Tribble, H

1987-06-01

The mechanism by which tumor allografts escape host immunologic attack was investigated. B16-BL6 cells (the bladder 6 subline of the B16 melanoma) (H-2b) were transfected with a gene (Dd) encoding an allogeneic class I major histocompatibility complex antigen. Clones that expressed Dd antigen were injected into the footpads of nonimmune syngeneic mice, syngeneic immune mice, and nude mice. Under conditions of immunologic selection a clone that contained multiple copies of the transfected gene formed variants that lacked the transfected gene. Primary tumors and pulmonary metastases of immunized mice and pulmonary metastases of nonimmunized mice had lost the Dd gene and, in most cases, all of the associated plasmid. In contrast, in immunodeficient nude mice, primary tumors and pulmonary metastases retained the Dd gene and the associated plasmid. Deletion of genes encoding cell surface antigens may be one of the mechanisms by which allogeneic tumors escape immunologic attack.

Adaptor Protein Complex-2 (AP-2) and Epsin-1 Mediate Protease-activated Receptor-1 Internalization via Phosphorylation- and Ubiquitination-dependent Sorting Signals*

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Chen, Buxin; Dores, Michael R.; Grimsey, Neil; Canto, Isabel; Barker, Breann L.; Trejo, JoAnn

2011-01-01

Signaling by protease-activated receptor-1 (PAR1), a G protein-coupled receptor (GPCR) for thrombin, is regulated by desensitization and internalization. PAR1 desensitization is mediated by β-arrestins, like most classic GPCRs. In contrast, internalization of PAR1 occurs through a clathrin- and dynamin-dependent pathway independent of β-arrestins. PAR1 displays two modes of internalization. Constitutive internalization of unactivated PAR1 is mediated by the clathrin adaptor protein complex-2 (AP-2), where the μ2-adaptin subunit binds directly to a tyrosine-based motif localized within the receptor C-tail domain. However, AP-2 depletion only partially inhibits agonist-induced internalization of PAR1, suggesting a function for other clathrin adaptors in this process. Here, we now report that AP-2 and epsin-1 are both critical mediators of agonist-stimulated PAR1 internalization. We show that ubiquitination of PAR1 and the ubiquitin-interacting motifs of epsin-1 are required for epsin-1-dependent internalization of activated PAR1. In addition, activation of PAR1 promotes epsin-1 de-ubiquitination, which may increase its endocytic adaptor activity to facilitate receptor internalization. AP-2 also regulates activated PAR1 internalization via recognition of distal C-tail phosphorylation sites rather than the canonical tyrosine-based motif. Thus, AP-2 and epsin-1 are both required to promote efficient internalization of activated PAR1 and recognize discrete receptor sorting signals. This study defines a new pathway for internalization of mammalian GPCRs. PMID:21965661

Histone Deacetylase Inhibitors Increase p27Kip1 by Affecting Its Ubiquitin-Dependent Degradation through Skp2 Downregulation

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Adriana Borriello

2016-01-01

Full Text Available Histone deacetylase inhibitors (HDACIs represent an intriguing class of pharmacologically active compounds. Currently, some HDACIs are FDA approved for cancer therapy and many others are in clinical trials, showing important clinical activities at well tolerated doses. HDACIs also interfere with the aging process and are involved in the control of inflammation and oxidative stress. In vitro, HDACIs induce different cellular responses including growth arrest, differentiation, and apoptosis. Here, we evaluated the effects of HDACIs on p27Kip1, a key cyclin-dependent kinase inhibitor (CKI. We observed that HDACI-dependent antiproliferative activity is associated with p27Kip1 accumulation due to a reduced protein degradation. p27Kip1 removal requires a preliminary ubiquitination step due to the Skp2-SCF E3 ligase complex. We demonstrated that HDACIs increase p27Kip1 stability through downregulation of Skp2 protein levels. Skp2 decline is only partially due to a reduced Skp2 gene expression. Conversely, the protein decrease is more profound and enduring compared to the changes of Skp2 transcript. This argues for HDACIs effects on Skp2 protein posttranslational modifications and/or on its removal. In summary, we demonstrate that HDACIs increase p27Kip1 by hampering its nuclear ubiquitination/degradation. The findings might be of relevance in the phenotypic effects of these compounds, including their anticancer and aging-modulating activities.

Distinct ubiquitin binding modes exhibited by SH3 domains: molecular determinants and functional implications.

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Jose L Ortega Roldan

Full Text Available SH3 domains constitute a new type of ubiquitin-binding domains. We previously showed that the third SH3 domain (SH3-C of CD2AP binds ubiquitin in an alternative orientation. We have determined the structure of the complex between first CD2AP SH3 domain and ubiquitin and performed a structural and mutational analysis to decipher the determinants of the SH3-C binding mode to ubiquitin. We found that the Phe-to-Tyr mutation in CD2AP and in the homologous CIN85 SH3-C domain does not abrogate ubiquitin binding, in contrast to previous hypothesis and our findings for the first two CD2AP SH3 domains. The similar alternative binding mode of the SH3-C domains of these related adaptor proteins is characterised by a higher affinity to C-terminal extended ubiquitin molecules. We conclude that CD2AP/CIN85 SH3-C domain interaction with ubiquitin constitutes a new ubiquitin-binding mode involved in a different cellular function and thus changes the previously established mechanism of EGF-dependent CD2AP/CIN85 mono-ubiquitination.

Immunoglobulin Heavy Chain Variable Region and Major Histocompatibility Region Genes Are Linked to Induced Graves' Disease in Females From Two Very Large Families of Recombinant Inbred Mice

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Aliesky, Holly; Banuelos, Bianca; Magana, Jessica; Williams, Robert W.; Rapoport, Basil

2014-01-01

Graves' hyperthyroidism is caused by antibodies to the TSH receptor (TSHR) that mimic thyroid stimulation by TSH. Stimulating TSHR antibodies and hyperthyroidism can be induced by immunizing mice with adenovirus expressing the human TSHR A-subunit. Prior analysis of induced Graves' disease in small families of recombinant inbred (RI) female mice demonstrated strong genetic control but did not resolve trait loci for TSHR antibodies or elevated serum T4. We investigated the genetic basis for induced Graves' disease in female mice of two large RI families and combined data with earlier findings to provide phenotypes for 178 genotypes. TSHR antibodies measured by inhibition of TSH binding to its receptor were highly significantly linked in the BXD set to the major histocompatibility region (chromosome 17), consistent with observations in 3 other RI families. In the LXS family, we detected linkage between T4 levels after TSHR-adenovirus immunization and the Ig heavy chain variable region (Igvh, chromosome 12). This observation is a key finding because components of the antigen binding region of Igs determine antibody specificity and have been previously linked to induced thyroid-stimulating antibodies. Data from the LXS family provide the first evidence in mice of a direct link between induced hyperthyroidism and Igvh genes. A role for major histocompatibility genes has now been established for genetic susceptibility to Graves' disease in both humans and mice. Future studies using arrays incorporating variation in the complex human Ig gene locus will be necessary to determine whether Igvh genes are also linked to Graves' disease in humans. PMID:25051451

Zika Virus Escapes NK Cell Detection by Upregulating Major Histocompatibility Complex Class I Molecules.

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Glasner, Ariella; Oiknine-Djian, Esther; Weisblum, Yiska; Diab, Mohammad; Panet, Amos; Wolf, Dana G; Mandelboim, Ofer

2017-11-15

NK cells are innate lymphocytes that participate in many immune processes encompassing cancer, bacterial and fungal infection, autoimmunity, and even pregnancy and that specialize in antiviral defense. NK cells express inhibitory and activating receptors and kill their targets when activating signals overpower inhibitory signals. The NK cell inhibitory receptors include a uniquely diverse array of proteins named killer cell immunoglobulin-like receptors (KIRs), the CD94 family, and the leukocyte immunoglobulin-like receptor (LIR) family. The NK cell inhibitory receptors recognize mostly major histocompatibility complex (MHC) class I (MHC-I) proteins. Zika virus has recently emerged as a major threat due to its association with birth defects and its pandemic potential. How Zika virus interacts with the immune system, and especially with NK cells, is unclear. Here we show that Zika virus infection is barely sensed by NK cells, since little or no increase in the expression of activating NK cell ligands was observed following Zika infection. In contrast, we demonstrate that Zika virus infection leads to the upregulation of MHC class I proteins and consequently to the inhibition of NK cell killing. Mechanistically, we show that MHC class I proteins are upregulated via the RIGI-IRF3 pathway and that this upregulation is mediated via beta interferon (IFN-β). Potentially, countering MHC class I upregulation during Zika virus infection could be used as a prophylactic treatment against Zika virus. IMPORTANCE NK cells are innate lymphocytes that recognize and eliminate various pathogens and are known mostly for their role in controlling viral infections. NK cells express inhibitory and activating receptors, and they kill or spare their targets based on the integration of inhibitory and activating signals. Zika virus has recently emerged as a major threat to humans due to its pandemic potential and its association with birth defects. The role of NK cells in Zika virus

Brucella abortus Inhibits Major Histocompatibility Complex Class II Expression and Antigen Processing through Interleukin-6 Secretion via Toll-Like Receptor 2▿

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Barrionuevo, Paula; Cassataro, Juliana; Delpino, M. Victoria; Zwerdling, Astrid; Pasquevich, Karina A.; Samartino, Clara García; Wallach, Jorge C.; Fossati, Carlos A.; Giambartolomei, Guillermo H.

2008-01-01

The strategies that allow Brucella abortus to survive inside macrophages for prolonged periods and to avoid the immunological surveillance of major histocompatibility complex class II (MHC-II)-restricted gamma interferon (IFN-γ)-producing CD4+ T lymphocytes are poorly understood. We report here that infection of THP-1 cells with B. abortus inhibited expression of MHC-II molecules and antigen (Ag) processing. Heat-killed B. abortus (HKBA) also induced both these phenomena, indicating the independence of bacterial viability and involvement of a structural component of the bacterium. Accordingly, outer membrane protein 19 (Omp19), a prototypical B. abortus lipoprotein, inhibited both MHC-II expression and Ag processing to the same extent as HKBA. Moreover, a synthetic lipohexapeptide that mimics the structure of the protein lipid moiety also inhibited MHC-II expression, indicating that any Brucella lipoprotein could down-modulate MHC-II expression and Ag processing. Inhibition of MHC-II expression and Ag processing by either HKBA or lipidated Omp19 (L-Omp19) depended on Toll-like receptor 2 and was mediated by interleukin-6. HKBA or L-Omp19 also inhibited MHC-II expression and Ag processing of human monocytes. In addition, exposure to the synthetic lipohexapeptide inhibited Ag-specific T-cell proliferation and IFN-γ production of peripheral blood mononuclear cells from Brucella-infected patients. Together, these results indicate that there is a mechanism by which B. abortus may prevent recognition by T cells to evade host immunity and establish a chronic infection. PMID:17984211

Low major histocompatibility complex class II DQA diversity in the Giant Panda (Ailuropoda melanoleuca

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Ruan Xiang-Dong

2007-06-01

Full Text Available Abstract Background The giant panda (Ailuropoda melanoleuca is one of the most endangered animals due to habitat fragmentation and loss. Although the captive breeding program for this species is now nearly two decades old, researches on the genetic background of such captive populations, especially on adaptive molecular polymorphism of major histocompatibility complex (MHC, are still limited. In this study, we characterized adaptive variation of the giant panda's MHC DQA gene by PCR amplification of its antigen-recognizing region (i.e. the exon 2 and subsequent single-strand conformational polymorphism (SSCP and sequence analyses. Results The results revealed a low level of DQA exon 2 diversity in this rare animal, presenting 6 alleles from 61 giant panda individuals. The observed polymorphism was restricted to 9 amino acid substitutions, all of which occurred at and adjacent to positions forming the functionally important antigen-binding sites. All the samples were in Hardy-Weinberg proportions. A significantly higher rate of non-synonymous than synonymous substitutions at the antigen-binding sites indicated positive selection for diversity in the locus. Conclusion The DQA allelic diversity of giant pandas was low relative to other vertebrates. Nonetheless, the pandas exhibited more alleles in DQA than those in DRB, suggesting the alpha chain genes would play a leading role when coping with certain pathogens and thus should be included in conservation genetic investigation. The microsatellite and MHC loci might predict long-term persistence potential and short-term survival ability, respectively. Consequently, it is recommended to utilize multiple suites of microsatellite markers and multiple MHC loci to detect overall genetic variation in order to design unbiased conservation strategies.

Major Histocompatibility Complex I Mediates Immunological Tolerance of the Trophoblast during Pregnancy and May Mediate Rejection during Parturition

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Anna Rapacz-Leonard

2014-01-01

Full Text Available During pregnancy in larger mammals, the maternal immune system must tolerate the fetus for months while resisting external infection. This tolerance is facilitated by immunological communication between the fetus and the mother, which is mediated by Major Histocompatibility Complex I (MHC I proteins, by leukocytes, and by the cytokines secreted by the leukocytes. Fetal-maternal immunological communication also supports pregnancy by inducing physiological changes in the mother. If the mother “misunderstands” the signal sent by the fetus during pregnancy, the fetus will be miscarried or delivered preterm. Unlike any other maternal organ, the placenta can express paternal antigens. At parturition, paternal antigens are known to be expressed in cows and may be expressed in horses, possibly so that the maternal immune system will reject the placenta and help to expel it. This review compares fetal-maternal crosstalk that is mediated by the immune system in three species with pregnancies that last for nine months or longer: humans, cattle, and horses. It raises the possibility that immunological communication early in pregnancy may prepare the mother for successful expulsion of fetal membranes at parturition.

Rates of ubiquitin conjugation increase when muscles atrophy, largely through activation of the N-end rule pathway

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Solomon, V.; Baracos, V.; Sarraf, P.; Goldberg, A. L.

1998-01-01

The rapid loss of muscle mass that accompanies many disease states, such as cancer or sepsis, is primarily a result of increased protein breakdown in muscle, and several observations have suggested an activation of the ubiquitin-proteasome system. Accordingly, in extracts of atrophying muscles from tumor-bearing or septic rats, rates of 125I-ubiquitin conjugation to endogenous proteins were found to be higher than in control extracts. On the other hand, in extracts of muscles from hypothyroid rats, where overall proteolysis is reduced below normal, the conjugation of 125I-ubiquitin to soluble proteins decreased by 50%, and treatment with triiodothyronine (T3) restored ubiquitination to control levels. Surprisingly, the N-end rule pathway, which selectively degrades proteins with basic or large hydrophobic N-terminal residues, was found to be responsible for most of these changes in ubiquitin conjugation. Competitive inhibitors of this pathway that specifically block the ubiquitin ligase, E3alpha, suppressed most of the increased ubiquitin conjugation in the muscle extracts from tumor-bearing and septic rats. These inhibitors also suppressed ubiquitination in normal extracts toward levels in hypothyroid extracts, which showed little E3alpha-dependent ubiquitination. Thus, the inhibitors eliminated most of the differences in ubiquitination under these different pathological conditions. Moreover, 125I-lysozyme, a model N-end rule substrate, was ubiquitinated more rapidly in extracts from tumor-bearing and septic rats, and more slowly in those from hypothyroid rats, than in controls. Thus, the rate of ubiquitin conjugation increases in atrophying muscles, and these hormone- and cytokine-dependent responses are in large part due to activation of the N-end rule pathway.

SUMO-targeted ubiquitin ligases.

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Sriramachandran, Annie M; Dohmen, R Jürgen

2014-01-01

Covalent posttranslational modification with SUMO (small ubiquitin-related modifier) modulates functions of a wide range of proteins in eukaryotic cells. Sumoylation affects the activity, interaction properties, subcellular localization and the stability of its substrate proteins. The recent discovery of a novel class of ubiquitin ligases (E3), termed ULS (E3-S) or STUbL, that recognize sumoylated proteins, links SUMO modification to the ubiquitin/proteasome system. Here we review recent insights into the properties and function of these ligases and their roles in regulating sumoylated proteins. This article is part of a Special Issue entitled: Ubiquitin-Proteasome System. Guest Editors: Thomas Sommer and Dieter H. Wolf. © 2013. Published by Elsevier B.V. All rights reserved.

Major histocompatibility complex-restricted self-recognition in responses to trinitrophenyl-Ficoll. A novel cell interaction pathway requiring self-recognition of accessory cell H-2 determinants by both T cells and B cells

International Nuclear Information System (INIS)

Hodes, R.J.; Hathcock, K.S.; Singer, A.

1983-01-01

In vitro primary antibody responses to limiting concentrations of trinitrophenyl (TNP)-Ficoll were shown to be T cell dependent, requiring the cooperation of T helper (TH) cells, B cells, and accessory cells. Under these conditions, TH cells derived from long-term radiation bone marrow chimeras were major histocompatibility complex (MHC) restricted in their ability to cooperate with accessory cells expressing host-type MHC determinants. The requirement for MHC-restricted self-recognition by TNP-Ficoll-reactive B cells was assessed under these T-dependent conditions. In the presence of competent TH cells, chimeric B cells were found to be MHC restricted, cooperating only with accessory cells that expressed host-type MHC products. In contrast, the soluble products of certain monoclonal T cell lines were able to directly activate B cells in response to TNP-Ficoll, bypassing any requirement for MHC-restricted self-recognition. These findings demonstrate the existence of a novel cell interaction pathway in which B cells as well as TH cells are each required to recognize self-MHC determinants on accessory cells, but are not required to recognize each other. They further demonstrate that the requirement for self-recognition by B cells may be bypassed in certain T-dependent activation pathways

UUCD: a family-based database of ubiquitin and ubiquitin-like conjugation.

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Gao, Tianshun; Liu, Zexian; Wang, Yongbo; Cheng, Han; Yang, Qing; Guo, Anyuan; Ren, Jian; Xue, Yu

2013-01-01

In this work, we developed a family-based database of UUCD (http://uucd.biocuckoo.org) for ubiquitin and ubiquitin-like conjugation, which is one of the most important post-translational modifications responsible for regulating a variety of cellular processes, through a similar E1 (ubiquitin-activating enzyme)-E2 (ubiquitin-conjugating enzyme)-E3 (ubiquitin-protein ligase) enzyme thioester cascade. Although extensive experimental efforts have been taken, an integrative data resource is still not available. From the scientific literature, 26 E1s, 105 E2s, 1003 E3s and 148 deubiquitination enzymes (DUBs) were collected and classified into 1, 3, 19 and 7 families, respectively. To computationally characterize potential enzymes in eukaryotes, we constructed 1, 1, 15 and 6 hidden Markov model (HMM) profiles for E1s, E2s, E3s and DUBs at the family level, separately. Moreover, the ortholog searches were conducted for E3 and DUB families without HMM profiles. Then the UUCD database was developed with 738 E1s, 2937 E2s, 46 631 E3s and 6647 DUBs of 70 eukaryotic species. The detailed annotations and classifications were also provided. The online service of UUCD was implemented in PHP + MySQL + JavaScript + Perl.

The effect of acetaminophen on ubiquitin homeostasis in Saccharomyces cerevisiae.

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Angelina Huseinovic

Full Text Available Acetaminophen (APAP, although considered a safe drug, is one of the major causes of acute liver failure by overdose, and therapeutic chronic use can cause serious health problems. Although the reactive APAP metabolite N-acetyl-p-benzoquinoneimine (NAPQI is clearly linked to liver toxicity, toxicity of APAP is also found without drug metabolism of APAP to NAPQI. To get more insight into mechanisms of APAP toxicity, a genome-wide screen in Saccharomyces cerevisiae for APAP-resistant deletion strains was performed. In this screen we identified genes related to the DNA damage response. Next, we investigated the link between genotype and APAP-induced toxicity or resistance by performing a more detailed screen with a library containing mutants of 1522 genes related to nuclear processes, like DNA repair and chromatin remodelling. We identified 233 strains that had an altered growth rate relative to wild type, of which 107 showed increased resistance to APAP and 126 showed increased sensitivity. Gene Ontology analysis identified ubiquitin homeostasis, regulation of transcription of RNA polymerase II genes, and the mitochondria-to-nucleus signalling pathway to be associated with APAP resistance, while histone exchange and modification, and vesicular transport were connected to APAP sensitivity. Indeed, we observed a link between ubiquitin levels and APAP resistance, whereby ubiquitin deficiency conferred resistance to APAP toxicity while ubiquitin overexpression resulted in sensitivity. The toxicity profile of various chemicals, APAP, and its positional isomer AMAP on a series of deletion strains with ubiquitin deficiency showed a unique resistance pattern for APAP. Furthermore, exposure to APAP increased the level of free ubiquitin and influenced the ubiquitination of proteins. Together, these results uncover a role for ubiquitin homeostasis in APAP-induced toxicity.

Genes of the major histocompatibility complex highlight interactions of the innate and adaptive immune system

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Barbara Lukasch

2017-08-01

Full Text Available Background A well-functioning immune defence is crucial for fitness, but our knowledge about the immune system and its complex interactions is still limited. Major histocompatibility complex (MHC molecules are involved in T-cell mediated adaptive immune responses, but MHC is also highly upregulated during the initial innate immune response. The aim of our study was therefore to determine to what extent the highly polymorphic MHC is involved in interactions of the innate and adaptive immune defence and if specific functional MHC alleles (FA or heterozygosity at the MHC are more important. Methods To do this we used captive house sparrows (Passer domesticus to survey MHC diversity and immune function controlling for several environmental factors. MHC class I alleles were identified using parallel amplicon sequencing and to mirror immune function, several immunological tests that correspond to the innate and adaptive immunity were conducted. Results Our results reveal that MHC was linked to all immune tests, highlighting its importance for the immune defence. While all innate responses were associated with one single FA, adaptive responses (cell-mediated and humoral were associated with several different alleles. Discussion We found that repeated injections of an antibody in nestlings and adults were linked to different FA and hence might affect different areas of the immune system. Also, individuals with a higher number of different FA produced a smaller secondary response, indicating a disadvantage of having numerous MHC alleles. These results demonstrate the complexity of the immune system in relation to the MHC and lay the foundation for other studies to further investigate this topic.

Genes of the major histocompatibility complex highlight interactions of the innate and adaptive immune system.

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Lukasch, Barbara; Westerdahl, Helena; Strandh, Maria; Winkler, Hans; Moodley, Yoshan; Knauer, Felix; Hoi, Herbert

2017-01-01

A well-functioning immune defence is crucial for fitness, but our knowledge about the immune system and its complex interactions is still limited. Major histocompatibility complex (MHC) molecules are involved in T-cell mediated adaptive immune responses, but MHC is also highly upregulated during the initial innate immune response. The aim of our study was therefore to determine to what extent the highly polymorphic MHC is involved in interactions of the innate and adaptive immune defence and if specific functional MHC alleles (FA) or heterozygosity at the MHC are more important. To do this we used captive house sparrows ( Passer domesticus ) to survey MHC diversity and immune function controlling for several environmental factors. MHC class I alleles were identified using parallel amplicon sequencing and to mirror immune function, several immunological tests that correspond to the innate and adaptive immunity were conducted. Our results reveal that MHC was linked to all immune tests, highlighting its importance for the immune defence. While all innate responses were associated with one single FA, adaptive responses (cell-mediated and humoral) were associated with several different alleles. We found that repeated injections of an antibody in nestlings and adults were linked to different FA and hence might affect different areas of the immune system. Also, individuals with a higher number of different FA produced a smaller secondary response, indicating a disadvantage of having numerous MHC alleles. These results demonstrate the complexity of the immune system in relation to the MHC and lay the foundation for other studies to further investigate this topic.

Selective Transgenic Expression of Mutant Ubiquitin in Purkinje Cell Stripes in the Cerebellum.

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Verheijen, Bert M; Gentier, Romina J G; Hermes, Denise J H P; van Leeuwen, Fred W; Hopkins, David A

2017-06-01

The ubiquitin-proteasome system (UPS) is one of the major mechanisms for protein breakdown in cells, targeting proteins for degradation by enzymatically conjugating them to ubiquitin molecules. Intracellular accumulation of ubiquitin-B +1 (UBB +1 ), a frameshift mutant of ubiquitin-B, is indicative of a dysfunctional UPS and has been implicated in several disorders, including neurodegenerative disease. UBB +1 -expressing transgenic mice display widespread labeling for UBB +1 in brain and exhibit behavioral deficits. Here, we show that UBB +1 is specifically expressed in a subset of parasagittal stripes of Purkinje cells in the cerebellar cortex of a UBB +1 -expressing mouse model. This expression pattern is reminiscent of that of the constitutively expressed Purkinje cell antigen HSP25, a small heat shock protein with neuroprotective properties.

Linear ubiquitination signals in adaptive immune responses.

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Ikeda, Fumiyo

2015-07-01

Ubiquitin can form eight different linkage types of chains using the intrinsic Met 1 residue or one of the seven intrinsic Lys residues. Each linkage type of ubiquitin chain has a distinct three-dimensional topology, functioning as a tag to attract specific signaling molecules, which are so-called ubiquitin readers, and regulates various biological functions. Ubiquitin chains linked via Met 1 in a head-to-tail manner are called linear ubiquitin chains. Linear ubiquitination plays an important role in the regulation of cellular signaling, including the best-characterized tumor necrosis factor (TNF)-induced canonical nuclear factor-κB (NF-κB) pathway. Linear ubiquitin chains are specifically generated by an E3 ligase complex called the linear ubiquitin chain assembly complex (LUBAC) and hydrolyzed by a deubiquitinase (DUB) called ovarian tumor (OTU) DUB with linear linkage specificity (OTULIN). LUBAC linearly ubiquitinates critical molecules in the TNF pathway, such as NEMO and RIPK1. The linear ubiquitin chains are then recognized by the ubiquitin readers, including NEMO, which control the TNF pathway. Accumulating evidence indicates an importance of the LUBAC complex in the regulation of apoptosis, development, and inflammation in mice. In this article, I focus on the role of linear ubiquitin chains in adaptive immune responses with an emphasis on the TNF-induced signaling pathways. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

H-2RIIBP, a member of the nuclear hormone receptor superfamily that binds to both the regulatory element of major histocompatibility class I genes and the estrogen response element.

OpenAIRE

Hamada, K; Gleason, S L; Levi, B Z; Hirschfeld, S; Appella, E; Ozato, K

1989-01-01

Transcription of major histocompatibility complex (MHC) class I genes is regulated by the conserved MHC class I regulatory element (CRE). The CRE has two factor-binding sites, region I and region II, both of which elicit enhancer function. By screening a mouse lambda gt 11 library with the CRE as a probe, we isolated a cDNA clone that encodes a protein capable of binding to region II of the CRE. This protein, H-2RIIBP (H-2 region II binding protein), bound to the native region II sequence, bu...

Ubiquitin--conserved protein or selfish gene?

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Catic, André; Ploegh, Hidde L

2005-11-01

The posttranslational modifier ubiquitin is encoded by a multigene family containing three primary members, which yield the precursor protein polyubiquitin and two ubiquitin moieties, Ub(L40) and Ub(S27), that are fused to the ribosomal proteins L40 and S27, respectively. The gene encoding polyubiquitin is highly conserved and, until now, those encoding Ub(L40) and Ub(S27) have been generally considered to be equally invariant. The evolution of the ribosomal ubiquitin moieties is, however, proving to be more dynamic. It seems that the genes encoding Ub(L40) and Ub(S27) are actively maintained by homologous recombination with the invariant polyubiquitin locus. Failure to recombine leads to deterioration of the sequence of the ribosomal ubiquitin moieties in several phyla, although this deterioration is evidently constrained by the structural requirements of the ubiquitin fold. Only a few amino acids in ubiquitin are vital for its function, and we propose that conservation of all three ubiquitin genes is driven not only by functional properties of the ubiquitin protein, but also by the propensity of the polyubiquitin locus to act as a 'selfish gene'.

Role of Ubiquitination in IGF-1 Receptor Signaling and Degradation

OpenAIRE

Sehat, Bita; Andersson, Sandra; Vasilcanu, Radu; Girnita, Leonard; Larsson, Olle

2007-01-01

BACKGROUND: The insulin-like growth factor 1 receptor (IGF-1R) plays numerous crucial roles in cancer biology. The majority of knowledge on IGF-1R signaling is concerned with its role in the activation of the canonical phosphatidyl inositol-3 kinase (PI3K)/Akt and MAPK/ERK pathways. However, the role of IGF-1R ubiquitination in modulating IGF-1R function is an area of current research. In light of this we sought to determine the relationship between IGF-1R phosphorylation, ubiquitination, and...

Introgression from domestic goat generated variation at the major histocompatibility complex of Alpine ibex.

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Christine Grossen

2014-06-01

Full Text Available The major histocompatibility complex (MHC is a crucial component of the vertebrate immune system and shows extremely high levels of genetic polymorphism. The extraordinary genetic variation is thought to be ancient polymorphisms maintained by balancing selection. However, introgression from related species was recently proposed as an additional mechanism. Here we provide evidence for introgression at the MHC in Alpine ibex (Capra ibex ibex. At a usually very polymorphic MHC exon involved in pathogen recognition (DRB exon 2, Alpine ibex carried only two alleles. We found that one of these DRB alleles is identical to a DRB allele of domestic goats (Capra aegagrus hircus. We sequenced 2489 bp of the coding and non-coding regions of the DRB gene and found that Alpine ibex homozygous for the goat-type DRB exon 2 allele showed nearly identical sequences (99.8% to a breed of domestic goats. Using Sanger and RAD sequencing, microsatellite and SNP chip data, we show that the chromosomal region containing the goat-type DRB allele has a signature of recent introgression in Alpine ibex. A region of approximately 750 kb including the DRB locus showed high rates of heterozygosity in individuals carrying one copy of the goat-type DRB allele. These individuals shared SNP alleles both with domestic goats and other Alpine ibex. In a survey of four Alpine ibex populations, we found that the region surrounding the DRB allele shows strong linkage disequilibria, strong sequence clustering and low diversity among haplotypes carrying the goat-type allele. Introgression at the MHC is likely adaptive and introgression critically increased MHC DRB diversity in the genetically impoverished Alpine ibex. Our finding contradicts the long-standing view that genetic variability at the MHC is solely a consequence of ancient trans-species polymorphism. Introgression is likely an underappreciated source of genetic diversity at the MHC and other loci under balancing selection.

Introgression from Domestic Goat Generated Variation at the Major Histocompatibility Complex of Alpine Ibex

Science.gov (United States)

Grossen, Christine; Keller, Lukas; Biebach, Iris; Croll, Daniel

2014-01-01

The major histocompatibility complex (MHC) is a crucial component of the vertebrate immune system and shows extremely high levels of genetic polymorphism. The extraordinary genetic variation is thought to be ancient polymorphisms maintained by balancing selection. However, introgression from related species was recently proposed as an additional mechanism. Here we provide evidence for introgression at the MHC in Alpine ibex (Capra ibex ibex). At a usually very polymorphic MHC exon involved in pathogen recognition (DRB exon 2), Alpine ibex carried only two alleles. We found that one of these DRB alleles is identical to a DRB allele of domestic goats (Capra aegagrus hircus). We sequenced 2489 bp of the coding and non-coding regions of the DRB gene and found that Alpine ibex homozygous for the goat-type DRB exon 2 allele showed nearly identical sequences (99.8%) to a breed of domestic goats. Using Sanger and RAD sequencing, microsatellite and SNP chip data, we show that the chromosomal region containing the goat-type DRB allele has a signature of recent introgression in Alpine ibex. A region of approximately 750 kb including the DRB locus showed high rates of heterozygosity in individuals carrying one copy of the goat-type DRB allele. These individuals shared SNP alleles both with domestic goats and other Alpine ibex. In a survey of four Alpine ibex populations, we found that the region surrounding the DRB allele shows strong linkage disequilibria, strong sequence clustering and low diversity among haplotypes carrying the goat-type allele. Introgression at the MHC is likely adaptive and introgression critically increased MHC DRB diversity in the genetically impoverished Alpine ibex. Our finding contradicts the long-standing view that genetic variability at the MHC is solely a consequence of ancient trans-species polymorphism. Introgression is likely an underappreciated source of genetic diversity at the MHC and other loci under balancing selection. PMID:24945814

TRIM37 defective in mulibrey nanism is a novel RING finger ubiquitin E3 ligase

International Nuclear Information System (INIS)

Kallijaervi, Jukka; Lahtinen, Ulla; Haemaelaeinen, Riikka; Lipsanen-Nyman, Marita; Palvimo, Jorma J.; Lehesjoki, Anna-Elina

2005-01-01

Mulibrey nanism is an autosomal recessive prenatal-onset growth disorder characterized by dysmorphic features, cardiomyopathy, and hepatomegaly. Mutations in TRIM37 encoding a tripartite motif (TRIM, RING-B-box-coiled-coil)-family protein underlie mulibrey nanism. We investigated the ubiquitin ligase activity predicted for the RING domain of TRIM37 by analyzing its autoubiquitination. Full-length TRIM37 and its TRIM domain were highly polyubiquitinated when co-expressed with ubiquitin. Polyubiquitination was decreased in a mutant protein with disrupted RING domain (Cys35Ser;Cys36Ser) and in the Leu76Pro mutant protein, a disease-associated missense mutation affecting the TRIM domain of TRIM37. Bacterially produced GST-TRIM domain fusion protein, but not its Cys35Ser;Cys36Ser or Leu76Pro mutants, were polyubiquitinated in cell-free conditions, implying RING-dependent modification. Ubiquitin was also identified as an interaction partner for TRIM37 in a yeast two-hybrid screen. Ectopically expressed TRIM37 rapidly formed aggregates that were ubiquitin-, proteasome subunit-, and chaperone-positive in immunofluorescence analysis, defining them as aggresomes. The Cys35Ser;Cys36Ser mutant and the Leu76Pro and Gly322Val patient mutant proteins were markedly less prone to aggregation, implying that aggresomal targeting reflects a physiological function of TRIM37. These findings suggest that TRIM37 acts as a TRIM domain-dependent E3 ubiquitin ligase and imply defective ubiquitin-dependent degradation of an as-yet-unidentified target protein in the pathogenesis of mulibrey nanism

Cyclophilin C Participates in the US2-Mediated Degradation of Major Histocompatibility Complex Class I Molecules.

Science.gov (United States)

Chapman, Daniel C; Stocki, Pawel; Williams, David B

2015-01-01

Human cytomegalovirus uses a variety of mechanisms to evade immune recognition through major histocompatibility complex class I molecules. One mechanism mediated by the immunoevasin protein US2 causes rapid disposal of newly synthesized class I molecules by the endoplasmic reticulum-associated degradation pathway. Although several components of this degradation pathway have been identified, there are still questions concerning how US2 targets class I molecules for degradation. In this study we identify cyclophilin C, a peptidyl prolyl isomerase of the endoplasmic reticulum, as a component of US2-mediated immune evasion. Cyclophilin C could be co-isolated with US2 and with the class I molecule HLA-A2. Furthermore, it was required at a particular expression level since depletion or overexpression of cyclophilin C impaired the degradation of class I molecules. To better characterize the involvement of cyclophilin C in class I degradation, we used LC-MS/MS to detect US2-interacting proteins that were influenced by cyclophilin C expression levels. We identified malectin, PDIA6, and TMEM33 as proteins that increased in association with US2 upon cyclophilin C knockdown. In subsequent validation all were shown to play a functional role in US2 degradation of class I molecules. This was specific to US2 rather than general ER-associated degradation since depletion of these proteins did not impede the degradation of a misfolded substrate, the null Hong Kong variant of α1-antitrypsin.

Association of a specific major histocompatibility complex class IIβ single nucleotide polymorphism with resistance to lactococcosis in rainbow trout, Oncorhynchus mykiss (Walbaum).

Science.gov (United States)

Colussi, S; Prearo, M; Bertuzzi, S A; Scanzio, T; Peletto, S; Favaro, L; Modesto, P; Maniaci, M G; Ru, G; Desiato, R; Acutis, P L

2015-01-01

Major histocompatibility complex (MHC) loci encode glycoproteins that bind to foreign peptides and initiate immune responses through their interaction with T cells. MHC class II molecules are heterodimers consisting of α and β chains encoded by extremely variable genes; variation in exon 2 is responsible for the majority of observed polymorphisms, mostly concentrated in the codons specifying the peptide-binding region. Lactococcus garvieae is the causative agent of lactococcosis, a warm-water bacterial infection pathogenic for cultured freshwater and marine fish. It causes considerable economic losses, limiting the profitability and development of fish industries in general and the intensive production of rainbow trout, Oncorhynchus mykiss (Walbaum), in particular. The disease is currently controlled with vaccines and antibiotics; however, vaccines have short-term efficacy, and increasing concerns regarding antibiotic residues have called for alternative strategies. To explore the involvement of the MHC class II β-1 domain as a candidate gene for resistance to lactococcosis, we exposed 400 rainbow trout to naturally contaminated water. One single nucleotide polymorphism (SNP) and one haplotype were associated with resistance (P trout resistant to lactococcosis. © 2014 John Wiley & Sons Ltd.

Two putative subunits of a peptide pump encoded in the human major histocompatability complex class 2 region

International Nuclear Information System (INIS)

Bahram, S.; Arnold, D.; Bresnahan, M.; Strominger, J.L.; Spies, T.

1991-01-01

The class 2 region of the human major histocompatibility complex (MHC) may encode several genes controlling the processing of endogenous antigen and the presentation of peptide epitopes by MHC class 1 molecules to cytotoxic T lymphocytes. A previously described peptide supply factor (PSF1) is a member of the multidrug-resistance family of transporters and may pump cytosolic peptides into the membrane-bound compartment where class 1 molecules assemble. A second transporter gene, PSF2, was identified 10 kilobases (kb) from PSF1, near the class 2 DOB gene. The complete sequences of PSF1 and PSF2 were determined from cDNA clones. The translation products are closely related in sequence and predicted secondary structure. Both contain a highly conserved ATP-binding fold and share 25% homology in a hydrophobic domain with a tentative number of eight membrane-spanning segments. Based on the principle dimeric organization of these two domains in other transporters, PSF1 and PSF2 may function as complementary subunits, independently as homodimers, or both. Taken together with previous genetic evidence, the coregulation of PSF1 and PSF2 by γ interferon and the to-some-degree coordinate transcription of these genes suggest a common role in peptide-loading of class 1 molecules, although a distinct function of PSF2 cannot be ruled out

Hard wiring of T cell receptor specificity for the major histocompatibility complex is underpinned by TCR adaptability

Energy Technology Data Exchange (ETDEWEB)

Burrows, Scott R.; Chen, Zhenjun; Archbold, Julia K.; Tynan, Fleur E.; Beddoe, Travis; Kjer-Nielsen, Lars; Miles, John J.; Khanna, Rajiv; Moss, Denis J.; Liu, Yu Chih; Gras, Stephanie; Kostenko, Lyudmila; Brennan, Rebekah M.; Clements, Craig S.; Brooks, Andrew G.; Purcell, Anthony W.; McCluskey, James; Rossjohn, Jamie (Monash); (Queensland Inst. of Med. Rsrch.); (Melbourne)

2010-07-07

{alpha}{beta} T cell receptors (TCRs) are genetically restricted to corecognize peptide antigens bound to self-major histocompatibility complex (pMHC) molecules; however, the basis for this MHC specificity remains unclear. Despite the current dogma, evaluation of the TCR-pMHC-I structural database shows that the nongermline-encoded complementarity-determining region (CDR)-3 loops often contact the MHC-I, and the germline-encoded CDR1 and -2 loops frequently participate in peptide-mediated interactions. Nevertheless, different TCRs adopt a roughly conserved docking mode over the pMHC-I, in which three MHC-I residues (65, 69, and 155) are invariably contacted by the TCR in one way or another. Nonetheless, the impact of mutations at these three positions, either individually or together, was not uniformly detrimental to TCR recognition of pHLA-B*0801 or pHLA-B*3508. Moreover, when TCR-pMHC-I recognition was impaired, this could be partially restored by expression of the CD8 coreceptor. The structure of a TCR-pMHC-I complex in which these three (65, 69, and 155) MHC-I positions were all mutated resulted in shifting of the TCR footprint relative to the cognate complex and formation of compensatory interactions. Collectively, our findings reveal the inherent adaptability of the TCR in maintaining peptide recognition while accommodating changes to the central docking site on the pMHC-I.

Major histocompatibility complex-unrestricted cytolytic activity of human T cells: analysis of precursor frequency and effector phenotype

International Nuclear Information System (INIS)

Patel, S.S.; Thiele, D.L.; Lipsky, P.E.

1987-01-01

The frequency and phenotype of human T cells that mediate major histocompatibility complex (MHC)-unrestricted cytolysis were analyzed. T cell clones were generated by culturing adherent cell-depleted peripheral blood mononuclear cells at a density of 0.3 cell/well with phytohemagglutinin, recombinant interleukin 2 (rIL-2), and irradiated autologous peripheral blood mononuclear cells and/or Epstein-Barr virus-transformed lymphoblastoid cell lines. All of the 198 clones generated by this method were T cells (CD2 + , CD3 + , CD4 + or CD2 + , CD3 + , CD8 + ) that possessed potent lytic activity against K562, an erythroleukemia line sensitive to lysis by human natural killer cells, and Cur, a renal carcinoma cell line resistant to human natural killer activity. Cytolysis, measured by 51 Cr release, was MHC-unrestricted, since the clones were able to lyse MHC class I or class II negative targets, as well as MHC class I and class II negative targets. Although the clones produced tissue necrosis factor/lymphotoxin-like molecules, lysis of Cur of K562 was not mediated by a soluble factor secreted by the clones. These data indicate that the capacity for MHC-unrestricted tumoricidal activity and expression of NKH1 and CD11b, but not CD 16, are properties common to all or nearly all human peripheral blood-derived T cell clones regardless of CD4 or CD8 phenotype

HUBUNGAN ANTARA PERTUMBUHAN DENGAN KEBERADAAN GEN TAHAN PENYAKIT MAJOR HISTOCOMPATIBILITY COMPLEX (MHC PADA IKAN MAS (Cyprinus carpio

Directory of Open Access Journals (Sweden)

Erma Primanita Hayuningtyas

2016-04-01

Full Text Available Wabah penyakit koi herpes virus (KHV di Indonesia yang terjadi sejak tahun 2002 merupakan salah satu faktor yang memicu kemerosotan produksi ikan mas budidaya. Pembentukan strain unggul ikan mas tahan KHV dapat menjadi solusi bagi permasalahan tersebut. Pemilihan genotip ikan mas tahan KHV dengan marka molekuler gen major histocompatibility complex class II (MHC-II, khususnya pada alel Cyca DAB 1*05 akan membantu dalam kegiatan seleksi. Penelitian ini bertujuan untuk mengetahui keberadaan gen MHC-II pada populasi dasar G0 ikan mas strain Rajadanu dan hubungannya dengan pertumbuhan (bobot. Metode deteksi keberadaan gen MHC-II pada dua kelompok ikan dengan ukuran berbeda dilakukan dengan teknik PCR. Hubungan antara pertumbuhan ikan mas dengan persentase kemunculan gen MHC-II dianalisis dengan menggunakan program SPSS (Statistical Package for the Social Sciences, sehingga diperoleh korelasi di antara keduanya. Hasil penelitian menunjukkan bahwa hubungan antara pertumbuhan dengan persentase keberadaan gen MHC-II berkorelasi negatif dengan nilai R = -0,742. Hal ini mengindikasikan bahwa semakin cepat pertumbuhan populasi ikan mas maka semakin sedikit persentase individu yang mempunyai gen MHC-II pada setiap populasi ikan mas. Sehingga populasi ikan mas yang pertumbuhannya lambat memiliki tingkat persentase positif MHC-II lebih tinggi (85,71%-100% dibandingkan populasi ikan mas yang pertumbuhannya cepat (42,86%-85,71%.

Regulation of Synaptic Structure by the Ubiquitin C-terminal Hydrolase UCH-L1

Science.gov (United States)

Cartier, Anna E.; Djakovic, Stevan N.; Salehi, Afshin; Wilson, Scott M.; Masliah, Eliezer; Patrick, Gentry N.

2009-01-01

UCH-L1 is a de-ubiquitinating enzyme that is selectively and abundantly expressed in the brain, and its activity is required for normal synaptic function. Here, we show that UCH-L1 functions in maintaining normal synaptic structure in hippocampal neurons. We have found that UCH-L1 activity is rapidly up-regulated by NMDA receptor activation which leads to an increase in the levels of free monomeric ubiquitin. Conversely, pharmacological inhibition of UCH-L1 significantly reduces monomeric ubiquitin levels and causes dramatic alterations in synaptic protein distribution and spine morphology. Inhibition of UCH-L1 activity increases spine size while decreasing spine density. Furthermore, there is a concomitant increase in the size of pre and postsynaptic protein clusters. Interestingly, however, ectopic expression of ubiquitin restores normal synaptic structure in UCH-L1 inhibited neurons. These findings point to a significant role of UCH-L1 in synaptic remodeling most likely by modulating free monomeric ubiquitin levels in an activity-dependent manner. PMID:19535597

Iron Loading Selectively Increases Hippocampal Levels of Ubiquitinated Proteins and Impairs Hippocampus-Dependent Memory.

Science.gov (United States)

Figueiredo, Luciana Silva; de Freitas, Betânia Souza; Garcia, Vanessa Athaíde; Dargél, Vinícius Ayub; Köbe, Luiza Machado; Kist, Luiza Wilges; Bogo, Maurício Reis; Schröder, Nadja

2016-11-01

Alterations of brain iron levels have been observed in a number of neurodegenerative disorders. We have previously demonstrated that iron overload in the neonatal period results in severe and persistent memory deficits in the adulthood. Protein degradation mediated by the ubiquitin-proteasome system (UPS) plays a central regulatory role in several cellular processes. Impairment of the UPS has been implicated in the pathogenesis of neurodegenerative disorders. Here, we examined the effects of iron exposure in the neonatal period (12th-14th day of postnatal life) on the expression of proteasome β-1, β-2, and β-5 subunits, and ubiquitinated proteins in brains of 15-day-old rats, to evaluate the immediate effect of the treatment, and in adulthood to assess long-lasting effects. Two different memory types, emotionally motivated conditioning and object recognition were assessed in adult animals. We found that iron administered in the neonatal period impairs both emotionally motivated and recognition memory. Polyubiquitinated protein levels were increased in the hippocampus, but not in the cortex, of adult animals treated with iron. Gene expression of subunits β1 and β5 was affected by age, being higher in the early stages of development in the hippocampus, accompanied by an age-related increase in polyubiquitinated protein levels in adults. In the cortex, gene expression of the three proteasome subunits was significantly higher in adulthood than in the neonatal period. These findings suggest that expression of proteasome subunits and activity are age-dependently regulated. Iron exposure in the neonatal period produces long-lasting harmful effects on the UPS functioning, which may be related with iron-induced memory impairment.

The Ubiquitin Binding Domain ZnF UBP Recognizes the C-Terminal Diglycine Motif of Unanchored Ubiquitin

Energy Technology Data Exchange (ETDEWEB)

Reyes-Turcu,F.; Horton, J.; Mullally, J.; Heroux, A.; Cheng, X.; Wilkinson, K.

2006-01-01

Ubiquitin is a highly versatile post-translational modification that controls virtually all types of cellular events. Over the past ten years we have learned that diverse forms of ubiquitin modifications and of ubiquitin binding modules co-exist in the cell, giving rise to complex networks of protein:protein interactions. A central problem that continues to puzzle ubiquitinologists is how cells translate this myriad of stimuli into highly specific responses. This is a classical signaling problem. Here, we draw parallels with the phosphorylation signaling pathway and we discuss the expanding repertoire of ubiquitin signals, signal tranducers and signaling-regulated E3 enzymes. We examine recent advances in the field, including a new mechanism of regulation of E3 ligases that relies on ubiquitination.

Ubiquitination of the common cytokine receptor γc and regulation of expression by an ubiquitination/deubiquitination machinery

International Nuclear Information System (INIS)

Gesbert, Franck; Malarde, Valerie; Dautry-Varsat, Alice

2005-01-01

The common cytokine receptor γ c is shared by the interleukin-2, -4, -7, -9, -15, and -21 receptors, and is essential for lymphocyte proliferation and survival. The regulation of γ c receptor expression level is therefore critical for the ability of cells to respond to these cytokines. We previously reported that γ c is efficiently constitutively internalized and addressed towards a degradation endocytic compartment. We show that γ c is ubiquitinated and also associated to ubiquitinated proteins. We report that the ubiquitin-ligase c-Cbl induces γ c down-regulation. In addition, the ubiquitin-hydrolase, DUB-2, counteracts the effect of c-Cbl on γ c expression. We show that an increase in DUB-2 expression correlates with an increased γ c half-life, resulting in the up-regulation of the receptor. Altogether, we show that γ c is the target of an ubiquitination mechanism and its expression level can be regulated through the activities of a couple of ubiquitin-ligase/ubiquitin-hydrolase enzymes, namely c-Cbl/DUB-2

Ubiquitination in apoptosis signaling

NARCIS (Netherlands)

van de Kooij, L.W.

2014-01-01

The work described in this thesis focuses on ubiquitination and protein degradation, with an emphasis on how these processes regulate apoptosis signaling. More specifically, our aims were: 1. To increase the understanding of ubiquitin-mediated regulation of apoptosis signaling. 2. To identify the E3

Identification of an estrogen receptor α non covalent ubiquitin-binding surface: role in 17β-estradiol-induced transcriptional activity.

Science.gov (United States)

Pesiri, Valeria; La Rosa, Piergiorgio; Stano, Pasquale; Acconcia, Filippo

2013-06-15

Ubiquitin (Ub)-binding domains (UBDs) located in Ub receptors decode the ubiquitination signal by non-covalently engaging the Ub modification on their binding partners and transduce the Ub signalling through Ub-based molecular interactions. In this way, inducible protein ubiquitination regulates diverse biological processes. The estrogen receptor alpha (ERα) is a ligand-activated transcription factor that mediates the pleiotropic effects of the sex hormone 17β-estradiol (E2). Fine regulation of E2 pleiotropic actions depends on E2-dependent ERα association with a plethora of binding partners and/or on the E2 modulation of receptor ubiquitination. Indeed, E2-induced ERα polyubiquitination triggers receptor degradation and transcriptional activity, and E2-dependent reduction in ERα monoubiquitination is crucial for E2 signalling. Monoubiquitinated proteins often contain UBDs, but whether non-covalent Ub-ERα binding could occur and play a role in E2-ERα signalling is unknown. Here, we report an Ub-binding surface within the ERα ligand binding domain that directs in vitro the receptor interaction with both ubiquitinated proteins and recombinant Ub chains. Mutational analysis reveals that ERα residues leucine 429 and alanine 430 are involved in Ub binding. Moreover, impairment of ERα association to ubiquitinated species strongly affects E2-induced ERα transcriptional activity. Considering the importance of UBDs in the Ub-based signalling network and the central role of different ERα binding partners in the modulation of E2-dependent effects, our discoveries provide novel insights into ERα activity that could also be relevant for ERα-dependent diseases.

The "adjuvant effect" of the polymorphic B-G antigens of the chicken major histocompatibility complex analyzed using purified molecules incorporated in liposomes

DEFF Research Database (Denmark)

Salomonsen, J; Eriksson, H; Skjødt, K

1991-01-01

The polymorphic B-G region of the chicken major histocompatibility complex has previously been shown to mediate an "adjuvant effect" on the humoral response to other erythrocyte alloantigens. We demonstrate here that B-G molecules purified with monoclonal antibodies exert this adjuvant effect...... on the production of alloantibodies to chicken class I (B-F) molecules, when the two are in the same liposome. The adjuvant effect may in part be mediated by antibodies, since the antibody response to B-G molecules occurs much faster than the response to B-F molecules, and conditions in which antibodies to B......-G are present increase the speed of the response to B-F molecules. We also found that the presence of B-G molecules in separate liposomes results in a lack of response to B-F molecules. In the light of this and other data, we consider the possible roles for the polymorphic B-G molecules, particularly...

Functional interchangeability of late domains, late domain cofactors and ubiquitin in viral budding.

Directory of Open Access Journals (Sweden)

Maria Zhadina

2010-10-01

Full Text Available The membrane scission event that separates nascent enveloped virions from host cell membranes often requires the ESCRT pathway, which can be engaged through the action of peptide motifs, termed late (L- domains, in viral proteins. Viral PTAP and YPDL-like L-domains bind directly to the ESCRT-I and ALIX components of the ESCRT pathway, while PPxY motifs bind Nedd4-like, HECT-domain containing, ubiquitin ligases (e.g. WWP1. It has been unclear precisely how ubiquitin ligase recruitment ultimately leads to particle release. Here, using a lysine-free viral Gag protein derived from the prototypic foamy virus (PFV, where attachment of ubiquitin to Gag can be controlled, we show that several different HECT domains can replace the WWP1 HECT domain in chimeric ubiquitin ligases and drive budding. Moreover, artificial recruitment of isolated HECT domains to Gag is sufficient to stimulate budding. Conversely, the HECT domain becomes dispensable if the other domains of WWP1 are directly fused to an ESCRT-1 protein. In each case where budding is driven by a HECT domain, its catalytic activity is essential, but Gag ubiquitination is dispensable, suggesting that ubiquitin ligation to trans-acting proteins drives budding. Paradoxically, however, we also demonstrate that direct fusion of a ubiquitin moiety to the C-terminus of PFV Gag can also promote budding, suggesting that ubiquitination of Gag can substitute for ubiquitination of trans-acting proteins. Depletion of Tsg101 and ALIX inhibits budding that is dependent on ubiquitin that is fused to Gag, or ligated to trans-acting proteins through the action of a PPxY motif. These studies underscore the flexibility in the ways that the ESCRT pathway can be engaged, and suggest a model in which the identity of the protein to which ubiquitin is attached is not critical for subsequent recruitment of ubiquitin-binding components of the ESCRT pathway and viral budding to proceed.

Ubiquitin-specific protease 14 regulates cell proliferation and apoptosis in oral squamous cell carcinoma.

Science.gov (United States)

Chen, Xiangyun; Wu, Jingjing; Chen, Yitian; Ye, Dongxia; Lei, Hu; Xu, Hanzhang; Yang, Li; Wu, Yingli; Gu, Wenli

2016-10-01

Ubiquitin-specific protease 14, a deubiquitinating enzyme, has been implicated in the tumorigenesis and progression of several cancers, but its role in oral squamous cell carcinoma remains to be elucidated. The aim of this study was to explore the expression pattern and roles of Ubiquitin-specific protease 14 in the occurrence and development of oral squamous cell carcinoma. Interestingly, Ubiquitin-specific protease 14 was overexpressed in oral cancer tissues and cell lines at both mRNA and protein levels. b-AP15, a specific inhibitor of Ubiquitin-specific protease 14, significantly inhibited the growth of cancer cells and increased cell apoptosis in a dose-dependent manner. Moreover, knockdown of Ubiquitin-specific protease 14 by shRNA significantly inhibited the proliferation and migration of cancer cells in vitro. Finally, using a xenograft mouse model of oral squamous cell carcinoma, knockdown of Ubiquitin-specific protease 14 markedly inhibited tumor growth and triggered the cancer cell apoptosis in vivo, supporting previous results. In conclusion, for the first time we have demonstrated the expression pattern of Ubiquitin-specific protease 14 in oral squamous cell carcinoma and verified a relationship with tumor growth and metastasis. These results may highlight new therapeutic strategies for tumor treatment, application of Ubiquitin-specific protease 14 selective inhibitor, such as b-AP15, or knockdown by shRNA. Collectively, Ubiquitin-specific protease 14 could be a potential therapeutic target for oral squamous cell carcinoma patients. Copyright © 2016 Elsevier Ltd. All rights reserved.

Regulation of DNA double-strand break repair by ubiquitin and ubiquitin-like modifiers

DEFF Research Database (Denmark)

Schwertman, Petra; Bekker-Jensen, Simon; Mailand, Niels

2016-01-01

DNA double-strand breaks (DSBs) are highly cytotoxic DNA lesions. The swift recognition and faithful repair of such damage is crucial for the maintenance of genomic stability, as well as for cell and organismal fitness. Signalling by ubiquitin, SUMO and other ubiquitin-like modifiers (UBLs...

Ligation of major histocompatibility complex class I antigens (MHC-I) prevents apoptosis induced by Fas or SAPK/JNK activation in T-lymphoma cells

DEFF Research Database (Denmark)

Lamberth, K; Claesson, M H

2001-01-01

Early apoptosis in Jurkat T-lymphoma cells was induced by agonistic anti-Fas Ab or by anisomycin which activates the stress kinases SAPK/JNK. Apoptosis was inhibited by ligation of major histocompatibility complex class I antigens (MHC-I). MHC-I ligation induced upregulation of the anti......-apoptotic Bcl-2 protein and stabilized the mitochondrial membrane potential (Deltapsim). MHC-I ligation also prevented downregulation of Bcl-2 and destabilization of Deltapsim induced by anti-Fas Ab treatment or anisomycin exposure. Studies on three different Jurkat cell mutants deficient for src p56(lck), ZAP......-70 kinase, or TCR/CD3 gamma-chain showed that the cells undergo apoptosis after Fas ligation. Anisomycin exposure induced apoptosis in the src p56(lck)-deficient cell line but not in the two other mutant cell lines. Simultaneous cross-linking of MHC-I and Fas ligation inhibited apoptosis in the ZAP...

Histone H1 couples initiation and amplification of ubiquitin signalling after DNA damage

DEFF Research Database (Denmark)

Thorslund, Tina; Ripplinger, Anita; Hoffmann, Saskia

2015-01-01

DNA double-strand breaks (DSBs) are highly cytotoxic DNA lesions that trigger non-proteolytic ubiquitylation of adjacent chromatin areas to generate binding sites for DNA repair factors. This depends on the sequential actions of the E3 ubiquitin ligases RNF8 and RNF168 (refs 1-6), and UBC13 (also...... known as UBE2N), an E2 ubiquitin-conjugating enzyme that specifically generates K63-linked ubiquitin chains. Whereas RNF168 is known to catalyse ubiquitylation of H2A-type histones, leading to the recruitment of repair factors such as 53BP1 (refs 8-10), the critical substrates of RNF8 and K63-linked...

Ubiquitination of the bacterial inositol phosphatase, SopB, regulates its biological activity at the plasma membrane.

LENUS (Irish Health Repository)

Knodler, Leigh A

2009-11-01

The Salmonella type III effector, SopB, is an inositol polyphosphate phosphatase that modulates host cell phospholipids at the plasma membrane and the nascent Salmonella-containing vacuole (SCV). Translocated SopB persists for many hours after infection and is ubiquitinated but the significance of this covalent modification has not been investigated. Here we identify by mass spectrometry six lysine residues of SopB that are mono-ubiquitinated. Substitution of these six lysine residues with arginine, SopB-K(6)R, almost completely eliminated SopB ubiquitination. We found that ubiquitination does not affect SopB stability or membrane association, or SopB-dependent events in SCV biogenesis. However, two spatially and temporally distinct events are dependent on ubiquitination, downregulation of SopB activity at the plasma membrane and prolonged retention of SopB on the SCV. Activation of the mammalian pro-survival kinase Akt\\/PKB, a downstream target of SopB, was intensified and prolonged after infection with the SopB-K(6)R mutant. At later times, fewer SCV were decorated with SopB-K(6)R compared with SopB. Instead SopB-K(6)R was present as discrete vesicles spread diffusely throughout the cell. Altogether, our data show that ubiquitination of SopB is not related to its intracellular stability but rather regulates its enzymatic activity at the plasma membrane and intracellular localization.

Ubiquitin-mediated proteolysis in Xenopus extract.

Science.gov (United States)

McDowell, Gary S; Philpott, Anna

2016-01-01

The small protein modifier, ubiquitin, can be covalently attached to proteins in the process of ubiquitylation, resulting in a variety of functional outcomes. In particular, the most commonly-associated and well-studied fate for proteins modified with ubiquitin is their ultimate destruction: degradation by the 26S proteasome via the ubiquitin-proteasome system, or digestion in lysosomes by proteolytic enzymes. From the earliest days of ubiquitylation research, a reliable and versatile "cell-in-a-test-tube" system has been employed in the form of cytoplasmic extracts from the eggs and embryos of the frog Xenopus laevis. Biochemical studies of ubiquitin and protein degradation using this system have led to significant advances particularly in the study of ubiquitin-mediated proteolysis, while the versatility of Xenopus as a developmental model has allowed investigation of the in vivo consequences of ubiquitylation. Here we describe the use and history of Xenopus extract in the study of ubiquitin-mediated protein degradation, and highlight the versatility of this system that has been exploited to uncover mechanisms and consequences of ubiquitylation and proteolysis.

The Ubiquitin-Specific Protease 14 (USP14) Is a Critical Regulator of Long-Term Memory Formation

Science.gov (United States)

Jarome, Timothy J.; Kwapis, Janine L.; Hallengren, Jada J.; Wilson, Scott M.; Helmstetter, Fred J.

2014-01-01

Numerous studies have suggested a role for ubiquitin-proteasome-mediated protein degradation in learning-dependent synaptic plasticity; however, very little is known about how protein degradation is regulated at the level of the proteasome during memory formation. The ubiquitin-specific protease 14 (USP14) is a proteasomal deubiquitinating enzyme…

The ubiquitin-proteasome system

Indian Academy of Sciences (India)

... the discovery of protein ubiquitination has led to the recognition of cellular proteolysis as a central area of research in biology. Eukaryotic proteins targeted for degradation by this pathway are first 'tagged' by multimers of a protein known as ubiquitin and are later proteolyzed by a giant enzyme known as the proteasome.

Autoimmunity and inflammation are independent of class II transactivator type PIV-dependent class II major histocompatibility complex expression in peripheral tissues during collagen-induced arthritis.

Science.gov (United States)

Waldburger, Jean-Marc; Palmer, Gaby; Seemayer, Christian; Lamacchia, Celine; Finckh, Axel; Christofilopoulos, Panayiotis; Baeten, Dominique; Reith, Walter; Gabay, Cem

2011-11-01

To determine the regulation of class II major histocompatibility complex (MHC) expression in fibroblast-like synoviocytes (FLS) in order to investigate their role as nonprofessional antigen-presenting cells in collagen-induced arthritis (CIA). Expression of class II MHC, class II MHC transactivator (CIITA), and Ciita isoforms PI, PIII, and PIV was examined by real-time quantitative polymerase chain reaction, immunohistochemistry, and flow cytometry in human synovial tissues, arthritic mouse joints, and human and murine FLS. CIA was induced in mice in which isoform PIV of Ciita was knocked out (PIV(-/-) ), in PIV(-/-) mice transgenic for CIITA in the thymus (K14 CIITA), and in their control littermates. HLA-DRA, total CIITA, and CIITA PIII messenger RNA levels were significantly increased in synovial tissue samples from patients with rheumatoid arthritis compared with the levels in tissue from patients with osteoarthritis. Human FLS expressed surface class II MHC via CIITA PIII and PIV, while class II MHC expression in murine FLS was entirely mediated by PIV. Mice with a targeted deletion of CIITA PIV lack CD4+ T cells and were protected against CIA. The expression of CIITA was restored in the thymus of PIV(-/-) K14 CIITA-transgenic mice, which had a normal CD4+ T cell repertoire and normal surface levels of class II MHC on professional antigen-presenting cells, but did not induce class II MHC on FLS. Synovial inflammation and immune responses against type II collagen were similar in PIV(-/-) K14 CIITA-transgenic mice and control mice with CIA, but bone erosion was significantly reduced in the absence of PIV. Overexpression of class II MHC is tightly correlated with CIITA expression in arthritic synovium and in FLS. Selective targeting of Ciita PIV in peripheral tissues abrogates class II MHC expression by murine FLS but does not protect against inflammation and autoimmune responses in CIA. Copyright © 2011 by the American College of Rheumatology.

Major-histocompatibility-complex-associated variation in secondary sexual traits of white-tailed deer (Odocoileus virginianus): evidence for good-genes advertisement.

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Ditchkoff, S S; Lochmiller, R L; Masters, R E; Hoofer, S R; Van Den Bussche, R A

2001-03-01

Good-genes hypotheses predict that development of secondary sexual characters can be an honest advertisement of heritable male quality. We explored this hypothesis using a cervid model (adult, male white-tailed deer, Odocoileus virginianus) to determine whether antler development could provide an honest signal of a male's genetic quality and condition to adversaries. We compared antler, morphometric, hormonal, and parasitic data collected from hunter-harvested deer to characteristics of the Mhc-DRB (Odvi), the most widely studied gene of the major histocompatibility complex (MHC) in Artiodactyla. We detected associations between genetic characteristics at Odvi-DRB and antler development and body mass, suggesting that antler development and body mass may be associated with pathogen resistance in deer and thus may be an honest signal of genetic quality. We also detected associations between Odvi-DRB characteristics and serum testosterone during the breeding season, suggesting that certain MHC characteristics may help deer cope with stresses related to breeding activity. In addition, we observed a negative relationship between degree of antler development and overall abundance of abomasal helminths. Our observations provide support for the hypothesis that antler development in white-tailed deer is an honest signal of quality.

Evolution of major histocompatibility complex class I and class II genes in the brown bear

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Kuduk Katarzyna

2012-10-01

Full Text Available Abstract Background Major histocompatibility complex (MHC proteins constitute an essential component of the vertebrate immune response, and are coded by the most polymorphic of the vertebrate genes. Here, we investigated sequence variation and evolution of MHC class I and class II DRB, DQA and DQB genes in the brown bear Ursus arctos to characterise the level of polymorphism, estimate the strength of positive selection acting on them, and assess the extent of gene orthology and trans-species polymorphism in Ursidae. Results We found 37 MHC class I, 16 MHC class II DRB, four DQB and two DQA alleles. We confirmed the expression of several loci: three MHC class I, two DRB, two DQB and one DQA. MHC class I also contained two clusters of non-expressed sequences. MHC class I and DRB allele frequencies differed between northern and southern populations of the Scandinavian brown bear. The rate of nonsynonymous substitutions (dN exceeded the rate of synonymous substitutions (dS at putative antigen binding sites of DRB and DQB loci and, marginally significantly, at MHC class I loci. Models of codon evolution supported positive selection at DRB and MHC class I loci. Both MHC class I and MHC class II sequences showed orthology to gene clusters found in the giant panda Ailuropoda melanoleuca. Conclusions Historical positive selection has acted on MHC class I, class II DRB and DQB, but not on the DQA locus. The signal of historical positive selection on the DRB locus was particularly strong, which may be a general feature of caniforms. The presence of MHC class I pseudogenes may indicate faster gene turnover in this class through the birth-and-death process. South–north population structure at MHC loci probably reflects origin of the populations from separate glacial refugia.

Evolution of major histocompatibility complex class I and class II genes in the brown bear.

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Kuduk, Katarzyna; Babik, Wiesław; Bojarska, Katarzyna; Sliwińska, Ewa B; Kindberg, Jonas; Taberlet, Pierre; Swenson, Jon E; Radwan, Jacek

2012-10-02

Major histocompatibility complex (MHC) proteins constitute an essential component of the vertebrate immune response, and are coded by the most polymorphic of the vertebrate genes. Here, we investigated sequence variation and evolution of MHC class I and class II DRB, DQA and DQB genes in the brown bear Ursus arctos to characterise the level of polymorphism, estimate the strength of positive selection acting on them, and assess the extent of gene orthology and trans-species polymorphism in Ursidae. We found 37 MHC class I, 16 MHC class II DRB, four DQB and two DQA alleles. We confirmed the expression of several loci: three MHC class I, two DRB, two DQB and one DQA. MHC class I also contained two clusters of non-expressed sequences. MHC class I and DRB allele frequencies differed between northern and southern populations of the Scandinavian brown bear. The rate of nonsynonymous substitutions (dN) exceeded the rate of synonymous substitutions (dS) at putative antigen binding sites of DRB and DQB loci and, marginally significantly, at MHC class I loci. Models of codon evolution supported positive selection at DRB and MHC class I loci. Both MHC class I and MHC class II sequences showed orthology to gene clusters found in the giant panda Ailuropoda melanoleuca. Historical positive selection has acted on MHC class I, class II DRB and DQB, but not on the DQA locus. The signal of historical positive selection on the DRB locus was particularly strong, which may be a general feature of caniforms. The presence of MHC class I pseudogenes may indicate faster gene turnover in this class through the birth-and-death process. South-north population structure at MHC loci probably reflects origin of the populations from separate glacial refugia.

Evolution of major histocompatibility complex class I and class II genes in the brown bear

Science.gov (United States)

2012-01-01

Background Major histocompatibility complex (MHC) proteins constitute an essential component of the vertebrate immune response, and are coded by the most polymorphic of the vertebrate genes. Here, we investigated sequence variation and evolution of MHC class I and class II DRB, DQA and DQB genes in the brown bear Ursus arctos to characterise the level of polymorphism, estimate the strength of positive selection acting on them, and assess the extent of gene orthology and trans-species polymorphism in Ursidae. Results We found 37 MHC class I, 16 MHC class II DRB, four DQB and two DQA alleles. We confirmed the expression of several loci: three MHC class I, two DRB, two DQB and one DQA. MHC class I also contained two clusters of non-expressed sequences. MHC class I and DRB allele frequencies differed between northern and southern populations of the Scandinavian brown bear. The rate of nonsynonymous substitutions (dN) exceeded the rate of synonymous substitutions (dS) at putative antigen binding sites of DRB and DQB loci and, marginally significantly, at MHC class I loci. Models of codon evolution supported positive selection at DRB and MHC class I loci. Both MHC class I and MHC class II sequences showed orthology to gene clusters found in the giant panda Ailuropoda melanoleuca. Conclusions Historical positive selection has acted on MHC class I, class II DRB and DQB, but not on the DQA locus. The signal of historical positive selection on the DRB locus was particularly strong, which may be a general feature of caniforms. The presence of MHC class I pseudogenes may indicate faster gene turnover in this class through the birth-and-death process. South–north population structure at MHC loci probably reflects origin of the populations from separate glacial refugia. PMID:23031405

Major Histocompatibility Complex Genes Map to Two Chromosomes in an Evolutionarily Ancient Reptile, the Tuatara Sphenodon punctatus.

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Miller, Hilary C; O'Meally, Denis; Ezaz, Tariq; Amemiya, Chris; Marshall-Graves, Jennifer A; Edwards, Scott

2015-05-07

Major histocompatibility complex (MHC) genes are a central component of the vertebrate immune system and usually exist in a single genomic region. However, considerable differences in MHC organization and size exist between different vertebrate lineages. Reptiles occupy a key evolutionary position for understanding how variation in MHC structure evolved in vertebrates, but information on the structure of the MHC region in reptiles is limited. In this study, we investigate the organization and cytogenetic location of MHC genes in the tuatara (Sphenodon punctatus), the sole extant representative of the early-diverging reptilian order Rhynchocephalia. Sequencing and mapping of 12 clones containing class I and II MHC genes from a bacterial artificial chromosome library indicated that the core MHC region is located on chromosome 13q. However, duplication and translocation of MHC genes outside of the core region was evident, because additional class I MHC genes were located on chromosome 4p. We found a total of seven class I sequences and 11 class II β sequences, with evidence for duplication and pseudogenization of genes within the tuatara lineage. The tuatara MHC is characterized by high repeat content and low gene density compared with other species and we found no antigen processing or MHC framework genes on the MHC gene-containing clones. Our findings indicate substantial differences in MHC organization in tuatara compared with mammalian and avian MHCs and highlight the dynamic nature of the MHC. Further sequencing and annotation of tuatara and other reptile MHCs will determine if the tuatara MHC is representative of nonavian reptiles in general. Copyright © 2015 Miller et al.

IFN-induced modulation of histocompatibility antigens on human cells. Background, mechanisms and perspectives

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Hokland, M; Basse, P; Justesen, J

1989-01-01

IFN proteins are a family of lymphokines with anti-viral effects. Several other effects of IFNs have also been described, including enhancement of natural killer (NK) cell activity, enhancement of cytotoxic T-lymphocyte activity, and enhancement of the expression of major histocompatibility compl...... to the classical anti-viral mechanism. This concept proposes that the MHC-enhancing effect of IFNs is a vital part of the immunological defense against virus infections and an integral part of the anti-viral effects of IFN proteins. Udgivelsesdato: 1988-Nov...

Structural Basis for Ubiquitin Recognition and Autoubiquitination by Rabex-5

International Nuclear Information System (INIS)

Lee, S.; Tsai, Y.; Mattera, R.; Smith, W.; Kostelansky, M.; Weissman, A.; Bonifacino, J.; Hurley, J.

2006-01-01

Rabex-5 is an exchange factor for Rab5, a master regulator of endosomal trafficking. Rabex-5 binds monoubiquitin, undergoes covalent ubiquitination and contains an intrinsic ubiquitin ligase activity, all of which require an N-terminal A20 zinc finger followed immediately by a helix. The structure of the N-terminal portion of Rabex-5 bound to ubiquitin at 2.5-Angstroms resolution shows that Rabex-5-ubiquitin interactions occur at two sites. The first site is a new type of ubiquitin-binding domain, an inverted ubiquitin-interacting motif, which binds with ∼29-μM affinity to the canonical Ile44 hydrophobic patch on ubiquitin. The second is a diaromatic patch on the A20 zinc finger, which binds with ∼22-μM affinity to a polar region centered on Asp58 of ubiquitin. The A20 zinc-finger diaromatic patch mediates ubiquitin-ligase activity by directly recruiting a ubiquitin-loaded ubiquitin-conjugating enzyme

Distribution of class ii major histocompatibility complex antigenexpressing cells in human dental pulp with carious lesions

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Tetiana Haniastuti

2012-09-01

Full Text Available Background: Dental caries is a bacterial infection which causes destruction of the hard tissues of the tooth. Exposure of the dentin to the oral environment as a result of caries inevitably results in a cellular response in the pulp. The major histocompatibility complex (MHC is a group of genes that code for cell-surface histocompatibility antigens. Cells expressing class II MHC molecules participate in the initial recognition and the processing of antigenic substances to serve as antigen-presenting cells. Purpose: The aim of the study was to elucidate the alteration in the distribution of class II MHC antigen-expressing cells in human dental pulp as carious lesions progressed toward the pulp. Methods: Fifteen third molars with caries at the occlusal site at various stages of decay and 5 intact third molars were extracted and used in this study. Before decalcifying with 10% EDTA solution (pH 7.4, all the samples were observed by micro-computed tomography to confirm the lesion condition three-dimensionally. The specimens were then processed for cryosection and immunohistochemistry using an anti-MHC class II monoclonal antibody. Results: Class II MHC antigen-expressing cells were found both in normal and carious specimens. In normal tooth, the class II MHC-immunopositive cells were observed mainly at the periphery of the pulp tissue. In teeth with caries, class II MHC-immunopositive cells were located predominantly subjacent to the carious lesions. As the caries progressed, the number of class II MHC antigen-expressing cells was increased. Conclusion: The depth of carious lesions affects the distribution of class II MHC antigen-expressing cells in the dental pulp.Latar belakang: Karies merupakan penyakit infeksi bakteri yang mengakibatkan destruksi jaringan keras gigi. Dentin yang terbuka akibat karies akan menginduksi respon imun seluler pada pulpa. Kompleks histokompatibilitas utama (MHC merupakan sekumpulan gen yang mengkode histokompatibilitas

Ubiquitination dynamics in the early-branching eukaryote Giardia intestinalis

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Niño, Carlos A; Chaparro, Jenny; Soffientini, Paolo; Polo, Simona; Wasserman, Moises

2013-01-01

Ubiquitination is a highly dynamic and versatile posttranslational modification that regulates protein function, stability, and interactions. To investigate the roles of ubiquitination in a primitive eukaryotic lineage, we utilized the early-branching eukaryote Giardia intestinalis. Using a combination of biochemical, immunofluorescence-based, and proteomics approaches, we assessed the ubiquitination status during the process of differentiation in Giardia. We observed that different types of ubiquitin modifications present specific cellular and temporal distribution throughout the Giardia life cycle from trophozoites to cyst maturation. Ubiquitin signal was detected in the wall of mature cysts, and enzymes implicated in cyst wall biogenesis were identified as substrates for ubiquitination. Interestingly, inhibition of proteasome activity did not affect trophozoite replication and differentiation, while it caused a decrease in cyst viability, arguing for proteasome involvement in cyst wall maturation. Using a proteomics approach, we identified around 200 high-confidence ubiquitinated candidates that vary their ubiquitination status during differentiation. Our results indicate that ubiquitination is critical for several cellular processes in this primitive eukaryote. PMID:23613346

Adaptive molecular evolution of the Major Histocompatibility Complex genes, DRA and DQA, in the genus Equus.

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Kamath, Pauline L; Getz, Wayne M

2011-05-18

Major Histocompatibility Complex (MHC) genes are central to vertebrate immune response and are believed to be under balancing selection by pathogens. This hypothesis has been supported by observations of extremely high polymorphism, elevated nonsynonymous to synonymous base pair substitution rates and trans-species polymorphisms at these loci. In equids, the organization and variability of this gene family has been described, however the full extent of diversity and selection is unknown. As selection is not expected to act uniformly on a functional gene, maximum likelihood codon-based models of selection that allow heterogeneity in selection across codon positions can be valuable for examining MHC gene evolution and the molecular basis for species adaptations. We investigated the evolution of two class II MHC genes of the Equine Lymphocyte Antigen (ELA), DRA and DQA, in the genus Equus with the addition of novel alleles identified in plains zebra (E. quagga, formerly E. burchelli). We found that both genes exhibited a high degree of polymorphism and inter-specific sharing of allele lineages. To our knowledge, DRA allelic diversity was discovered to be higher than has ever been observed in vertebrates. Evidence was also found to support a duplication of the DQA locus. Selection analyses, evaluated in terms of relative rates of nonsynonymous to synonymous mutations (dN/dS) averaged over the gene region, indicated that the majority of codon sites were conserved and under purifying selection (dN

Adaptive molecular evolution of the Major Histocompatibility Complex genes, DRA and DQA, in the genus Equus

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Getz Wayne M

2011-05-01

Full Text Available Abstract Background Major Histocompatibility Complex (MHC genes are central to vertebrate immune response and are believed to be under balancing selection by pathogens. This hypothesis has been supported by observations of extremely high polymorphism, elevated nonsynonymous to synonymous base pair substitution rates and trans-species polymorphisms at these loci. In equids, the organization and variability of this gene family has been described, however the full extent of diversity and selection is unknown. As selection is not expected to act uniformly on a functional gene, maximum likelihood codon-based models of selection that allow heterogeneity in selection across codon positions can be valuable for examining MHC gene evolution and the molecular basis for species adaptations. Results We investigated the evolution of two class II MHC genes of the Equine Lymphocyte Antigen (ELA, DRA and DQA, in the genus Equus with the addition of novel alleles identified in plains zebra (E. quagga, formerly E. burchelli. We found that both genes exhibited a high degree of polymorphism and inter-specific sharing of allele lineages. To our knowledge, DRA allelic diversity was discovered to be higher than has ever been observed in vertebrates. Evidence was also found to support a duplication of the DQA locus. Selection analyses, evaluated in terms of relative rates of nonsynonymous to synonymous mutations (dN/dS averaged over the gene region, indicated that the majority of codon sites were conserved and under purifying selection (dN dS. However, the most likely evolutionary codon models allowed for variable rates of selection across codon sites at both loci and, at the DQA, supported the hypothesis of positive selection acting on specific sites. Conclusions Observations of elevated genetic diversity and trans-species polymorphisms supported the conclusion that balancing selection may be acting on these loci. Furthermore, at the DQA, positive selection was

E2-EPF UCP Possesses E3 Ubiquitin Ligase Activity via Its Cysteine 118 Residue.

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Lim, Jung Hwa; Shin, Hee Won; Chung, Kyung-Sook; Kim, Nam-Soon; Kim, Ju Hee; Jung, Hong-Ryul; Im, Dong-Soo; Jung, Cho-Rok

Here, we show that E2-EPF ubiquitin carrier protein (UCP) elongated E3-independent polyubiquitin chains on the lysine residues of von Hippel-Lindau protein (pVHL) and its own lysine residues both in vitro and in vivo. The initiation of the ubiquitin reaction depended on not only Lys11 linkage but also the Lys6, Lys48 and Lys63 residues of ubiquitin, which were involved in polyubiquitin chain formation on UCP itself. UCP self-association occurred through the UBC domain, which also contributed to the interaction with pVHL. The polyubiquitin chains appeared on the N-terminus of UCP in vivo, which indicated that the N-terminus of UCP contains target lysines for polyubiquitination. The Lys76 residue of UCP was the most critical site for auto-ubiquitination, whereas the polyubiquitin chain formation on pVHL occurred on all three of its lysines (Lys159, Lys171 and Lys196). A UCP mutant in which Cys118 was changed to alanine (UCPC118A) did not form a polyubiquitin chain but did strongly accumulate mono- and di-ubiquitin via auto-ubiquitination. Polyubiquitin chain formation required the coordination of Cys95 and Cys118 between two interacting molecules. The mechanism of the polyubiquitin chain reaction of UCP may involve the transfer of ubiquitin from Cys95 to Cys118 by trans-thiolation, with polyubiquitin chains forming at Cys118 by reversible thioester bonding. The polyubiquitin chains are then moved to the lysine residues of the substrate by irreversible isopeptide bonding. During the elongation of the ubiquitin chain, an active Cys118 residue is required in both parts of UCP, namely, the catalytic enzyme and the substrate. In conclusion, UCP possesses not only E2 ubiquitin conjugating enzyme activity but also E3 ubiquitin ligase activity, and Cys118 is critical for polyubiquitin chain formation.

E2-EPF UCP Possesses E3 Ubiquitin Ligase Activity via Its Cysteine 118 Residue.

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Jung Hwa Lim

Full Text Available Here, we show that E2-EPF ubiquitin carrier protein (UCP elongated E3-independent polyubiquitin chains on the lysine residues of von Hippel-Lindau protein (pVHL and its own lysine residues both in vitro and in vivo. The initiation of the ubiquitin reaction depended on not only Lys11 linkage but also the Lys6, Lys48 and Lys63 residues of ubiquitin, which were involved in polyubiquitin chain formation on UCP itself. UCP self-association occurred through the UBC domain, which also contributed to the interaction with pVHL. The polyubiquitin chains appeared on the N-terminus of UCP in vivo, which indicated that the N-terminus of UCP contains target lysines for polyubiquitination. The Lys76 residue of UCP was the most critical site for auto-ubiquitination, whereas the polyubiquitin chain formation on pVHL occurred on all three of its lysines (Lys159, Lys171 and Lys196. A UCP mutant in which Cys118 was changed to alanine (UCPC118A did not form a polyubiquitin chain but did strongly accumulate mono- and di-ubiquitin via auto-ubiquitination. Polyubiquitin chain formation required the coordination of Cys95 and Cys118 between two interacting molecules. The mechanism of the polyubiquitin chain reaction of UCP may involve the transfer of ubiquitin from Cys95 to Cys118 by trans-thiolation, with polyubiquitin chains forming at Cys118 by reversible thioester bonding. The polyubiquitin chains are then moved to the lysine residues of the substrate by irreversible isopeptide bonding. During the elongation of the ubiquitin chain, an active Cys118 residue is required in both parts of UCP, namely, the catalytic enzyme and the substrate. In conclusion, UCP possesses not only E2 ubiquitin conjugating enzyme activity but also E3 ubiquitin ligase activity, and Cys118 is critical for polyubiquitin chain formation.

Heterologous SUMO-2/3-ubiquitin chains optimize IκBα degradation and NF-κB activity.

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Fabienne Aillet

Full Text Available The NF-κB pathway is regulated by SUMOylation at least at three levels: the inhibitory molecule IκBα, the IKK subunit γ/NEMO and the p52 precursor p100. Here we investigate the role of SUMO-2/3 in the degradation of IκBα and activation of NF-κB mediated by TNFα. We found that under conditions of deficient SUMOylation, an important delay in both TNFα-mediated proteolysis of IκBα and NF-κB dependent transcription occurs. In vitro and ex vivo approaches, including the use of ubiquitin-traps (TUBEs, revealed the formation of chains on IκBα containing SUMO-2/3 and ubiquitin after TNFα stimulation. The integration of SUMO-2/3 appears to promote the formation of ubiquitin chains on IκBα after activation of the TNFα signalling pathway. Furthermore, heterologous chains of SUMO-2/3 and ubiquitin promote a more efficient degradation of IκBα by the 26S proteasome in vitro compared to chains of either SUMO-2/3 or ubiquitin alone. Consistently, Ubc9 silencing reduced the capture of IκBα modified with SUMO-ubiquitin hybrid chains that display a defective proteasome-mediated degradation. Thus, hybrid SUMO-2/3-ubiquitin chains increase the susceptibility of modified IκBα to the action of 26S proteasome, contributing to the optimal control of NF-κB activity after TNFα-stimulation.

Signal one and two blockade are both critical for non-myeloablative murine HSCT across a major histocompatibility complex barrier.

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Kia J Langford-Smith

Full Text Available Non-myeloablative allogeneic haematopoietic stem cell transplantation (HSCT is rarely achievable clinically, except where donor cells have selective advantages. Murine non-myeloablative conditioning regimens have limited clinical success, partly through use of clinically unachievable cell doses or strain combinations permitting allograft acceptance using immunosuppression alone. We found that reducing busulfan conditioning in murine syngeneic HSCT, increases bone marrow (BM:blood SDF-1 ratio and total donor cells homing to BM, but reduces the proportion of donor cells engrafting. Despite this, syngeneic engraftment is achievable with non-myeloablative busulfan (25 mg/kg and higher cell doses induce increased chimerism. Therefore we investigated regimens promoting initial donor cell engraftment in the major histocompatibility complex barrier mismatched CBA to C57BL/6 allo-transplant model. This requires full myeloablation and immunosuppression with non-depleting anti-CD4/CD8 blocking antibodies to achieve engraftment of low cell doses, and rejects with reduced intensity conditioning (≤75 mg/kg busulfan. We compared increased antibody treatment, G-CSF, niche disruption and high cell dose, using reduced intensity busulfan and CD4/8 blockade in this model. Most treatments increased initial donor engraftment, but only addition of co-stimulatory blockade permitted long-term engraftment with reduced intensity or non-myeloablative conditioning, suggesting that signal 1 and 2 T-cell blockade is more important than early BM niche engraftment for transplant success.

Signal one and two blockade are both critical for non-myeloablative murine HSCT across a major histocompatibility complex barrier.

Science.gov (United States)

Langford-Smith, Kia J; Sandiford, Zara; Langford-Smith, Alex; Wilkinson, Fiona L; Jones, Simon A; Wraith, J Ed; Wynn, Robert F; Bigger, Brian W

2013-01-01

Non-myeloablative allogeneic haematopoietic stem cell transplantation (HSCT) is rarely achievable clinically, except where donor cells have selective advantages. Murine non-myeloablative conditioning regimens have limited clinical success, partly through use of clinically unachievable cell doses or strain combinations permitting allograft acceptance using immunosuppression alone. We found that reducing busulfan conditioning in murine syngeneic HSCT, increases bone marrow (BM):blood SDF-1 ratio and total donor cells homing to BM, but reduces the proportion of donor cells engrafting. Despite this, syngeneic engraftment is achievable with non-myeloablative busulfan (25 mg/kg) and higher cell doses induce increased chimerism. Therefore we investigated regimens promoting initial donor cell engraftment in the major histocompatibility complex barrier mismatched CBA to C57BL/6 allo-transplant model. This requires full myeloablation and immunosuppression with non-depleting anti-CD4/CD8 blocking antibodies to achieve engraftment of low cell doses, and rejects with reduced intensity conditioning (≤75 mg/kg busulfan). We compared increased antibody treatment, G-CSF, niche disruption and high cell dose, using reduced intensity busulfan and CD4/8 blockade in this model. Most treatments increased initial donor engraftment, but only addition of co-stimulatory blockade permitted long-term engraftment with reduced intensity or non-myeloablative conditioning, suggesting that signal 1 and 2 T-cell blockade is more important than early BM niche engraftment for transplant success.

The T-Cell Receptor Can Bind to the Peptide-Bound Major Histocompatibility Complex and Uncomplexed β₂-Microglobulin through Distinct Binding Sites

DEFF Research Database (Denmark)

Merkle, Patrick S.; Irving, Melita; Hongjian, Song

2017-01-01

from molecular dynamics simulations. Using a biological assay based on TCR gene-engineered primary human T cells, we did not observe a significant effect of β2m on T-cell cytotoxicity, suggesting an alternate role for β2m binding. Overall, we show that binding of β2m to the TCR occurs in vitro and......T-Cell receptor (TCR)-mediated recognition of the peptide-bound major histocompatibility complex (pMHC) initiates an adaptive immune response against antigen-presenting target cells. The recognition events take place at the TCR-pMHC interface, and their effects on TCR conformation and dynamics...... are controversial. Here, we have measured the time-resolved hydrogen/deuterium exchange (HDX) of a soluble TCR in the presence and absence of its cognate pMHC by mass spectrometry to delineate the impact of pMHC binding on solution-phase structural dynamics in the TCR. Our results demonstrate that while TCR...

A Review on Ubiquitination of Neurotrophin Receptors: Facts and Perspectives

Science.gov (United States)

Sánchez-Sánchez, Julia; Arévalo, Juan Carlos

2017-01-01

Ubiquitination is a reversible post-translational modification involved in a plethora of different physiological functions. Among the substrates that are ubiquitinated, neurotrophin receptors (TrkA, TrkB, TrkC, and p75NTR) have been studied recently. TrkA is the most studied receptor in terms of its ubiquitination, and different E3 ubiquitin ligases and deubiquitinases have been implicated in its ubiquitination, whereas not much is known about the other neurotrophin receptors aside from their ubiquitination. Additional studies are needed that focus on the ubiquitination of TrkB, TrkC, and p75NTR in order to further understand the role of ubiquitination in their physiological and pathological functions. Here we review what is currently known regarding the ubiquitination of neurotrophin receptors and its physiological and pathological relevance. PMID:28335430

Ubiquitin Signaling: Extreme Conservation as a Source of Diversity

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Alice Zuin

2014-07-01

Full Text Available Around 2 × 103–2.5 × 103 million years ago, a unicellular organism with radically novel features, ancestor of all eukaryotes, dwelt the earth. This organism, commonly referred as the last eukaryotic common ancestor, contained in its proteome the same functionally capable ubiquitin molecule that all eukaryotic species contain today. The fact that ubiquitin protein has virtually not changed during all eukaryotic evolution contrasts with the high expansion of the ubiquitin system, constituted by hundreds of enzymes, ubiquitin-interacting proteins, protein complexes, and cofactors. Interestingly, the simplest genetic arrangement encoding a fully-equipped ubiquitin signaling system is constituted by five genes organized in an operon-like cluster, and is found in archaea. How did ubiquitin achieve the status of central element in eukaryotic physiology? We analyze here the features of the ubiquitin molecule and the network that it conforms, and propose notions to explain the complexity of the ubiquitin signaling system in eukaryotic cells.

Activation of Stat-3 is involved in the induction of apoptosis after ligation of major histocompatibility complex class I molecules on human Jurkat T cells

DEFF Research Database (Denmark)

Skov, S; Nielsen, M; Bregenholt, S

1998-01-01

Activation of Janus tyrosine kinases (Jak) and Signal transducers and activators of transcription (Stat) after ligation of major histocompatibility complex class I (MHC-I) was explored in Jurkat T cells. Cross-linking of MHC-I mediated tyrosine phosphorylation of Tyk2, but not Jak1, Jak2, and Jak3......-probe derived from the interferon-gamma activated site (GAS) in the c-fos promoter, a common DNA sequence for Stat protein binding. An association between hSIE and Stat-3 after MHC-I ligation was directly demonstrated by precipitating Stat-3 from nuclear extracts with biotinylated hSIE probe and avidin......-coupled agarose. To investigate the function of the activated Stat-3, Jurkat T cells were transiently transfected with a Stat-3 isoform lacking the transactivating domain. This dominant-negative acting Stat-3 isoform significantly inhibited apoptosis induced by ligation of MHC-I. In conclusion, our data suggest...

454 sequencing reveals extreme complexity of the class II Major Histocompatibility Complex in the collared flycatcher

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Gustafsson Lars

2010-12-01

Full Text Available Abstract Background Because of their functional significance, the Major Histocompatibility Complex (MHC class I and II genes have been the subject of continuous interest in the fields of ecology, evolution and conservation. In some vertebrate groups MHC consists of multiple loci with similar alleles; therefore, the multiple loci must be genotyped simultaneously. In such complex systems, understanding of the evolutionary patterns and their causes has been limited due to challenges posed by genotyping. Results Here we used 454 amplicon sequencing to characterize MHC class IIB exon 2 variation in the collared flycatcher, an important organism in evolutionary and immuno-ecological studies. On the basis of over 152,000 sequencing reads we identified 194 putative alleles in 237 individuals. We found an extreme complexity of the MHC class IIB in the collared flycatchers, with our estimates pointing to the presence of at least nine expressed loci and a large, though difficult to estimate precisely, number of pseudogene loci. Many similar alleles occurred in the pseudogenes indicating either a series of recent duplications or extensive concerted evolution. The expressed alleles showed unambiguous signals of historical selection and the occurrence of apparent interlocus exchange of alleles. Placing the collared flycatcher's MHC sequences in the context of passerine diversity revealed transspecific MHC class II evolution within the Muscicapidae family. Conclusions 454 amplicon sequencing is an effective tool for advancing our understanding of the MHC class II structure and evolutionary patterns in Passeriformes. We found a highly dynamic pattern of evolution of MHC class IIB genes with strong signals of selection and pronounced sequence divergence in expressed genes, in contrast to the apparent sequence homogenization in pseudogenes. We show that next generation sequencing offers a universal, affordable method for the characterization and, in perspective

The dynamics of histone H2A ubiquitination in HeLa cells exposed to rapamycin, ethanol, hydroxyurea, ER stress, heat shock and DNA damage.

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Nakata, Shiori; Watanabe, Tadashi; Nakagawa, Koji; Takeda, Hiroshi; Ito, Akihiro; Fujimuro, Masahiro

2016-03-25

Polyubiquitination plays key roles in proteasome-dependent and independent cellular events, whereas monoubiquitination is involved in gene expression, DNA repair, protein-protein interaction, and protein trafficking. We previously developed an FK2 antibody, which specifically recognizes poly-Ub moieties but not free Ub. To elucidate the role of Ub conjugation in response to cellular stress, we used FK2 to investigate whether chemical stress (rapamycin, ethanol, or hydroxyurea), ER stress (thapsigargin or tunicamycin), heat shock or DNA damage (H2O2 or methyl methanesulfonate) affect the formation of Ub conjugates including histone H2A (hH2A) ubiquitination. First, we found that all forms of stress tested increased poly-ubiquitinated proteins in HeLa cells. Furthermore, rapamycin and hydroxyurea treatment, and ER stress increased ubiquitination of hH2A, while methyl methanesulfonate (MMS) treatment induced deubiquitination of hH2A. The ethanol and H2O2 treatments, and heat shock transiently induced hH2A de-ubiquitination, although deubiquitinated hH2A were ubiquitinated again by subsequent cultivation. We also revealed that FK2 reacts with not only polyubiquitinated proteins but also mono-ubiquitinated hH2A. With the exception of MMS, all forms of stress tested increased the acetylation of K5-hH2A, K9-hH3 and K8-hH4 in addition to ubiquitination. K118 and K119 of hH2A were ubiquitinated in cells under normal conditions, and K119 was the major ubiquitination site. The MMS-treatment and heat shock induced the deubiquitination of both K118 and K119-histone H2A. Interestingly, MMS treatment did not affect cell HeLa cell viability expressing double-mutant hH2A (KK118,119AA-hH2A), while heat shock slightly but significantly decreased viability of double-mutant hH2A expressing cells, indicating that ubiquitination of both sites associates with recovery from heat shock but not MMS treatment. Thus, we characterized FK2 reactivity and demonstrated that various stresses alter

Stealing the spotlight: CUL4-DDB1 ubiquitin ligase docks WD40-repeat proteins to destroy

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Zhang Hui

2007-02-01

Full Text Available Abstract Recent investigation of Cullin 4 (CUL4 has ushered this class of multiprotein ubiquitin E3 ligases to center stage as critical regulators of diverse processes including cell cycle regulation, developmental patterning, DNA replication, DNA damage and repair, and epigenetic control of gene expression. CUL4 associates with DNA Damage Binding protein 1 (DDB1 to assemble an ubiquitin E3 ligase that targets protein substrates for ubiquitin-dependent proteolysis. CUL4 ligase activity is also regulated by the covalent attachment of the ubiquitin-like protein NEDD8 to CUL4, or neddylation, and the COP9 signalosome complex (CSN that removes this important modification. Recently, multiple WD40-repeat proteins (WDR were found to interact with DDB1 and serve as the substrate-recognition subunits of the CUL4-DDB1 ubiquitin ligase. As more than 150–300 WDR proteins exist in the human genome, these findings impact a wide array of biological processes through CUL4 ligase-mediated proteolysis. Here, we review the recent progress in understanding the mechanism of CUL4 ubiquitin E3 ligase and discuss the architecture of CUL4-assembled E3 ubiquitin ligase complexes by comparison to CUL1-based E3s (SCF. Then, we will review several examples to highlight the critical roles of CUL4 ubiquitin ligase in genome stability, cell cycle regulation, and histone lysine methylation. Together, these studies provide insights into the mechanism of this novel ubiquitin ligase in the regulation of important biological processes.

Data on mass spectrometry-based proteomics for studying the involvement of CYLD in the ubiquitination events downstream of EGFR activation

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Virginia Sanchez-Quiles

2018-06-01

Full Text Available The present data article corresponds to the proteomic data of the involvement of Cylindromatosis protein (CYLD in the ubiquitination signaling initiated by EGF stimulation. CYLD tumor suppressor protein has Lys63-chain deubiquitinase activity that has been proved essential for the negative regulation of crucial signaling mechanisms, namely the NFkB pathway. Previous results have suggested the involvement of CYLD in the EGF-dependent signal transduction as well, showing its engagement within the tyrosine-phosphorylated complexes formed following the addition of the growth factor. EGFR signaling participates in central cellular processes and its tight regulation, partly through ubiquitination cascades, is decisive for a balanced cellular homeostasis. We carried out the substitution of the endogenous pool of ubiquitin for a His-FLAG-tagged ubiquitin (Stable Ubiquitin Exchange, StUbEx, in combination with the shRNA silencing of CYLD and SILAC-labeling on HeLa cells. The subsequent tandem affinity purification of ubiquitinated proteins in control and CYLD-depleted cells was followed by mass spectrometric analysis. Therefore, we present an unbiased study investigating the impact of CYLD in the EGF-dependent ubiquitination. The data supplied herein is related to the research article entitled “Cylindromatosis tumor suppressor protein (CYLD deubiquitinase is necessary for proper ubiquitination and degradation of the epidermal growth factor receptor” (Sanchez-Quiles et al., 2017 [1]. We provide the associated mass spectrometry raw files, excel tables and gene ontology enrichments. The data have been deposited in the ProteomeXchange with the identifier PXD003423.

Motional properties of unfolded ubiquitin: a model for a random coil protein

Energy Technology Data Exchange (ETDEWEB)

Wirmer, Julia [Johann Wolfgang GoeUniversityFrankfurt, Institute for Organic Chemistry and Chemical Biology, Center for Biomolecular Magnetic Resonance (Germany); Peti, Wolfgang [Brown University, Department of Molecular Pharmacology, Physiology and Biotechnology (United States); Schwalbe, Harald [Johann Wolfgang GoeUniversityFrankfurt, Institute for Organic Chemistry and Chemical Biology, Center for Biomolecular Magnetic Resonance (Germany)], E-mail: schwalbe@nmr.uni-frankfurt.de

2006-07-15

The characterization of unfolded states of proteins has recently attracted considerable interest, as the residual structure present in these states may play a crucial role in determining their folding and misfolding behavior. Here, we investigated the dynamics in the denatured state of ubiquitin in 8 M urea at pH2. Under these conditions, ubiquitin does not have any detectable local residual structure, and uniform {sup 15}N relaxation rates along the sequence indicate the absence of motional restrictions caused by residual secondary structure and/or long-range interactions. A comparison of different models to predict relaxation data in unfolded proteins suggests that the subnanosecond dynamics in unfolded states depend on segmental motions only and do not show a dependence on the residue type but for proline and glycine residues.

BAG3 Expression in Glioblastoma Cells Promotes Accumulation of Ubiquitinated Clients in an Hsp70-dependent Manner*

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Gentilella, Antonio; Khalili, Kamel

2011-01-01

Disposal of damaged proteins and protein aggregates is a prerequisite for the maintenance of cellular homeostasis and impairment of this disposal can lead to a broad range of pathological conditions, most notably in brain-associated disorders including Parkinson and Alzheimer diseases, and cancer. In this respect, the Protein Quality Control (PQC) pathway plays a central role in the clearance of damaged proteins. The Hsc/Hsp70-co-chaperone BAG3 has been described as a new and critical component of the PQC in several cellular contexts. For example, the expression of BAG3 in the rodent brain correlates with the engagement of protein degradation machineries in response to proteotoxic stress. Nevertheless, little is known about the molecular events assisted by BAG3. Here we show that ectopic expression of BAG3 in glioblastoma cells leads to the activation of an HSF1-driven stress response, as attested by transcriptional activation of BAG3 and Hsp70. BAG3 overexpression determines an accumulation of ubiquitinated proteins and this event requires the N-terminal region, WW domain of BAG3 and the association of BAG3 with Hsp70. The ubiquitination mainly occurs on BAG3-client proteins and the inhibition of proteasomal activity results in a further accumulation of ubiquitinated clients. At the cellular level, overexpression of BAG3 in glioblastoma cell lines, but not in non-glial cells, results in a remarkable decrease in colony formation capacity and this effect is reverted when the binding of BAG3 to Hsp70 is impaired. These observations provide the first evidence for an involvement of BAG3 in the ubiquitination and turnover of its partners. PMID:21233200

Ubiquitin C-terminal electrophiles are activity-based probes for identification and mechanistic study of ubiquitin conjugating machinery.

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Love, Kerry Routenberg; Pandya, Renuka K; Spooner, Eric; Ploegh, Hidde L

2009-04-17

Protein modification by ubiquitin (Ub) and ubiquitin-like modifiers (Ubl) requires the action of activating (E1), conjugating (E2), and ligating (E3) enzymes and is a key step in the specific destruction of proteins. Deubiquitinating enzymes (DUBs) deconjugate substrates modified with Ub/Ubl's and recycle Ub inside the cell. Genome mining based on sequence homology to proteins with known function has assigned many enzymes to this pathway without confirmation of either conjugating or DUB activity. Function-dependent methodologies are still the most useful for rapid identification or assessment of biological activity of expressed proteins from cells. Activity-based protein profiling uses chemical probes that are active-site-directed for the classification of protein activities in complex mixtures. Here we show that the design and use of an expanded set of Ub-based electrophilic probes allowed us to recover and identify members of each enzyme class in the ubiquitin-proteasome system, including E3 ligases and DUBs with previously unverified activity. We show that epitope-tagged Ub-electrophilic probes can be used as activity-based probes for E3 ligase identification by in vitro labeling and activity studies of purified enzymes identified from complex mixtures in cell lysate. Furthermore, the reactivity of our probe with the HECT domain of the E3 Ub ligase ARF-BP1 suggests that multiple cysteines may be in the vicinity of the E2-binding site and are capable of the transfer of Ub to self or to a substrate protein.

The Ubiquitin-associated (UBA) 1 Domain of Schizosaccharomyces pombe Rhp23 Is Essential for the Recognition of Ubiquitin-proteasome System Substrates Both in Vitro and in Vivo*

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Medina, Bethan; Paraskevopoulos, Konstantinos; Boehringer, Jonas; Sznajder, Anna; Robertson, Morag; Endicott, Jane; Gordon, Colin

2012-01-01

The ubiquitin-proteasome system is essential for maintaining a functional cell. Not only does it remove incorrectly folded proteins, it also regulates protein levels to ensure their appropriate spatial and temporal distribution. Proteins marked for degradation by the addition of Lys48-linked ubiquitin (Ub) chains are recognized by shuttle factors and transported to the 26 S proteasome. One of these shuttle factors, Schizosaccharomyces pombe Rhp23, has an unusual domain architecture. It comprises an N-terminal ubiquitin-like domain that can recognize the proteasome followed by two ubiquitin-associated (UBA) domains, termed UBA1 and UBA2, which can bind Ub. This architecture is conserved up to humans, suggesting that both domains are important for Rhp23 function. Such an extent of conservation raises the question as to why, in contrast to all other shuttle proteins, does Rhp23 require two UBA domains? We performed in vitro Ub binding assays using domain swap chimeric proteins and mutated domains in isolation as well as in the context of the full-length protein to reveal that the Ub binding properties of the UBA domains are context-dependent. In vivo, the internal Rhp23 UBA1 domain provides sufficient Ub recognition for the protein to function without UBA2. PMID:23038266

Genetic wealth, population health: Major histocompatibility complex variation in captive and wild ring-tailed lemurs (Lemur catta).

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Grogan, Kathleen E; Sauther, Michelle L; Cuozzo, Frank P; Drea, Christine M

2017-10-01

Across species, diversity at the major histocompatibility complex (MHC) is critical to individual disease resistance and, hence, to population health; however, MHC diversity can be reduced in small, fragmented, or isolated populations. Given the need for comparative studies of functional genetic diversity, we investigated whether MHC diversity differs between populations which are open, that is experiencing gene flow, versus populations which are closed, that is isolated from other populations. Using the endangered ring-tailed lemur ( Lemur catta ) as a model, we compared two populations under long-term study: a relatively "open," wild population ( n  = 180) derived from Bezà Mahafaly Special Reserve, Madagascar (2003-2013) and a "closed," captive population ( n  = 121) derived from the Duke Lemur Center (DLC, 1980-2013) and from the Indianapolis and Cincinnati Zoos (2012). For all animals, we assessed MHC-DRB diversity and, across populations, we compared the number of unique MHC-DRB alleles and their distributions. Wild individuals possessed more MHC-DRB alleles than did captive individuals, and overall, the wild population had more unique MHC-DRB alleles that were more evenly distributed than did the captive population. Despite management efforts to maintain or increase genetic diversity in the DLC population, MHC diversity remained static from 1980 to 2010. Since 2010, however, captive-breeding efforts resulted in the MHC diversity of offspring increasing to a level commensurate with that found in wild individuals. Therefore, loss of genetic diversity in lemurs, owing to small founder populations or reduced gene flow, can be mitigated by managed breeding efforts. Quantifying MHC diversity within individuals and between populations is the necessary first step to identifying potential improvements to captive management and conservation plans.

Novel full-length major histocompatibility complex class I allele discovery and haplotype definition in pig-tailed macaques.

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Semler, Matthew R; Wiseman, Roger W; Karl, Julie A; Graham, Michael E; Gieger, Samantha M; O'Connor, David H

2017-11-13

Pig-tailed macaques (Macaca nemestrina, Mane) are important models for human immunodeficiency virus (HIV) studies. Their infectability with minimally modified HIV makes them a uniquely valuable animal model to mimic human infection with HIV and progression to acquired immunodeficiency syndrome (AIDS). However, variation in the pig-tailed macaque major histocompatibility complex (MHC) and the impact of individual transcripts on the pathogenesis of HIV and other infectious diseases is understudied compared to that of rhesus and cynomolgus macaques. In this study, we used Pacific Biosciences single-molecule real-time circular consensus sequencing to describe full-length MHC class I (MHC-I) transcripts for 194 pig-tailed macaques from three breeding centers. We then used the full-length sequences to infer Mane-A and Mane-B haplotypes containing groups of MHC-I transcripts that co-segregate due to physical linkage. In total, we characterized full-length open reading frames (ORFs) for 313 Mane-A, Mane-B, and Mane-I sequences that defined 86 Mane-A and 106 Mane-B MHC-I haplotypes. Pacific Biosciences technology allows us to resolve these Mane-A and Mane-B haplotypes to the level of synonymous allelic variants. The newly defined haplotypes and transcript sequences containing full-length ORFs provide an important resource for infectious disease researchers as certain MHC haplotypes have been shown to provide exceptional control of simian immunodeficiency virus (SIV) replication and prevention of AIDS-like disease in nonhuman primates. The increased allelic resolution provided by Pacific Biosciences sequencing also benefits transplant research by allowing researchers to more specifically match haplotypes between donors and recipients to the level of nonsynonymous allelic variation, thus reducing the risk of graft-versus-host disease.

Smad3 recruits the anaphase-promoting complex for ubiquitination and degradation of SnoN

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Stroschein, Shannon L.; Bonni, Shirin; Wrana, Jeffrey L.; Luo, Kunxin

2001-09-11

Smad proteins mediate transforming growth factor-b signaling to regulate cell growth and differentiation. SnoN is an important negative regulator of TGFb signaling that functions to maintain the repressed state of TGFb target genes in the absence of ligand. Upon TGFb stimulation, Smad3 and Smad2 translocate into the nucleus and induce a rapid degradation of SnoN, allowing activation of TGFb target genes. Here we show that Smad2- or Smad3-induced degradation of SnoN requires the ubiquitin-dependent proteasome and can be mediated by the anaphase promoting complex (APC) and the UbcH5 family of ubiquitin conjugating enzymes. Smad3 and to a lesser extent, Smad2, interact with both the APC and SnoN, resulting in the recruitment of the APC to SnoN and subsequent ubiquitination of SnoN in a destruction box-dependent manner. In addition to the destruction box, efficient degradation of SnoN also requires the Smad3 binding site in SnoN as well as key lysine residues necessary for ubiquitin attachment. Mutation of either the Smad3 binding site or lysine residues results in stabilization of SnoN and in enhanced antagonism of TGFb signaling. Our studies elucidate an important pathway for the degradation of SnoN and reveal a novel role of the APC in regulation of TGFb signaling.

Smad3 recruits the anaphase-promoting complex for ubiquitination and degradation of SnoN

International Nuclear Information System (INIS)

Stroschein, Shannon L.; Bonni, Shirin; Wrana, Jeffrey L.; Luo, Kunxin

2001-01-01

Smad proteins mediate transforming growth factor-b signaling to regulate cell growth and differentiation. SnoN is an important negative regulator of TGFb signaling that functions to maintain the repressed state of TGFb target genes in the absence of ligand. Upon TGFb stimulation, Smad3 and Smad2 translocate into the nucleus and induce a rapid degradation of SnoN, allowing activation of TGFb target genes. Here we show that Smad2- or Smad3-induced degradation of SnoN requires the ubiquitin-dependent proteasome and can be mediated by the anaphase promoting complex (APC) and the UbcH5 family of ubiquitin conjugating enzymes. Smad3 and to a lesser extent, Smad2, interact with both the APC and SnoN, resulting in the recruitment of the APC to SnoN and subsequent ubiquitination of SnoN in a destruction box-dependent manner. In addition to the destruction box, efficient degradation of SnoN also requires the Smad3 binding site in SnoN as well as key lysine residues necessary for ubiquitin attachment. Mutation of either the Smad3 binding site or lysine residues results in stabilization of SnoN and in enhanced antagonism of TGFb signaling. Our studies elucidate an important pathway for the degradation of SnoN and reveal a novel role of the APC in regulation of TGFb signaling

Puromycin induces SUMO and ubiquitin redistribution upon proteasome inhibition

International Nuclear Information System (INIS)

Matsumoto, Hotaru; Saitoh, Hisato

2016-01-01

We have previously reported the co-localization of O-propargyl-puromycin (OP-Puro) with SUMO-2/3 and ubiquitin at promyelocytic leukemia-nuclear bodies (PML-NBs) in the presence of the proteasome inhibitor MG132, implying a role for the ubiquitin family in sequestering OP-puromycylated immature polypeptides to the nucleus during impaired proteasome activity. Here, we found that as expected puromycin induced SUMO-1/2/3 accumulation with ubiquitin at multiple nuclear foci in HeLa cells when co-exposed to MG132. Co-administration of puromycin and MG132 also facilitated redistribution of PML and the SUMO-targeted ubiquitin ligase RNF4 concurrently with SUMO-2/3. As removal of the drugs from the medium led to disappearance of the SUMO-2/3-ubiquitin nuclear foci, our findings indicated that nuclear assembly/disassembly of SUMO-2/3 and ubiquitin was pharmacologically manipulable, supporting our previous observation on OP-Puro, which predicted the ubiquitin family function in sequestrating aberrant proteins to the nucleus. -- Highlights: •Puromycin exhibits the O-propargyl-puromycin effect. •Puromycin induces SUMO redistribution upon proteasome inhibition. •Ubiquitin and RNF4 accumulate at PML-nuclear bodies with SUMO-2/3. •The ubiquitin family may function in nuclear sequestration of immature proteins.

Puromycin induces SUMO and ubiquitin redistribution upon proteasome inhibition

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Matsumoto, Hotaru [Course for Biological Sciences, Faculty of Science, Kumamoto University, Kumamoto (Japan); Saitoh, Hisato, E-mail: hisa@kumamoto-u.ac.jp [Course for Biological Sciences, Faculty of Science, Kumamoto University, Kumamoto (Japan); Department of Biological Sciences, Graduate School of Science and Technology, Kumamoto University, Kumamoto (Japan)

2016-07-29

We have previously reported the co-localization of O-propargyl-puromycin (OP-Puro) with SUMO-2/3 and ubiquitin at promyelocytic leukemia-nuclear bodies (PML-NBs) in the presence of the proteasome inhibitor MG132, implying a role for the ubiquitin family in sequestering OP-puromycylated immature polypeptides to the nucleus during impaired proteasome activity. Here, we found that as expected puromycin induced SUMO-1/2/3 accumulation with ubiquitin at multiple nuclear foci in HeLa cells when co-exposed to MG132. Co-administration of puromycin and MG132 also facilitated redistribution of PML and the SUMO-targeted ubiquitin ligase RNF4 concurrently with SUMO-2/3. As removal of the drugs from the medium led to disappearance of the SUMO-2/3-ubiquitin nuclear foci, our findings indicated that nuclear assembly/disassembly of SUMO-2/3 and ubiquitin was pharmacologically manipulable, supporting our previous observation on OP-Puro, which predicted the ubiquitin family function in sequestrating aberrant proteins to the nucleus. -- Highlights: •Puromycin exhibits the O-propargyl-puromycin effect. •Puromycin induces SUMO redistribution upon proteasome inhibition. •Ubiquitin and RNF4 accumulate at PML-nuclear bodies with SUMO-2/3. •The ubiquitin family may function in nuclear sequestration of immature proteins.

Regulation of G Protein-Coupled Receptors by Ubiquitination

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Kamila Skieterska

2017-04-01

Full Text Available G protein-coupled receptors (GPCRs comprise the largest family of membrane receptors that control many cellular processes and consequently often serve as drug targets. These receptors undergo a strict regulation by mechanisms such as internalization and desensitization, which are strongly influenced by posttranslational modifications. Ubiquitination is a posttranslational modification with a broad range of functions that is currently gaining increased appreciation as a regulator of GPCR activity. The role of ubiquitination in directing GPCRs for lysosomal degradation has already been well-established. Furthermore, this modification can also play a role in targeting membrane and endoplasmic reticulum-associated receptors to the proteasome. Most recently, ubiquitination was also shown to be involved in GPCR signaling. In this review, we present current knowledge on the molecular basis of GPCR regulation by ubiquitination, and highlight the importance of E3 ubiquitin ligases, deubiquitinating enzymes and β-arrestins. Finally, we discuss classical and newly-discovered functions of ubiquitination in controlling GPCR activity.

Mastermind-Like 1 Is Ubiquitinated: Functional Consequences for Notch Signaling.

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Mozhgan Farshbaf

Full Text Available Early studies demonstrated the involvement of ubiquitination of the Notch intracellular domain for rapid turnover of the transcriptional complex at Notch target genes. It was shown that this ubiquitination was promoted by the co-activator Mastermind like 1 (MAML1. MAML1 also contains numerous lysine residues that may also be ubiquitinated and necessary for protein regulation. In this study, we show that over-expressed MAML1 is ubiquitinated and identify eight conserved lysine residues which are required for ubiquitination. We also show that p300 stimulates ubiquitination and that Notch inhibits ubiquitination. Furthermore, we show that a mutant MAML1 that has decreased ubiquitination shows increased output from a HES1 reporter gene assay. Therefore, we speculate that ubiquitination of MAML1 might be a mechanism to maintain low levels of the protein until needed for transcriptional activation. In summary, this study identifies that MAML1 is ubiquitinated in the absence of Notch signaling to maintain low levels of MAML1 in the cell. Our data supports the notion that a precise and tight regulation of the Notch pathway is required for this signaling pathway.

Ubiquitin domain proteins in disease

DEFF Research Database (Denmark)

Klausen, Louise Kjær; Schulze, Andrea; Seeger, Michael

2007-01-01

The human genome encodes several ubiquitin-like (UBL) domain proteins (UDPs). Members of this protein family are involved in a variety of cellular functions and many are connected to the ubiquitin proteasome system, an essential pathway for protein degradation in eukaryotic cells. Despite...... and cancer. Publication history: Republished from Current BioData's Targeted Proteins database (TPdb; http://www.targetedproteinsdb.com)....

Linear ubiquitin chain induces apoptosis and inhibits tumor growth.

Science.gov (United States)

Qin, Zhoushuai; Jiang, Wandong; Wang, Guifen; Sun, Ying; Xiao, Wei

2018-01-01

Ubiquitination of proliferating cell nuclear antigen (PCNA) plays an important role in DNA damage response. Ectopic expression of PCNA fused at either terminus with ubiquitin (Ub) lacking two C-terminal glycine residues induces translesion DNA synthesis which resembles synthesis mediated by PCNA monoubiquitination. PCNA fused with Ub containing the C-terminal Gly residues at the C-terminus can be further polyubiquitinated in a Gly-dependent manner, which inhibits cell proliferation and induces ATR-dependent replication checkpoint. In this study, we surprisingly found that PCNA fused to a head-to-tail linear Ub chain induces apoptosis in a Ub chain length-dependent manner. Further investigation revealed that the apoptotic effect is actually induced by the linear Ub chain independently from PCNA, as the Ub chain fused to GFP or an epitope tag still efficiently induces apoptosis. It is revealed that the artificial linear Ub chain differs from endogenously encoded linear Ub chains in that its Ubs contain a Ub-G76S substitution, making the Ub chain resistant to cleavage by deubiquitination enzymes. We demonstrated in this study that ectopic expression of the artificial Ub chain alone in cultured human cancer cells is sufficient to inhibit tumor growth in a xenograft mouse model, making the linear Ub chain a putative anti-cancer agent.

Impairment of social behavior and communication in mice lacking the Uba6-dependent ubiquitin activation system.

Science.gov (United States)

Lee, Ji Yeon; Kwak, Minseok; Lee, Peter C W

2015-03-15

The Uba6-Use1 ubiquitin enzyme cascade is a poorly understood arm of the ubiquitin-proteasome system required for mouse development. Recently, we reported that Uba6 brain-specific knockout (termed NKO) mice display abnormal social behavior and neuronal development due to a decreased spine density and accumulation of Ube3a and Shank3. To better characterize a potential role for NKO mice in autism spectrum disorders (ASDs), we performed a comprehensive behavioral characterization of the social behavior and communication of NKO mice. Our behavioral results confirmed that NKO mice display social impairments, as indicated by fewer vocalizations and decreased social interaction. We conclude that UBA6 NKO mice represent a novel ASD mouse model of anti-social and less verbal behavioral symptoms. Copyright © 2014 Elsevier B.V. All rights reserved.

The ubiquitin family meets the Fanconi anemia proteins.

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Renaudin, Xavier; Koch Lerner, Leticia; Menck, Carlos Frederico Martins; Rosselli, Filippo

2016-01-01

Fanconi anaemia (FA) is a hereditary disorder characterized by bone marrow failure, developmental defects, predisposition to cancer and chromosomal abnormalities. FA is caused by biallelic mutations that inactivate genes encoding proteins involved in replication stress-associated DNA damage responses. The 20 FANC proteins identified to date constitute the FANC pathway. A key event in this pathway involves the monoubiquitination of the FANCD2-FANCI heterodimer by the collective action of at least 10 different proteins assembled in the FANC core complex. The FANC core complex-mediated monoubiquitination of FANCD2-FANCI is essential to assemble the heterodimer in subnuclear, chromatin-associated, foci and to regulate the process of DNA repair as well as the rescue of stalled replication forks. Several recent works have demonstrated that the activity of the FANC pathway is linked to several other protein post-translational modifications from the ubiquitin-like family, including SUMO and NEDD8. These modifications are related to DNA damage responses but may also affect other cellular functions potentially related to the clinical phenotypes of the syndrome. This review summarizes the interplay between the ubiquitin and ubiquitin-like proteins and the FANC proteins that constitute a major pathway for the surveillance of the genomic integrity and addresses the implications of their interactions in maintaining genome stability. Copyright © 2016 Elsevier B.V. All rights reserved.

Time-of-day- and light-dependent expression of ubiquitin protein ligase E3 component N-recognin 4 (UBR4 in the suprachiasmatic nucleus circadian clock.

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Harrod H Ling

Full Text Available Circadian rhythms of behavior and physiology are driven by the biological clock that operates endogenously but can also be entrained to the light-dark cycle of the environment. In mammals, the master circadian pacemaker is located in the suprachiasmatic nucleus (SCN, which is composed of individual cellular oscillators that are driven by a set of core clock genes interacting in transcriptional/translational feedback loops. Light signals can trigger molecular events in the SCN that ultimately impact on the phase of expression of core clock genes to reset the master pacemaker. While transcriptional regulation has received much attention in the field of circadian biology in the past, other mechanisms including targeted protein degradation likely contribute to the clock timing and entrainment process. In the present study, proteome-wide screens of the murine SCN led to the identification of ubiquitin protein ligase E3 component N-recognin 4 (UBR4, a novel E3 ubiquitin ligase component of the N-end rule pathway, as a time-of-day-dependent and light-inducible protein. The spatial and temporal expression pattern of UBR4 in the SCN was subsequently characterized by immunofluorescence microscopy. UBR4 is expressed across the entire rostrocaudal extent of the SCN in a time-of-day-dependent fashion. UBR4 is localized exclusively to arginine vasopressin (AVP-expressing neurons of the SCN shell. Upon photic stimulation in the early subjective night, the number of UBR4-expressing cells within the SCN increases. This study is the first to identify a novel E3 ubiquitin ligase component, UBR4, in the murine SCN and to implicate the N-end rule degradation pathway as a potential player in regulating core clock mechanisms and photic entrainment.

A unique deubiquitinase that deconjugates phosphoribosyl-linked protein ubiquitination

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Qiu, Jiazhang; Yu, Kaiwen; Fei, Xiaowen; Liu, Yao; Nakayasu, Ernesto S.; Piehowski, Paul D.; Shaw, Jared B.; Puvar, Kedar; Das, Chittaranjan; Liu, Xiaoyun; Luo, Zhao-Qing

2017-05-12

Ubiquitination regulates many aspects of host immunity and thus is a common target for infectious agents. Recent studies revealed that members of the SidE effector family of the bacterial pathogen Legionella pneumophila attacked several small GTPases associated with the endoplasmic reticulum by a novel ubiquitination mechanism that does not require the E1 and E2 enzymes of the host ubiquitination machinery. Following ubiquitin activation by ADP- ribosylation via a mono-ADP-ribosylation motif, ADP-ribosylated ubiquitin is cleaved by a phosphodiesterasedomainwithinSdeA,whichisconcomitantwiththelinkof phosphoribosylated ubiquitin to serine residues in the substrate. Here we demonstrate that the activity of SidEs is regulated by SidJ, another effector encoded by a gene situated in the locus coding for three members of the SidE family (SdeC, SdeB and SdeA). SidJ functions to remove ubiquitin from SidEs-modified substrates by cleaving the phosphodiester bond that links phosphoribosylated ubiquitin to protein substrates. Further, the deubiquitinase activity of SidJ is essential for its role in L. pneumophila infection. Finally, the activity of SidJ is required for efficiently reducing the abundance of ubiquitinated Rab33b in infected cells within a few hours after bacterial uptake. Our results establish SidJ as a deubiquitinase that functions to impose temporal regulation of the activity of the SidE effectors. The identification of SidJ may shed light on future study of signaling cascades mediated by this unique ubiquitination that also potentially regulates cellular processes in eukaryotic cells.

NEMO binds ubiquitinated TANK-binding kinase 1 (TBK1 to regulate innate immune responses to RNA viruses.

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Lingyan Wang

Full Text Available RIG-I-like receptors (RLR are intracellular sensors utilized by nearly all cell types for recognition of viral RNA, initiation of antiviral defense, and induction of type I interferons (IFN. TBK1 is a critical kinase implicated in RLR-dependent IFN transcription. Posttranslational modification of TBK1 by K63-linked ubiquitin is required for RLR driven signaling. However, the TBK1 ubiquitin acceptor sites and the function of ubiquitinated TBK1 in the signaling cascade are unknown. We now show that TBK1 is ubiquitinated on residues K69, K154, and K372 in response to infection with RNA virus. The K69 and K154 residues are critical for innate antiviral responses and IFN production. Ubiquitinated TBK1 recruits the downstream adaptor NEMO through ubiquitin binding domains. The assembly of the NEMO/TBK1 complex on the mitochondrial protein MAVS leads to activation of TBK1 kinase activity and phosphorylation of the transcription factor, interferon response factor 3. The combined results refine current views of RLR signaling, define the role of TBK1 polyubiquitination, and detail the mechanisms involved in signalosome assembly.

Genetic variation of the major histocompatibility complex (MHC class II B gene in the threatened Hume's pheasant, Syrmaticus humiae.

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Weicai Chen

Full Text Available Major histocompatibility complex (MHC genes are the most polymorphic genes in vertebrates and encode molecules that play a crucial role in pathogen resistance. As a result of their diversity, they have received much attention in the fields of evolutionary and conservation biology. Here, we described the genetic variation of MHC class II B (MHCIIB exon 2 in a wild population of Hume's pheasant (Syrmaticus humiae, which has suffered a dramatic decline in population over the last three decades across its ranges in the face of heavy exploitation and habitat loss. Twenty-four distinct alleles were found in 73 S. humiae specimens. We found seven shared alleles among four geographical groups as well as six rare MHCIIB alleles. Most individuals displayed between one to five alleles, suggesting that there are at least three MHCIIB loci of the Hume's pheasant. The dN ⁄ dS ratio at putative antigen-binding sites (ABS was significantly greater than one, indicating balancing selection is acting on MHCIIB exon 2. Additionally, recombination and gene conversion contributed to generating MHCIIB diversity in the Hume's pheasant. One to three recombination events and seventy-five significant gene conversion events were observed within the Hume's pheasant MHCIIB loci. The phylogenetic tree and network analysis revealed that the Hume's pheasant alleles do not cluster together, but are scattered through the tree or network indicating a trans-species evolutionary mode. These findings revealed the evolution of the Hume's pheasant MHC after suffering extreme habitat fragmentation.

Generation of a genomic tiling array of the human Major Histocompatibility Complex (MHC and its application for DNA methylation analysis

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Ottaviani Diego

2008-05-01

Full Text Available Abstract Background The major histocompatibility complex (MHC is essential for human immunity and is highly associated with common diseases, including cancer. While the genetics of the MHC has been studied intensively for many decades, very little is known about the epigenetics of this most polymorphic and disease-associated region of the genome. Methods To facilitate comprehensive epigenetic analyses of this region, we have generated a genomic tiling array of 2 Kb resolution covering the entire 4 Mb MHC region. The array has been designed to be compatible with chromatin immunoprecipitation (ChIP, methylated DNA immunoprecipitation (MeDIP, array comparative genomic hybridization (aCGH and expression profiling, including of non-coding RNAs. The array comprises 7832 features, consisting of two replicates of both forward and reverse strands of MHC amplicons and appropriate controls. Results Using MeDIP, we demonstrate the application of the MHC array for DNA methylation profiling and the identification of tissue-specific differentially methylated regions (tDMRs. Based on the analysis of two tissues and two cell types, we identified 90 tDMRs within the MHC and describe their characterisation. Conclusion A tiling array covering the MHC region was developed and validated. Its successful application for DNA methylation profiling indicates that this array represents a useful tool for molecular analyses of the MHC in the context of medical genomics.

The human ubiquitin-conjugating enzyme Cdc34 controls cellular proliferation through regulation of p27Kip1 protein levels

International Nuclear Information System (INIS)

Butz, Nicole; Ruetz, Stephan; Natt, Francois; Hall, Jonathan; Weiler, Jan; Mestan, Juergen; Ducarre, Monique; Grossenbacher, Rita; Hauser, Patrick; Kempf, Dominique; Hofmann, Francesco

2005-01-01

Ubiquitin-mediated degradation of the cyclin-dependent kinase inhibitor p27 Kip1 was shown to be required for the activation of key cyclin-dependent kinases, thereby triggering the onset of DNA replication and cell cycle progression. Although the SCF Skp2 ubiquitin ligase has been reported to mediate p27 Kip1 degradation, the nature of the human ubiquitin-conjugating enzyme involved in this process has not yet been determined at the cellular level. Here, we show that antisense oligonucleotides targeting the human ubiquitin-conjugating enzyme Cdc34 downregulate its expression, inhibit the degradation of p27 Kip1 , and prevent cellular proliferation. Elevation of p27 Kip1 protein level is found to be the sole requirement for the inhibition of cellular proliferation induced upon downregulation of Cdc34. Indeed, reducing the expression of p27 Kip1 with a specific antisense oligonucleotide is sufficient to reverse the anti-proliferative phenotype elicited by the Cdc34 antisense. Furthermore, downregulation of Cdc34 is found to specifically increase the abundance of the SCF Skp2 ubiquitin ligase substrate p27 Kip1 , but has no concomitant effect on the level of IkBα and β-catenin, which are known substrates of a closely related SCF ligase

The tomato Fni3 lysine-63-specific ubiquitin-conjugating enzyme and suv ubiquitin E2 variant positively regulate plant immunity.

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Mural, Ravi V; Liu, Yao; Rosebrock, Tracy R; Brady, Jennifer J; Hamera, Sadia; Connor, Richard A; Martin, Gregory B; Zeng, Lirong

2013-09-01

The activation of an immune response in tomato (Solanum lycopersicum) against Pseudomonas syringae relies on the recognition of E3 ligase-deficient forms of AvrPtoB by the host protein kinase, Fen. To investigate the mechanisms by which Fen-mediated immunity is regulated, we characterize in this study a Fen-interacting protein, Fni3, and its cofactor, S. lycoperiscum Uev (Suv). Fni3 encodes a homolog of the Ubc13-type ubiquitin-conjugating enzyme that catalyzes exclusively Lys-63-linked ubiquitination, whereas Suv is a ubiquitin-conjugating enzyme variant. The C-terminal region of Fen was necessary for interaction with Fni3, and this interaction was required for cell death triggered by overexpression of Fen in Nicotiana benthamiana leaves. Fni3 was shown to be an active E2 enzyme, but Suv displayed no ubiquitin-conjugating activity; Fni3 and Suv together directed Lys-63-linked ubiquitination. Decreased expression of Fni3, another tomato Ubc13 homolog, Sl-Ubc13-2, or Suv in N. benthamiana leaves diminished cell death associated with Fen-mediated immunity and cell death elicited by several other resistance (R) proteins and their cognate effectors. We also discovered that coexpression of Fen and other R proteins/effectors with a Fni3 mutant that is compromised for ubiquitin-conjugating activity diminished the cell death. These results suggest that Fni3/Sl-Ubc13-2 and Suv regulate the immune response mediated by Fen and other R proteins through Lys-63-linked ubiquitination.

The N-end rule pathway catalyzes a major fraction of the protein degradation in skeletal muscle

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Solomon, V.; Lecker, S. H.; Goldberg, A. L.

1998-01-01

In skeletal muscle, overall protein degradation involves the ubiquitin-proteasome system. One property of a protein that leads to rapid ubiquitin-dependent degradation is the presence of a basic, acidic, or bulky hydrophobic residue at its N terminus. However, in normal cells, substrates for this N-end rule pathway, which involves ubiquitin carrier protein (E2) E214k and ubiquitin-protein ligase (E3) E3alpha, have remained unclear. Surprisingly, in soluble extracts of rabbit muscle, we found that competitive inhibitors of E3alpha markedly inhibited the 125I-ubiquitin conjugation and ATP-dependent degradation of endogenous proteins. These inhibitors appear to selectively inhibit E3alpha, since they blocked degradation of 125I-lysozyme, a model N-end rule substrate, but did not affect the degradation of proteins whose ubiquitination involved other E3s. The addition of several E2s or E3alpha to the muscle extracts stimulated overall proteolysis and ubiquitination, but only the stimulation by E3alpha or E214k was sensitive to these inhibitors. A similar general inhibition of ubiquitin conjugation to endogenous proteins was observed with a dominant negative inhibitor of E214k. Certain substrates of the N-end rule pathway are degraded after their tRNA-dependent arginylation. We found that adding RNase A to muscle extracts reduced the ATP-dependent proteolysis of endogenous proteins, and supplying tRNA partially restored this process. Finally, although in muscle extracts the N-end rule pathway catalyzes most ubiquitin conjugation, it makes only a minor contribution to overall protein ubiquitination in HeLa cell extracts.

Expression and clinical value of the soluble major histocompatibility complex class I-related chain A molecule in the serum of patients with renal tumors.

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Zhao, Y-K; Jia, C-M; Yuan, G-J; Liu, W; Qiu, Y; Zhu, Q-G

2015-06-29

We investigated the expression and clinical value of the soluble major histocompatibility complex class I-related chain A (sMICA) molecule in the serum of patients with renal tumors. Sixty patients diagnosed with renal tumors were enrolled in the experimental group, whereas 20 healthy volunteers served as the control group. The sMICA levels were measured via enzyme-linked immunosorbent assay, and the results were analyzed in combination with data from pathol-ogy examination. The experimental group had a statistically significant higher sMICA level (P < 0.05) than the control group. The sMICA level was higher in patients with malignant tumors than in those with be-nign tumors. We also observed a positive relationship among different tumor-node-metastasis (TNM) pathological stages with more advanced diseases exhibiting higher sMICA levels. As a tumor-associated antigen, MICA has a close relationship with renal tumorigenesis and immune es-cape. Our results indicated that sMICA levels were related to tumor pathol-ogy, TNM stage, and metastasis. Therefore, sMICA might be a potential marker for tumor characteristics, prognosis, and recurrence prediction.

Healthy and unhealthy dependence: implications for major depression.

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Schulte, Fiona S; Mongrain, Myriam; Flora, David B

2008-09-01

To examine the contribution of varying levels of dependency to Axis I and Axis II disorders, and to the recurrence of major depression in a graduate student sample diagnosed with a history of the disorder. At Time 1, participants were interviewed to confirm a current or past episode of major depression along with the presence of Axis II and other current or past Axis I disorders. Various measures of dependency were administered including the Depressive Experiences Questionnaire (DEQ; Blatt, D'Afflitti, & Quinlan, 1976), the 3-Vector Dependency Inventory (3VDI; Pincus & Gurtman, 1995), and the Personal Style Inventory (PSI; Robins et al., 1994). Participants were interviewed 20 months later to determine the recurrence of a depressive episode. A factor analysis conducted on scale scores for each dependency measure resulted in three factors labelled 'unhealthy', 'intermediate', and 'healthy' dependence. Controlling for history of major depression, structural equation modelling found 'unhealthy' dependence to be the only predictor of recurrences of major depression and Axis II disorders, while 'healthy' dependence was related to fewer depressive symptoms. These results have important implications for the conceptualization of the dependency construct.

Dengue Virus Genome Uncoating Requires Ubiquitination

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Laura A. Byk

2016-06-01

Full Text Available The process of genome release or uncoating after viral entry is one of the least-studied steps in the flavivirus life cycle. Flaviviruses are mainly arthropod-borne viruses, including emerging and reemerging pathogens such as dengue, Zika, and West Nile viruses. Currently, dengue virus is one of the most significant human viral pathogens transmitted by mosquitoes and is responsible for about 390 million infections every year around the world. Here, we examined for the first time molecular aspects of dengue virus genome uncoating. We followed the fate of the capsid protein and RNA genome early during infection and found that capsid is degraded after viral internalization by the host ubiquitin-proteasome system. However, proteasome activity and capsid degradation were not necessary to free the genome for initial viral translation. Unexpectedly, genome uncoating was blocked by inhibiting ubiquitination. Using different assays to bypass entry and evaluate the first rounds of viral translation, a narrow window of time during infection that requires ubiquitination but not proteasome activity was identified. In this regard, ubiquitin E1-activating enzyme inhibition was sufficient to stabilize the incoming viral genome in the cytoplasm of infected cells, causing its retention in either endosomes or nucleocapsids. Our data support a model in which dengue virus genome uncoating requires a nondegradative ubiquitination step, providing new insights into this crucial but understudied viral process.

TDP-43 in Familial and Sporadic Frontotemporal Lobar Degeneration with Ubiquitin Inclusions

NARCIS (Netherlands)

Cairns, Nigel J.; Neumann, Manuela; Bigio, Eileen H.; Holm, Ida E.; Troost, Dirk; Hatanpaa, Kimmo J.; Foong, Chan; White, Charles L.; Schneider, Julie A.; Kretzschmar, Hans A.; Carter, Deborah; Taylor-Reinwald, Lisa; Paulsmeyer, Katherine; Strider, Jeffrey; Gitcho, Michael; Goate, Alison M.; Morris, John C.; Mishrall, Manjari; Kwong, Linda K.; Stieber, Anna; Xu, Yan; Forman, Mark S.; Trojanowski, John Q.; Lee, Virginia M.-Y.; Mackenzie, Ian R. A.

2007-01-01

TAR DNA-binding protein 43 (TDP-43) is a major pathological protein of sporadic and familial frontotemporal lobar degeneration with ubiquitin-positive, tau-negative inclusions (FTLD-U) with or without motor neuron disease (MND). Thus, TDP-43 defines a novel class of neurodegenerative diseases called

ρ0 Cells Feature De-Ubiquitination of SLC Transporters and Increased Levels and Fluxes of Amino Acids

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André Bordinassi Medina

2017-04-01

Full Text Available Solute carrier (SLC transporters are a diverse group of membrane transporter proteins that regulate the cellular flux and distribution of endogenous and xenobiotic compounds. Post-translational modifications (PTMs, such as ubiquitination, have recently emerged as one of the major regulatory mechanisms in protein function and localization. Previously, we showed that SLC amino acid transporters were on average 6-fold de-ubiquitinated and increased amino acid levels were detected in ρ0 cells (lacking mitochondrial DNA, mtDNA compared to parental cells. Here, we elucidated the altered functionality of SLC transporters and their dynamic ubiquitination status by measuring the uptake of several isotopically labeled amino acids in both human osteosarcoma 143B.TK- and ρ0 cells. Our pulse chase analysis indicated that de-ubiquitinated amino acid transporters in ρ0 cells were accompanied by an increased transport rate, which leads to higher levels of amino acids in the cell. Finding SLC transport enhancers is an aim of the pharmaceutical industry in order to compensate for loss of function mutations in these genes. Thus, the ubiquitination status of SLC transporters could be an indicator for their functionality, but evidence for a direct connection between de-ubiquitination and transporter activity has to be further elucidated.

Proteomes and Ubiquitylomes Analysis Reveals the Involvement of Ubiquitination in Protein Degradation in Petunias1

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Liu, Juanxu; Wei, Qian; Wang, Rongmin; Yang, Weiyuan; Ma, Yueyue; Chen, Guoju

2017-01-01

Petal senescence is a complex programmed process. It has been demonstrated previously that treatment with ethylene, a plant hormone involved in senescence, can extensively alter transcriptome and proteome profiles in plants. However, little is known regarding the impact of ethylene on posttranslational modification (PTM) or the association between PTM and the proteome. Protein degradation is one of the hallmarks of senescence, and ubiquitination, a major PTM in eukaryotes, plays important roles in protein degradation. In this study, we first obtained reference petunia (Petunia hybrida) transcriptome data via RNA sequencing. Next, we quantitatively investigated the petunia proteome and ubiquitylome and the association between them in petunia corollas following ethylene treatment. In total, 51,799 unigenes, 3,606 proteins, and 2,270 ubiquitination sites were quantified 16 h after ethylene treatment. Treatment with ethylene resulted in 14,448 down-regulated and 6,303 up-regulated unigenes (absolute log2 fold change > 1 and false discovery rate petunia. Several putative ubiquitin ligases were up-regulated at the protein and transcription levels. Our results showed that the global proteome and ubiquitylome were negatively correlated and that ubiquitination could be involved in the degradation of proteins during ethylene-mediated corolla senescence in petunia. Ethylene regulates hormone signaling transduction pathways at both the protein and ubiquitination levels in petunia corollas. In addition, our results revealed that ethylene increases the ubiquitination levels of proteins involved in endoplasmic reticulum-associated degradation. PMID:27810942

Met1-linked Ubiquitination in Immune Signalling

DEFF Research Database (Denmark)

Fiil, Berthe Katrine; Gyrd-Hansen, Mads

2014-01-01

Methionine 1-linked ubiquitin chains (Met1-Ub), or linear ubiquitin, has emerged as a central post-translational modification in innate immune signalling. Molecular machinery that assembles, senses and, more recently, disassembles Met1-Ub has been identified, and technical advances have enabled...... identification of physiological substrates for Met1-Ub in response to activation of innate immune receptors. These discoveries have significantly advanced our understanding of how non-degradative ubiquitin modifications control pro-inflammatory responses mediated by nuclear factor κB and mitogen...

Cooperativity of the SUMO and Ubiquitin Pathways in Genome Stability

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Minghua Nie

2016-02-01

Full Text Available Covalent attachment of ubiquitin (Ub or SUMO to DNA repair proteins plays critical roles in maintaining genome stability. These structurally related polypeptides can be viewed as distinct road signs, with each being read by specific protein interaction motifs. Therefore, via their interactions with selective readers in the proteome, ubiquitin and SUMO can elicit distinct cellular responses, such as directing DNA lesions into different repair pathways. On the other hand, through the action of the SUMO-targeted ubiquitin ligase (STUbL family proteins, ubiquitin and SUMO can cooperate in the form of a hybrid signal. These mixed SUMO-ubiquitin chains recruit “effector” proteins such as the AAA+ ATPase Cdc48/p97-Ufd1-Npl4 complex that contain both ubiquitin and SUMO interaction motifs. This review will summarize recent key findings on collaborative and distinct roles that ubiquitin and SUMO play in orchestrating DNA damage responses.

Polarisation of major histocompatibility complex II host genotype with pathogenesis of European Brown Hare syndrome virus.

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Christos Iacovakis

Full Text Available A study was conducted in order to determine the occurrence of European Brown Hare Syndrome virus (EBHSV in Denmark and possible relation between disease pathogenesis and Major Histocompatibility Complex (MHC host genotype. Liver samples were examined from 170 brown hares (hunted, found sick or dead, collected between 2004 and 2009. Macroscopical and histopathological findings consistent with EBHS were detected in 24 (14.1% hares; 35 (20.6% had liver lesions not typical of the syndrome, 50 (29.4% had lesions in other tissues and 61 (35.9% had no lesions. Sixty five (38.2% of 170 samples were found to be EBHSV-positive (RT-PCR, VP60 gene. In order to investigate associations between viral pathogenesis and host genotype, variation within the exon 2 DQA gene of MHC was assessed. DQA exon 2 analysis revealed the occurrence of seven different alleles in Denmark. Consistent with other populations examined so far in Europe, observed heterozygosity of DQA (H o = 0.1180 was lower than expected (H e = 0.5835. The overall variation for both nucleotide and amino acid differences (2.9% and 14.9%, respectively were lower in Denmark than those assessed in other European countries (8.3% and 16.9%, respectively. Within the peptide binding region codons the number of nonsynonymous substitutions (dN was much higher than synonymous substitutions (dS, which would be expected for MHC alleles under balancing selection. Allele frequencies did not significantly differ between EBHSV-positive and -negative hares. However, allele Leeu-DQA*30 was detected in significantly higher (P = 0.000006 frequency among the positive hares found dead with severe histopathological lesions than among those found sick or apparently healthy. In contrast, the latter group was characterized by a higher frequency of the allele Leeu-DQA*14 as well as the proportion of heterozygous individuals (P = 0.000006 and P = 0.027. These data reveal a polarisation between EBHSV

Key Role of Toll-Like Receptor 2 in the Inflammatory Response and Major Histocompatibility Complex Class II Downregulation in Brucella abortus-Infected Alveolar Macrophages

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Ferrero, Mariana C.; Hielpos, M. Soledad; Carvalho, Natalia B.; Barrionuevo, Paula; Corsetti, Patricia P.; Giambartolomei, Guillermo H.; Oliveira, Sergio C.

2014-01-01

Alveolar macrophages (AM) seem to constitute the main cellular target of inhaled brucellae. Here, we show that Brucella abortus invades and replicates in murine AM without inducing cytotoxicity. B. abortus infection induced a statistically significant increase of tumor necrosis factor alpha (TNF-α), CXCL1 or keratinocyte chemoattractant (KC), interleukin-1β (IL-1β), IL-6, and IL-12 in AM from C57BL/6 mice and BALB/c mice, but these responses were generally weaker and/or delayed compared to those elicited in peritoneal macrophages. Studies using knockout mice for TLR2, TLR4, and TLR9 revealed that TNF-α and KC responses were mediated by TLR2 recognition. Brucella infection reduced in a multiplicity of infection-dependent manner the expression of major histocompatibility complex class II (MHC-II) molecules induced by gamma interferon (IFN-γ) in AM. The same phenomenon was induced by incubation with heat-killed B. abortus (HKBA) or the lipidated form of the 19-kDa outer membrane protein of Brucella (L-Omp19), and it was shown to be mediated by TLR2 recognition. In contrast, no significant downregulation of MHC-II was induced by either unlipidated Omp19 or Brucella LPS. In a functional assay, treatment of AM with either L-Omp19 or HKBA reduced the MHC-II-restricted presentation of OVA peptides to specific T cells. One week after intratracheal infection, viable B. abortus was detected in AM from both wild-type and TLR2 KO mice, but CFU counts were higher in the latter. These results suggest that B. abortus survives in AM after inhalatory infection in spite of a certain degree of immune control exerted by the TLR2-mediated inflammatory response. Both the modest nature of the latter and the modulation of MHC-II expression by the bacterium may contribute to such survival. PMID:24478078

Low Major Histocompatibility Complex Class II Variation in the Endangered Indo-Pacific Humpback Dolphin (Sousa chinensis): Inferences About the Role of Balancing Selection.

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Zhang, Xiyang; Lin, Wenzhi; Zhou, Ruilian; Gui, Duan; Yu, Xinjian; Wu, Yuping

2016-03-01

It has been widely reported that the major histocompatibility complex (MHC) is under balancing selection due to its immune function across terrestrial and aquatic mammals. The comprehensive studies at MHC and other neutral loci could give us a synthetic evaluation about the major force determining genetic diversity of species. Previously, a low level of genetic diversity has been reported among the Indo-Pacific humpback dolphin (Sousa chinensis) in the Pearl River Estuary (PRE) using both mitochondrial marker and microsatellite loci. Here, the expression and sequence polymorphism of 2 MHC class II genes (DQB and DRB) in 32 S. chinensis from PRE collected between 2003 and 2011 were investigated. High ratios of non-synonymous to synonymous substitution rates, codon-based selection analysis, and trans-species polymorphism (TSP) support the hypothesis that balancing selection acted on S. chinensis MHC sequences. However, only 2 haplotypes were detected at either DQB or DRB loci. Moreover, the lack of deviation from the Hardy-Weinberg expectation at DRB locus combined with the relatively low heterozygosity at both DQB locus and microsatellite loci suggested that balancing selection might not be sufficient, which further suggested that genetic drift associated with historical bottlenecks was not mitigated by balancing selection in terms of the loss of MHC and neutral variation in S. chinensis. The combined results highlighted the importance of maintaining the genetic diversity of the endangered S. chinensis. © The American Genetic Association 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Engineering chimeric human and mouse major histocompatibility complex (MHC) class I tetramers for the production of T-cell receptor (TCR) mimic antibodies

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Bentley, Carol; Yates, Jenna; Salimi, Maryam; Greig, Jenny; Wiblin, Sarah; Hassanali, Tasneem; Banham, Alison H.

2017-01-01

Therapeutic monoclonal antibodies targeting cell surface or secreted antigens are among the most effective classes of novel immunotherapies. However, the majority of human proteins and established cancer biomarkers are intracellular. Peptides derived from these intracellular proteins are presented on the cell surface by major histocompatibility complex class I (MHC-I) and can be targeted by a novel class of T-cell receptor mimic (TCRm) antibodies that recognise similar epitopes to T-cell receptors. Humoural immune responses to MHC-I tetramers rarely generate TCRm antibodies and many antibodies recognise the α3 domain of MHC-I and β2 microglobulin (β2m) that are not directly involved in presenting the target peptide. Here we describe the production of functional chimeric human-murine HLA-A2-H2Dd tetramers and modifications that increase their bacterial expression and refolding efficiency. These chimeric tetramers were successfully used to generate TCRm antibodies against two epitopes derived from wild type tumour suppressor p53 (RMPEAAPPV and GLAPPQHLIRV) that have been used in vaccination studies. Immunisation with chimeric tetramers yielded no antibodies recognising the human α3 domain and β2m and generated TCRm antibodies capable of specifically recognising the target peptide/MHC-I complex in fully human tetramers and on the cell surface of peptide pulsed T2 cells. Chimeric tetramers represent novel immunogens for TCRm antibody production and may also improve the yield of tetramers for groups using these reagents to monitor CD8 T-cell immune responses in HLA-A2 transgenic mouse models of immunotherapy. PMID:28448627

Principles of ubiquitin and SUMO modifications in DNA repair

NARCIS (Netherlands)

Bergink, Steven; Jentsch, Stefan

2009-01-01

With the discovery in the late 1980s that the DNA-repair gene RAD6 encodes a ubiquitin-conjugating enzyme, it became clear that protein modification by ubiquitin conjugation has a much broader significance than had previously been assumed. Now, two decades later, ubiquitin and its cousin SUMO are

Ubiquitin-specific protease 5 is required for the efficient repair of DNA double-strand breaks.

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Satoshi Nakajima

Full Text Available During the DNA damage response (DDR, ubiquitination plays an important role in the recruitment and regulation of repair proteins. However, little is known about elimination of the ubiquitination signal after repair is completed. Here we show that the ubiquitin-specific protease 5 (USP5, a deubiquitinating enzyme, is involved in the elimination of the ubiquitin signal from damaged sites and is required for efficient DNA double-strand break (DSB repair. Depletion of USP5 sensitizes cells to DNA damaging agents, produces DSBs, causes delayed disappearance of γH2AX foci after Bleocin treatment, and influences DSB repair efficiency in the homologous recombination pathway but not in the non-homologous end joining pathway. USP5 co-localizes to DSBs induced by laser micro-irradiation in a RAD18-dependent manner. Importantly, polyubiquitin chains at sites of DNA damage remained for longer periods in USP5-depleted cells. Our results show that disassembly of polyubiquitin chains by USP5 at sites of damage is important for efficient DSB repair.

Amino acids and insulin act additively to regulate components of the ubiquitin-proteasome pathway in C2C12 myotubes

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Lomax Michael A

2007-03-01

Full Text Available Abstract Background The ubiquitin-proteasome system is the predominant pathway for myofibrillar proteolysis but a previous study in C2C12 myotubes only observed alterations in lysosome-dependent proteolysis in response to complete starvation of amino acids or leucine from the media. Here, we determined the interaction between insulin and amino acids in the regulation of myotube proteolysis Results Incubation of C2C12 myotubes with 0.2 × physiological amino acids concentration (0.2 × PC AA, relative to 1.0 × PC AA, significantly increased total proteolysis and the expression of 14-kDa E2 ubiquitin conjugating enzyme (p Conclusion In a C2C12 myotube model of myofibrillar protein turnover, amino acid limitation increases proteolysis in a ubiquitin-proteasome-dependent manner. Increasing amino acids or leucine alone, act additively with insulin to down regulate proteolysis and expression of components of ubiquitin-proteasome pathway. The effects of amino acids on proteolysis but not insulin and leucine, are blocked by inhibition of the mTOR signalling pathway.

The Regulation of Tumor Suppressor p63 by the Ubiquitin-Proteasome System

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Stephen R. Armstrong

2016-12-01

Full Text Available The protein p63 has been identified as a homolog of the tumor suppressor protein p53 and is capable of inducing apoptosis, cell cycle arrest, or senescence. p63 has at least six isoforms, which can be divided into two major groups: the TAp63 variants that contain the N-terminal transactivation domain and the ΔNp63 variants that lack the N-terminal transactivation domain. The TAp63 variants are generally considered to be tumor suppressors involved in activating apoptosis and suppressing metastasis. ΔNp63 variants cannot induce apoptosis but can act as dominant negative inhibitors to block the function of TAp53, TAp73, and TAp63. p63 is rarely mutated in human tumors and is predominately regulated at the post-translational level by phosphorylation and ubiquitination. This review focuses primarily on regulation of p63 by the ubiquitin E-3 ligase family of enzymes via ubiquitination and proteasome-mediated degradation, and introduces a new key regulator of the p63 protein.

Protosappanin B protects PC12 cells against oxygen-glucose deprivation-induced neuronal death by maintaining mitochondrial homeostasis via induction of ubiquitin-dependent p53 protein degradation.

Science.gov (United States)

Zeng, Ke-Wu; Liao, Li-Xi; Zhao, Ming-Bo; Song, Fang-Jiao; Yu, Qian; Jiang, Yong; Tu, Peng-Fei

2015-03-15

Protosappanin B (PTB) is a bioactive dibenzoxocin derivative isolated from Caesalpinia sappan L. Here, we investigated the neuroprotective effects and the potential mechanisms of PTB on oxygen-glucose deprivation (OGD)-injured PC12 cells. Results showed that PTB significantly increased cell viability, inhibited cell apoptosis and up-regulated the expression of growth-associated protein 43 (a marker of neural outgrowth). Moreover, our study revealed that PTB effectively maintained mitochondrial homeostasis by up-regulation of mitochondrial membrane potential (MMP), inhibition of cytochrome c release from mitochondria and inactivation of mitochondrial caspase-9/3 apoptosis pathway. Further study showed that PTB significantly promoted cytoplasmic component degradation of p53 protein, a key negative regulator for mitochondrial function, resulting in a release of Bcl-2 from p53-Bcl-2 complex and an enhancing translocation of Bcl-2 to mitochondrial outer membrane. Finally, we found the degradation of p53 protein was induced by PTB via activation of a MDM2-dependent ubiquitination process. Taken together, our findings provided a new viewpoint of neuronal protection strategy for anoxia and ischemic injury with natural small molecular dibenzoxocin derivative by activating ubiquitin-dependent p53 protein degradation as well as increasing mitochondrial function. Copyright © 2015 Elsevier B.V. All rights reserved.

Selection, trans-species polymorphism, and locus identification of major histocompatibility complex class IIβ alleles of New World ranid frogs

Science.gov (United States)

Kiemnec-Tyburczy, Karen M.; Richmond, Jonathan Q.; Savage, Anna E.; Zamudio, Kelly R.

2010-01-01

Genes encoded by the major histocompatibility complex (MHC) play key roles in the vertebrate immune system. However, our understanding of the evolutionary processes and underlying genetic mechanisms shaping these genes is limited in many taxa, including amphibians, a group currently impacted by emerging infectious diseases. To further elucidate the evolution of the MHC in frogs (anurans) and develop tools for population genetics, we surveyed allelic diversity of the MHC class II ??1 domain in both genomic and complementary DNA of seven New World species in the genus Rana (Lithobates). To assign locus affiliation to our alleles, we used a "gene walking" technique to obtain intron 2 sequences that flanked MHC class II?? exon 2. Two distinct intron sequences were recovered, suggesting the presence of at least two class II?? loci in Rana. We designed a primer pair that successfully amplified an orthologous locus from all seven Rana species. In total, we recovered 13 alleles and documented trans-species polymorphism for four of the alleles. We also found quantitative evidence of selection acting on amino acid residues that are putatively involved in peptide binding and structural stability of the ??1 domain of anurans. Our results indicated that primer mismatch can result in polymerase chain reaction (PCR) bias, which influences the number of alleles that are recovered. Using a single locus may minimize PCR bias caused by primer mismatch, and the gene walking technique was an effective approach for generating single-copy orthologous markers necessary for future studies of MHC allelic variation in natural amphibian populations. ?? 2010 Springer-Verlag.

Use of 8-methoxypsoralen and ultraviolet-A pretreated platelet concentrates to prevent alloimmunization against class I major histocompatibility antigens

International Nuclear Information System (INIS)

Grana, N.H.; Kao, K.J.

1991-01-01

The use of 8-methoxypsoralen (8-MOP) and UV-A irradiation to inactivate contaminating donor leukocytes in platelet concentrates and to prevent primary alloimmunization against donor class I major histocompatibility (MHC) antigens in mice was investigated. CBA/CaH-T6J mice with the H2k haplotype and BALB/cByJ mice with the H2d haplotype were used as donors and recipients, respectively. The mixed leukocyte reaction between these two strains of mice showed that treatment of spleen cells with 500 ng/mL 8-MOP and 5J/cm2 UV-A inhibited 99% of responder and 92% of stimulator function. There was no measurable loss of platelet aggregating activity after the treatment. After two weekly transfusions of platelets without any treatment, 93% of control mice (n = 15) developed anti-H2k antibody. In contrast, only 33% of mice (n = 15) receiving platelets treated with 8-MOP and UV-A became alloimmunized. After six weekly platelet transfusions, all mice became alloimmunized. Nevertheless, the mean titers of anti-H2k antibody in sera of the treated groups were significantly lower than the control groups. One hour posttransfusion recoveries of 51Cr-labeled donor platelets were also higher in mice transfused with the treated platelets. Thus, the pretreatment of platelet concentrates with 8-MOP and UV-A irradiation effectively reduced the alloantigenicity of class I MHC molecules. The implication of this finding in relation to the mechanism by which donor leukocytes allosensitize recipients is discussed

Detection of ubiquitinated huntingtin species in intracellular aggregates

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Katrin eJuenemann

2015-01-01

Full Text Available Protein conformation diseases, including polyglutamine diseases, result from the accumulation and aggregation of misfolded proteins. Huntington’s disease is one of nine diseases caused by an expanded polyglutamine repeat within the affected protein and is hallmarked by intracellular inclusion bodies composed of aggregated N-terminal huntingtin fragments and other sequestered proteins. Fluorescence microscopy and filter trap assay are conventional methods to study protein aggregates, but cannot be used to analyze the presence and levels of post-translational modifications of aggregated huntingtin such as ubiquitination. Ubiquitination of proteins can be a signal for degradation and intracellular localization, but also affects protein activity and protein-protein interactions. The function of ubiquitination relies on its mono- and polymeric isoforms attached to protein substrates. Studying the ubiquitination pattern of aggregated huntingtin fragments offers an important possibility to understand huntingtin degradation and aggregation processes within the cell. For the identification of aggregated huntingtin and its ubiquitinated species, solubilization of the cellular aggregates is mandatory. Here we describe methods to identify post-translational modifications such as ubiquitination of aggregated mutant huntingtin. This approach is specifically described for use with mammalian cell culture and is suitable to study other disease-related proteins prone to aggregate.

Involvement of the major histocompatibility complex region in the genetic regulation of circulating CD8 T-cell numbers in humans.

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Cruz, E; Vieira, J; Gonçalves, R; Alves, H; Almeida, S; Rodrigues, P; Lacerda, R; Porto, G

2004-07-01

Variability in T-lymphocyte numbers is partially explained by a genetic regulation. From studies in animal models, it is known that the Major Histocompatibility Complex (MHC) is involved in this regulation. In humans, this has not been shown yet. The objective of the present study was to test the hypothesis that genes in the MHC region influence the regulation of T-lymphocyte numbers. Two approaches were used. Association studies between T-cell counts (CD4(+) and CD8(+)) or total lymphocyte counts and HLA class I alleles (A and B) or mutations in the HFE (C282Y and H63D), the hemochromatosis gene, in an unrelated population (n = 264). A second approach was a sibpair correlation analysis of the same T-cell counts in relation to HLA-HFE haplotypes in subjects belonging to 48 hemochromatosis families (n = 456 sibpairs). In the normal population, results showed a strong statistically significant association of the HLA-A*01 with high numbers of CD8(+) T cells and a less powerful association with the HLA-A*24 with low numbers of CD8(+) T cells. Sibpair correlations revealed the most significant correlation for CD8(+) T-cell numbers for sibpairs with HLA-HFE-identical haplotypes. This was not observed for CD4(+) T cells. These results show that the MHC region is involved in the genetic regulation of CD8(+) T-cell numbers in humans. Identification of genes responsible for this control may have important biological and clinical implications.

SNP association mapping across the extended major histocompatibility complex and risk of B-cell precursor acute lymphoblastic leukemia in children.

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Kevin Y Urayama

Full Text Available The extended major histocompatibility complex (xMHC is the most gene-dense region of the genome and harbors a disproportionately large number of genes involved in immune function. The postulated role of infection in the causation of childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL suggests that the xMHC may make an important contribution to the risk of this disease. We conducted association mapping across an approximately 4 megabase region of the xMHC using a validated panel of single nucleotide polymorphisms (SNPs in childhood BCP-ALL cases (n=567 enrolled in the Northern California Childhood Leukemia Study (NCCLS compared with population controls (n=892. Logistic regression analyses of 1,145 SNPs, adjusted for age, sex, and Hispanic ethnicity indicated potential associations between several SNPs and childhood BCP-ALL. After accounting for multiple comparisons, one of these included a statistically significant increased risk associated with rs9296068 (OR=1.40, 95% CI=1.19-1.66, corrected p=0.036, located in proximity to HLA-DOA. Sliding window haplotype analysis identified an additional locus located in the extended class I region in proximity to TRIM27 tagged by a haplotype comprising rs1237485, rs3118361, and rs2032502 (corrected global p=0.046. Our findings suggest that susceptibility to childhood BCP-ALL is influenced by genetic variation within the xMHC and indicate at least two important regions for future evaluation.

Anti-GBM disease after nephrectomy for xanthogranulomatous pyelonephritis in a patient expressing HLA DR15 major histocompatibility antigens: a case report.

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O'Hagan, Emma; Mallett, Tamara; Convery, Mairead; McKeever, Karl

2015-01-01

Antiglomerular basement membrane (anti-GBM) antibody disease is uncommon in the pediatric population. There are no cases in the literature describing the development of anti-GBM disease following XGP or nephrectomy. We report the case of a 7-year-old boy with no past history of urological illness, treated with antimicrobials and nephrectomy for diffuse, unilateral xanthogranulomatous pyelonephritis (XGP). Renal function and ultrasound scan of the contralateral kidney postoperatively were normal. Three months later, the child represented in acute renal failure with rapidly progressive glomerulonephritis requiring hemodialysis. Renal biopsy showed severe crescentic glomerulonephritis with 95% of glomeruli demonstrating circumferential cellular crescents. Strong linear IgG staining of the glomerular basement membranes was present, in keeping with anti-GBM disease. Circulating anti-GBM antibodies were positive. Treatment with plasma exchange, methylprednisolone, and cyclophosphamide led to normalization of anti-GBM antibody titers. Frequency of hemodialysis was reduced as renal function improved, and he is currently independent of dialysis with estimated glomerular filtration rate 20.7 mls/min/1.73 m 2 . Case studies in the adult literature have reported the development of a rapidly progressive anti-GBM antibody-induced glomerulonephritis following renal surgery where patients expressed HLA DR2/HLA DR15 major histocompatibility (MHC) antigens. Of note, our patient also expresses the HLA DR15 MHC antigen.

Integrated Genomic Analysis of the Ubiquitin Pathway across Cancer Types

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Zhongqi Ge

2018-04-01

Full Text Available Summary: Protein ubiquitination is a dynamic and reversible process of adding single ubiquitin molecules or various ubiquitin chains to target proteins. Here, using multidimensional omic data of 9,125 tumor samples across 33 cancer types from The Cancer Genome Atlas, we perform comprehensive molecular characterization of 929 ubiquitin-related genes and 95 deubiquitinase genes. Among them, we systematically identify top somatic driver candidates, including mutated FBXW7 with cancer-type-specific patterns and amplified MDM2 showing a mutually exclusive pattern with BRAF mutations. Ubiquitin pathway genes tend to be upregulated in cancer mediated by diverse mechanisms. By integrating pan-cancer multiomic data, we identify a group of tumor samples that exhibit worse prognosis. These samples are consistently associated with the upregulation of cell-cycle and DNA repair pathways, characterized by mutated TP53, MYC/TERT amplification, and APC/PTEN deletion. Our analysis highlights the importance of the ubiquitin pathway in cancer development and lays a foundation for developing relevant therapeutic strategies. : Ge et al. analyze a cohort of 9,125 TCGA samples across 33 cancer types to provide a comprehensive characterization of the ubiquitin pathway. They detect somatic driver candidates in the ubiquitin pathway and identify a cluster of patients with poor survival, highlighting the importance of this pathway in cancer development. Keywords: ubiquitin pathway, pan-cancer analysis, The Cancer Genome Atlas, tumor subtype, cancer prognosis, therapeutic targets, biomarker, FBXW7

Development of a rapid in vitro protein refolding assay which discriminates between peptide-bound and peptide-free forms of recombinant porcine major histocompatibility class I complex (SLA-I)

DEFF Research Database (Denmark)

Oleksiewicz, M.B.; Kristensen, B.; Ladekjaer-Mikkelsen, A.S.

2002-01-01

The extracellular domains of swine leukocyte antigen class I (SLA-I, major histocompatibility complex protein class 1) were cloned and sequenced for two haplotypes (114 and H7) which do not share any alleles based on serological typing, and which are the most important in Danish farmed pigs....... The extracellular domain of SLA-I was connected to porcine beta2 microglobulin by glycine-rich linkers. The engineered sin.-le-chain proteins, consisting of fused SLA-I and beta2 microglobulin, were overexpressed as inclusion bodies in Escherichia coli. Also, variants were made of the single-chain proteins......, by linking them through glycine-rich linkers to peptides representing T-cell epitopes from classical swine fever virus (CSFV) and foot-and-mouth disease virus (FMDV). An in vitro refold assay was developed, using a monoclonal anti-SLA antibody (PT85A) to gauge refolding. The single best-defined, SLA...

Toponomics analysis of functional interactions of the ubiquitin ligase PAM (Protein Associated with Myc) during spinal nociceptive processing.

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Pierre, Sandra; Maeurer, Christian; Coste, Ovidiu; Becker, Wiebke; Schmidtko, Achim; Holland, Sabrina; Wittpoth, Claus; Geisslinger, Gerd; Scholich, Klaus

2008-12-01

Protein associated with Myc (PAM) is a giant E3 ubiquitin ligase of 510 kDa. Although the role of PAM during neuronal development is well established, very little is known about its function in the regulation of synaptic strength. Here we used multiepitope ligand cartography (MELC) to study protein network profiles associated with PAM during the modulation of synaptic strength. MELC is a novel imaging technology that utilizes biomathematical tools to describe protein networks after consecutive immunohistochemical visualization of up to 100 proteins on the same sample. As an in vivo model to modulate synaptic strength we used the formalin test, a common model for acute and inflammatory pain. MELC analysis was performed with 37 different antibodies or fluorescence tags on spinal cord slices and led to the identification of 1390 PAM-related motifs that distinguish untreated and formalin-treated spinal cords. The majority of these motifs related to ubiquitin-dependent processes and/or the actin cytoskeleton. We detected an intermittent colocalization of PAM and ubiquitin with TSC2, a known substrate of PAM, and the glutamate receptors mGluR5 and GLUR1. Importantly these complexes were detected exclusively in the presence of F-actin. A direct PAM/F-actin interaction was confirmed by colocalization and cosedimentation. The binding of PAM toward F-actin varied strongly between the PAM splice forms found in rat spinal cords. PAM did not ubiquitylate actin or alter actin polymerization and depolymerization. However, F-actin decreased the ubiquitin ligase activity of purified PAM. Because PAM activation is known to involve its translocation, the binding of PAM to F-actin may serve to control its subcellular localization as well as its activity. Taken together we show that defining protein network profiles by topological proteomics analysis is a useful tool to identify previously unknown protein/protein interactions that underlie synaptic processes.

Sperm competition, but not major histocompatibility divergence, drives differential fertilization success between alternative reproductive tactics in Chinook salmon.

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Lehnert, S J; Helou, L; Pitcher, T E; Heath, J W; Heath, D D

2018-01-01

Post-copulatory sexual selection processes, including sperm competition and cryptic female choice (CFC), can operate based on major histocompatibility (MH) genes. We investigated sperm competition between male alternative reproductive tactics [jack (sneaker) and hooknose (guard)] of Chinook salmon (Oncorhynchus tshawytscha). Using a full factorial design, we examined in vitro competitive fertilization success of paired jack and hooknose males at three time points after sperm activation (0, 15 and 60 s) to test for male competition, CFC and time effects on male fertilization success. We also examined egg-mediated CFC at two MH genes by examining both the relationship between competitive fertilization success and MH divergence as well as inheritance patterns of MH alleles in resulting offspring. We found that jacks sired more offspring than hooknose males at 0 s post-activation; however, jack fertilization success declined over time post-activation, suggesting a trade-off between sperm speed and longevity. Enhanced fertilization success of jacks (presumably via higher sperm quality) may serve to increase sneaker tactic competitiveness relative to dominant hooknose males. We also found evidence of egg-mediated CFC (i.e. female × male interaction) influencing competitive fertilization success; however, CFC was not acting on the MH genes as we found no relationship between fertilization success and MH II β 1 or MH I α 1 divergence and we found no deviations from Mendelian inheritance of MH alleles in the offspring. Our study provides insight into evolutionary mechanisms influencing variation in male mating success within alternative reproductive tactics, thus underscoring different strategies that males can adopt to attain success. © 2017 European Society For Evolutionary Biology. Journal of Evolutionary Biology © 2017 European Society For Evolutionary Biology.

Role of major histocompatibility complex class II in resistance of mice to naturally acquired infection with Syphacia obvelata

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Stewart, Patricia W.; Chapes, Stephen K.

2003-01-01

Genetics plays a substantial role in host resistance in many host-parasite interactions. We examined the prevalence of naturally acquired infection with Syphacia obvelata in a number of mouse strains housed in a non-barrier facility. These mice, which included cross-bred and congenic, inbred strains on various genetic backgrounds, differ in the loci for the immune function genes--major histocompatibility complex class II (MHCII), toll-like receptor 4 (Tlr4), and solute carrier family 11, member 1 (Slc11a1)--which allowed comparisons of the impact of these genes on resistance to pinworm infection. Male and female mice of various ages were sampled over an 18-month period; infection was determined by use of the cellophane tape test. Results indicated that mice that were MHCII+/+ had a significantly lower prevalence of infection than did mice that were MHCII-/-. Differences were not seen between male and female mice. Although MHCII+/+ mice had an age-associated decrease in infection prevalence, such decrease was not seen in MHCII-/- mice. In contrast, infection prevalence in mice with the normal Tlr4 gene (Tlr4(LPS-n/LPS-n)) gene did not differ significantly compared with that in mice that were homozygous for either the point mutation (Tlr4(LPS-d/LPS-d)) or deletion (Tlr4(LPS-del/LPS-del)) of that gene. Likewise, the presence (Sle11a1r/r) or absence (Slc11a1s/s) of functional alleles for Slc11a1 had no effect on the prevalence of infection with S. obvelata. In conclusion, presence of MHCII, but not Tlr4 or Slc11a1 significantly influences prevalence of naturally acquired infection with S. obvelata. These data justify further comprehensive analyses of the immune components that are involved in pinworm resistance.

Immunomodulation of glioma cells after gene therapy: induction of major histocompatibility complex class I but not class II antigen in vitro.

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Parsa, A T; Chi, J H; Hurley, P T; Jeyapalan, S A; Bruce, J N

2001-09-01

Acquired immunity has been demonstrated in Fischer rats bearing syngeneic 9L tumors after herpes simplex virus (HSV) thymidine kinase (TK) gene transfection and ganciclovir treatment. The nature of this immunity in rats and its relevance to the HSV TK/ganciclovir protocol for human subjects remain to be determined. In this study, levels of major histocompatibility complex (MHC) Class I and II antigen expression were measured before and after HSV TK transfection, in an effort to document immunomodulatory changes caused by gene therapy. Tumor cells from the 9L gliosarcoma cell line, three primary human glioma cultures, and the human glioma cell line U87 MG were transduced with HSV TK vector-containing supernatant from fibroblast-producing cells (titer of 5 x 10(6) colony-forming units/ml) and selected in G418 medium for neomycin resistance. Clones were pooled or individually selected for cell-killing assays with ganciclovir, to confirm TK expression (10(3) cells/well in a 96-well dish). Northern analyses using MHC Class I and Class II complementary deoxyribonucleic acid probes were performed on blots containing total ribonucleic acid from wild-type tumor cells and HSV TK transfectants. A beta-actin complementary deoxyribonucleic acid probe served as an internal control. Cell surface expression was confirmed with flow cytometry. The induction of MHC Class I was tested for cycloheximide and genistein sensitivity. All cell cultures exhibited increases in MHC Class I but not MHC Class II expression, as determined by Northern analysis densitometry and flow cytometry. Cycloheximide treatment did not diminish the up-regulation of MHC Class I after retroviral transfection, implicating a signal transduction pathway that does not require ongoing protein synthesis. Genistein pretreatment of cell cultures did diminish the up-regulation of MHC Class I, implicating a tyrosine kinase in the signaling cascade. Induction of MHC Class I in rat and human glioma cells after HSV TK

Characterization of major histocompatibility complex (MHC DRB exon 2 and DRA exon 3 fragments in a primary terrestrial rabies vector (Procyon lotor.

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Sarrah Castillo

Full Text Available The major histocompatibility complex (MHC presents a unique system to explore links between genetic diversity and pathogens, as diversity within MHC is maintained in part by pathogen driven selection. While the majority of wildlife MHC studies have investigated species that are of conservation concern, here we characterize MHC variation in a common and broadly distributed species, the North American raccoon (Procyon lotor. Raccoons host an array of broadly distributed wildlife diseases (e.g., canine distemper, parvovirus and raccoon rabies virus and present important human health risks as they persist in high densities and in close proximity to humans and livestock. To further explore how genetic variation influences the spread and maintenance of disease in raccoons we characterized a fragment of MHC class II DRA exon 3 (250 bp and DRB exon 2 (228 bp. MHC DRA was found to be functionally monomorphic in the 32 individuals screened; whereas DRB exon 2 revealed 66 unique alleles among the 246 individuals screened. Between two and four alleles were observed in each individual suggesting we were amplifying a duplicated DRB locus. Nucleotide differences between DRB alleles ranged from 1 to 36 bp (0.4-15.8% divergence and translated into 1 to 21 (1.3-27.6% divergence amino acid differences. We detected a significant excess of nonsynonymous substitutions at the peptide binding region (P = 0.005, indicating that DRB exon 2 in raccoons has been influenced by positive selection. These data will form the basis of continued analyses into the spatial and temporal relationship of the raccoon rabies virus and the immunogenetic response in its primary host.

Ubiquitin-Like Protein from Human Placental Extract Exhibits Collagenase Activity

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De, Debashree; Datta Chakraborty, Piyali; Mitra, Jyotirmoy; Sharma, Kanika; Mandal, Somnath; Das, Aneesha; Chakrabarti, Saikat; Bhattacharyya, Debasish

2013-01-01

An aqueous extract of human placenta exhibits strong gelatinase/collagenase activity in zymography. 2-D gel electrophoresis of the extract with gelatin zymography in the second dimension displayed a single spot, identified as ubiquitin-like component upon MALDI/TOF MS/MS analysis. Immunoblot indicated presence of ubiquitin and absence of collagenase in the extract. Collagenase activity of the ubiquitin-like component was confirmed from the change in solubility of collagen in aqueous buffer, degradation of collagen by size-exclusion HPLC and atomic force microscopy. Quantification with DQ-gelatin showed that the extract contains 0.04 U/ml of collagenase activity that was inhibited up to 95% by ubiquitin antibody. Ubiquitin from bovine erythrocytes demonstrated mild collagenase activity. Bioinformatics studies suggest that placental ubiquitin and collagenase follow structurally divergent evolution. This thermostable intrinsic collagenase activity of placental extract might have wide physiological relevance in degrading and remodeling collagen as it is used as a drug for wound healing and pelvic inflammatory diseases. PMID:23555718

Degradation signals for ubiquitin system proteolysis in Saccharomyces cerevisiae.

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Gilon, T; Chomsky, O; Kulka, R G

1998-01-01

Combinations of different ubiquitin-conjugating (Ubc) enzymes and other factors constitute subsidiary pathways of the ubiquitin system, each of which ubiquitinates a specific subset of proteins. There is evidence that certain sequence elements or structural motifs of target proteins are degradation signals which mark them for ubiquitination by a particular branch of the ubiquitin system and for subsequent degradation. Our aim was to devise a way of searching systematically for degradation signals and to determine to which ubiquitin system subpathways they direct the proteins. We have constructed two reporter gene libraries based on the lacZ or URA3 genes which, in Saccharomyces cerevisiae, express fusion proteins with a wide variety of C-terminal extensions. From these, we have isolated clones producing unstable fusion proteins which are stabilized in various ubc mutants. Among these are 10 clones whose products are stabilized in ubc6, ubc7 or ubc6ubc7 double mutants. The C-terminal extensions of these clones, which vary in length from 16 to 50 amino acid residues, are presumed to contain degradation signals channeling proteins for degradation via the UBC6 and/or UBC7 subpathways of the ubiquitin system. Some of these C-terminal tails share similar sequence motifs, and a feature common to almost all of these sequences is a highly hydrophobic region such as is usually located inside globular proteins or inserted into membranes. PMID:9582269

Level of ubiquitinated histone H2B in chromatin is coupled to ongoing transcription

International Nuclear Information System (INIS)

Davie, J.R.; Murphy, L.C.

1990-01-01

The relationship between transcription and ubiquitination of the histones was investigated. Previous studies have shown that ubiquitinated (u) histone H2B and, to a lesser extend, mono- and polyubiquitinated histone H2A are enriched in transcriptionally active gene-enriched chromatin fractions. Here, the authors show that treatment of T-47D-5 human breast cancer cells with actinomycin D or 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole, inhibitors of heterogeneous nuclear RNA synthesis, selectively reduced the level of uH2B, but not uH2A, uH2A.Z, or polyubiquitinated H2A, in chromatin. Treatment of the cells with low levels of actinomycin D slightly reduced the level of uH2B, suggesting that inhibition of ribosomal RNA synthesis does not have a profound effect on the level of uH2B in chromatin. These results demonstrate that maintenance of the levels of uH2B in chromatin is dependent upon ongoing transcription, particularly the synthesis of hnRNA. Thus, histone H2B would be ubiquitinated when the nucleosome was opened during transcription. Ubiquitination of histone H2B may impede nucleosome refolding, facilitating subsequent rounds of transcription

End-joining inhibition at telomeres requires the translocase and polySUMO-dependent ubiquitin ligase Uls1.

Science.gov (United States)

Lescasse, Rachel; Pobiega, Sabrina; Callebaut, Isabelle; Marcand, Stéphane

2013-03-20

In eukaryotes, permanent inhibition of the non-homologous end joining (NHEJ) repair pathway at telomeres ensures that chromosome ends do not fuse. In budding yeast, binding of Rap1 to telomere repeats establishes NHEJ inhibition. Here, we show that the Uls1 protein is required for the maintenance of NHEJ inhibition at telomeres. Uls1 protein is a non-essential Swi2/Snf2-related translocase and a Small Ubiquitin-related Modifier (SUMO)-Targeted Ubiquitin Ligase (STUbL) with unknown targets. Loss of Uls1 results in telomere-telomere fusions. Uls1 requirement is alleviated by the absence of poly-SUMO chains and by rap1 alleles lacking SUMOylation sites. Furthermore, Uls1 limits the accumulation of Rap1 poly-SUMO conjugates. We propose that one of Uls1 functions is to clear non-functional poly-SUMOylated Rap1 molecules from telomeres to ensure the continuous efficiency of NHEJ inhibition. Since Uls1 is the only known STUbL with a translocase activity, it can be the general molecular sweeper for the clearance of poly-SUMOylated proteins on DNA in eukaryotes.

Applicability of major histocompatibility complex DRB1 alleles as markers to detect vertebrate hybridization: a case study from Iberian ibex × domestic goat in southern Spain

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Alasaad Samer

2012-09-01

Full Text Available Abstract Background Hybridization between closely related wild and domestic species is of great concern because it can alter the evolutionary integrity of the affected populations. The high allelic variability of Major Histocompatibility Complex (MHC loci usually excludes them from being used in studies to detect hybridization events. However, if a the parental species don’t share alleles, and b one of the parental species possesses an exceptionally low number of alleles (to facilitate analysis, then even MHC loci have the potential to detect hybrids. Results By genotyping the exon2 of the MHC class II DRB1 locus, we were able to detect hybridization between domestic goats (Capra hircus and free-ranging Iberian ibex (Capra pyrenaica hispanica by molecular means. Conclusions This is the first documentation of a Capra pyrenaica × Capra hircus hybridization, which presented us the opportunity to test the applicability of MHC loci as new, simple, cost-effective, and time-saving approach to detect hybridization between wild species and their domesticated relatives, thus adding value to MHC genes role in animal conservation and management.

Expression Profiling of WSSV ORF 199 and Shrimp Ubiquitin Conjugating Enzyme in WSSV Infected

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K. Jeena

2012-08-01

Full Text Available White spot syndrome virus (WSSV is one of the major viral pathogens affecting shrimp aquaculture. Four proteins, WSSV199, WSSV 222, WSSV 249 and WSSV 403, from WSSV are predicted to encode a RING-H2 domain, which in presence of ubiquitin conjugating enzyme (E2 in shrimp can function as viral E3 ligase and modulate the host ubiquitin proteasome pathway. Modulation of host ubiquitin proteasome pathway by viral proteins is implicated in viral pathogenesis. In the present study, a time course expression profile analysis of WSSV Open Reading Frame (ORF 199 and Penaeus monodon ubiquitin conjugating enzyme (PmUbc was carried out at 0, 3, 6, 12, 24, 48 and 72 h post WSSV challenge by semi-quantitative RT-PCR as well as Real Time PCR. EF1α was used as reference control to normalize the expression levels. A significant increase in PmUbc expression at 24 h post infection (h.p.i was observed followed by a decline till 72 h.p.i. Expression of WSSV199 was observed at 24 h.p.i in WSSV infected P. monodon. Since the up-regulation of PmUbc was observed at 24 h.p.i where WSSV199 expression was detected, it can be speculated that these proteins might interact with host ubiquitination pathway for viral pathogenesis. However, further studies need to be carried out to unfold the molecular mechanism of interaction between host and virus to devise efficient control strategies for this chaos in the shrimp culture industry.

Ubiquitination of HTLV-I Tax in response to DNA damage regulates nuclear complex formation and nuclear export

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Marriott Susan J

2007-12-01

Full Text Available Abstract Background The HTLV-I oncoprotein, Tax, is a pleiotropic protein whose activity is partially regulated by its ability to interact with, and perturb the functions of, numerous cellular proteins. Tax is predominantly a nuclear protein that localizes to nuclear foci known as Tax Speckled Structures (TSS. We recently reported that the localization of Tax and its interactions with cellular proteins are altered in response to various forms of genotoxic and cellular stress. The level of cytoplasmic Tax increases in response to stress and this relocalization depends upon the interaction of Tax with CRM1. Cellular pathways and signals that regulate the subcellular localization of Tax remain to be determined. However, post-translational modifications including sumoylation and ubiquitination are known to influence the subcellular localization of Tax and its interactions with cellular proteins. The sumoylated form of Tax exists predominantly in the nucleus while ubiquitinated Tax exists predominantly in the cytoplasm. Therefore, we hypothesized that post-translational modifications of Tax that occur in response to DNA damage regulate the localization of Tax and its interactions with cellular proteins. Results We found a significant increase in mono-ubiquitination of Tax in response to UV irradiation. Mutation of specific lysine residues (K280 and K284 within Tax inhibited DNA damage-induced ubiquitination. In contrast to wild-type Tax, which undergoes transient nucleocytoplasmic shuttling in response to DNA damage, the K280 and K284 mutants were retained in nuclear foci following UV irradiation and remained co-localized with the cellular TSS protein, sc35. Conclusion This study demonstrates that the localization of Tax, and its interactions with cellular proteins, are dynamic following DNA damage and depend on the post-translational modification status of Tax. Specifically, DNA damage induces the ubiquitination of Tax at K280 and K284

Dengue Virus Genome Uncoating Requires Ubiquitination.

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Byk, Laura A; Iglesias, Néstor G; De Maio, Federico A; Gebhard, Leopoldo G; Rossi, Mario; Gamarnik, Andrea V

2016-06-28

The process of genome release or uncoating after viral entry is one of the least-studied steps in the flavivirus life cycle. Flaviviruses are mainly arthropod-borne viruses, including emerging and reemerging pathogens such as dengue, Zika, and West Nile viruses. Currently, dengue virus is one of the most significant human viral pathogens transmitted by mosquitoes and is responsible for about 390 million infections every year around the world. Here, we examined for the first time molecular aspects of dengue virus genome uncoating. We followed the fate of the capsid protein and RNA genome early during infection and found that capsid is degraded after viral internalization by the host ubiquitin-proteasome system. However, proteasome activity and capsid degradation were not necessary to free the genome for initial viral translation. Unexpectedly, genome uncoating was blocked by inhibiting ubiquitination. Using different assays to bypass entry and evaluate the first rounds of viral translation, a narrow window of time during infection that requires ubiquitination but not proteasome activity was identified. In this regard, ubiquitin E1-activating enzyme inhibition was sufficient to stabilize the incoming viral genome in the cytoplasm of infected cells, causing its retention in either endosomes or nucleocapsids. Our data support a model in which dengue virus genome uncoating requires a nondegradative ubiquitination step, providing new insights into this crucial but understudied viral process. Dengue is the most significant arthropod-borne viral infection in humans. Although the number of cases increases every year, there are no approved therapeutics available for the treatment of dengue infection, and many basic aspects of the viral biology remain elusive. After entry, the viral membrane must fuse with the endosomal membrane to deliver the viral genome into the cytoplasm for translation and replication. A great deal of information has been obtained in the last decade

Ubiquitination of basal VEGFR2 regulates signal transduction and endothelial function

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Gina A. Smith

2017-10-01

Full Text Available Cell surface receptors can undergo recycling or proteolysis but the cellular decision-making events that sort between these pathways remain poorly defined. Vascular endothelial growth factor A (VEGF-A and vascular endothelial growth factor receptor 2 (VEGFR2 regulate signal transduction and angiogenesis, but how signaling and proteolysis is regulated is not well understood. Here, we provide evidence that a pathway requiring the E1 ubiquitin-activating enzyme UBA1 controls basal VEGFR2 levels, hence metering plasma membrane receptor availability for the VEGF-A-regulated endothelial cell response. VEGFR2 undergoes VEGF-A-independent constitutive degradation via a UBA1-dependent ubiquitin-linked pathway. Depletion of UBA1 increased VEGFR2 recycling from endosome-to-plasma membrane and decreased proteolysis. Increased membrane receptor availability after UBA1 depletion elevated VEGF-A-stimulated activation of key signaling enzymes such as PLCγ1 and ERK1/2. Although UBA1 depletion caused an overall decrease in endothelial cell proliferation, surviving cells showed greater VEGF-A-stimulated responses such as cell migration and tubulogenesis. Our study now suggests that a ubiquitin-linked pathway regulates the balance between receptor recycling and degradation which in turn impacts on the intensity and duration of VEGF-A-stimulated signal transduction and the endothelial response.

Short communication Identification of genetic variation in the major ...

African Journals Online (AJOL)

EXPER

2016-10-31

Oct 31, 2016 ... DRB1 with Hin1I was more frequent in the Kivircik and White .... evolution of class I duplication blocks, diversity and complexity from shark to man. ... The great diversity of major histocompatibility complex class II genes in ...

Rsp5 ubiquitin ligase is required for protein trafficking in Saccharomyces cerevisiae COPI mutants.

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Katarzyna Jarmoszewicz

Full Text Available Retrograde trafficking from the Golgi to the endoplasmic reticulum (ER depends on the formation of vesicles coated with the multiprotein complex COPI. In Saccharomyces cerevisiae ubiquitinated derivatives of several COPI subunits have been identified. The importance of this modification of COPI proteins is unknown. With the exception of the Sec27 protein (β'COP neither the ubiquitin ligase responsible for ubiquitination of COPI subunits nor the importance of this modification are known. Here we find that the ubiquitin ligase mutation, rsp5-1, has a negative effect that is additive with ret1-1 and sec28Δ mutations, in genes encoding α- and ε-COP, respectively. The double ret1-1 rsp5-1 mutant is also more severely defective in the Golgi-to-ER trafficking compared to the single ret1-1, secreting more of the ER chaperone Kar2p, localizing Rer1p mostly to the vacuole, and increasing sensitivity to neomycin. Overexpression of ubiquitin in ret1-1 rsp5-1 mutant suppresses vacuolar accumulation of Rer1p. We found that the effect of rsp5 mutation on the Golgi-to-ER trafficking is similar to that of sla1Δ mutation in a gene encoding actin cytoskeleton proteins, an Rsp5p substrate. Additionally, Rsp5 and Sla1 proteins were found by co-immunoprecipitation in a complex containing COPI subunits. Together, our results show that Rsp5 ligase plays a role in regulating retrograde Golgi-to-ER trafficking.

The Fbw7 tumor suppressor targets KLF5 for ubiquitin-mediated degradation and suppresses breast cell proliferation.

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Zhao, Dong; Zheng, Han-Qiu; Zhou, Zhongmei; Chen, Ceshi

2010-06-01

Fbw7 is a tumor suppressor frequently inactivated in cancers. The KLF5 transcription factor promotes breast cell proliferation and tumorigenesis through upregulating FGF-BP. The KLF5 protein degrades rapidly through the ubiquitin proteasome pathway. Here, we show that the Skp1-CUL1-Fbw7 E3 ubiquitin ligase complex (SCF(Fbw7)) targets KLF5 for ubiquitin-mediated degradation in a GSK3beta-mediated KLF5 phosphorylation-dependent manner. Mutation of the critical S303 residue in the KLF5 Cdc4 phospho-degrons motif ((303)SPPSS) abolishes the protein interaction, ubiquitination, and degradation by Fbw7. Inactivation of endogenous Fbw7 remarkably increases the endogenous KLF5 protein abundances. Endogenous Fbw7 suppresses the FGF-BP gene expression and breast cell proliferation through targeting KLF5 for degradation. These findings suggest that Fbw7 inhibits breast cell proliferation at least partially through targeting KLF5 for proteolysis. This new regulatory mechanism of KLF5 degradation may result in useful diagnostic and therapeutic targets for breast cancer and other cancers. Copyright 2010 AACR.

The Tomato Fni3 Lysine-63–Specific Ubiquitin-Conjugating Enzyme and Suv Ubiquitin E2 Variant Positively Regulate Plant Immunity[C][W

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Mural, Ravi V.; Liu, Yao; Rosebrock, Tracy R.; Brady, Jennifer J.; Hamera, Sadia; Connor, Richard A.; Martin, Gregory B.; Zeng, Lirong

2013-01-01

The activation of an immune response in tomato (Solanum lycopersicum) against Pseudomonas syringae relies on the recognition of E3 ligase–deficient forms of AvrPtoB by the host protein kinase, Fen. To investigate the mechanisms by which Fen-mediated immunity is regulated, we characterize in this study a Fen-interacting protein, Fni3, and its cofactor, S. lycoperiscum Uev (Suv). Fni3 encodes a homolog of the Ubc13-type ubiquitin-conjugating enzyme that catalyzes exclusively Lys-63–linked ubiquitination, whereas Suv is a ubiquitin-conjugating enzyme variant. The C-terminal region of Fen was necessary for interaction with Fni3, and this interaction was required for cell death triggered by overexpression of Fen in Nicotiana benthamiana leaves. Fni3 was shown to be an active E2 enzyme, but Suv displayed no ubiquitin-conjugating activity; Fni3 and Suv together directed Lys-63–linked ubiquitination. Decreased expression of Fni3, another tomato Ubc13 homolog, Sl-Ubc13-2, or Suv in N. benthamiana leaves diminished cell death associated with Fen-mediated immunity and cell death elicited by several other resistance (R) proteins and their cognate effectors. We also discovered that coexpression of Fen and other R proteins/effectors with a Fni3 mutant that is compromised for ubiquitin-conjugating activity diminished the cell death. These results suggest that Fni3/Sl-Ubc13-2 and Suv regulate the immune response mediated by Fen and other R proteins through Lys-63–linked ubiquitination. PMID:24076975

Skp2 regulates androgen receptor through ubiquitin-mediated degradation independent of Akt/mTOR pathways in prostate cancer.

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Li, Bo; Lu, Wenfu; Yang, Qing; Yu, Xiuping; Matusik, Robert J; Chen, Zhenbang

2014-04-01

The intervention of advanced prostate cancer (PCa) in patients has been commonly depending on androgen deprivation therapy. Despite of tremendous research efforts, however, molecular mechanisms on AR regulation remain poorly understood, particularly for castration resistant prostate cancer (CRPC). Targeting AR and associated factors is considered an effective strategy in PCa treatment. Human prostate cancer cells were used in this study. Manipulations of Skp2 expression were achieved by Skp2 shRNA/siRNA or overexpression of plasmids. Dual luciferase reporter assay was applied for AR activity assessment. Western blot, ubiquitination assay, immunoprecipitation, and immunofluorescence were applied to detect the proteins. Our results demonstrated that Skp2 directly involves the regulation of AR expression through ubiquitination-mediated degradation. Skp2 interacted with AR protein in PCa cells, and enforced expression of Skp2 resulted in a decreased level and activity of AR. By contrast, Skp2 knockdown increased the protein accumulation and activity of AR. Importantly, changes of AR contributed by Skp2 led to subsequent alterations of PSA level in PCa cells. AR ubiquitination was significantly increased upon Skp2 overexpression but greatly reduced upon Skp2 knockdown. AR mutant at K847R abrogated Skp2-mediated ubiquitination of AR. NVP-BEZ235, a dual PI3K/mTOR inhibitor, remarkably inhibited Skp2 level with a striking elevation of AR. The results indicate that Skp2 is an E3 ligase for proteasome-dependent AR degradation, and K847 on AR is the recognition site for Skp2-mediated ubiquitination. Our findings reveal an essential role of Skp2 in AR signaling. © 2013 Wiley Periodicals, Inc.

Nickel compounds induce histone ubiquitination by inhibiting histone deubiquitinating enzyme activity

International Nuclear Information System (INIS)

Ke Qingdong; Ellen, Thomas P.; Costa, Max

2008-01-01

Nickel (Ni) compounds are known carcinogens but underlying mechanisms are not clear. Epigenetic changes are likely to play an important role in nickel ion carcinogenesis. Previous studies have shown epigenetic effects of nickel ions, including the loss of histone acetylation and a pronounced increase in dimethylated H3K9 in nickel-exposed cells. In this study, we demonstrated that both water-soluble and insoluble nickel compounds induce histone ubiquitination (uH2A and uH2B) in a variety of cell lines. Investigations of the mechanism by which nickel increases histone ubiquitination in cells reveal that nickel does not affect cellular levels of the substrates of this modification, i.e., ubiquitin, histones, and other non-histone ubiquitinated proteins. In vitro ubiquitination and deubiquitination assays have been developed to further investigate possible effects of nickel on enzymes responsible for histone ubiquitination. Results from the in vitro assays demonstrate that the presence of nickel did not affect the levels of ubiquitinated histones in the ubiquitinating assay. Instead, the addition of nickel significantly prevents loss of uH2A and uH2B in the deubiquitinating assay, suggesting that nickel-induced histone ubiquitination is the result of inhibition of (a) putative deubiquitinating enzyme(s). Additional supporting evidence comes from the comparison of the response to nickel ions with a known deubiquitinating enzyme inhibitor, iodoacetamide (IAA). This study is the first to demonstrate such effects of nickel ions on histone ubiquitination. It also sheds light on the possible mechanisms involved in altering the steady state of this modification. The study provides further evidence that supports the notion that nickel ions alter epigenetic homeostasis in cells, which may lead to altered programs of gene expression and carcinogenesis

Sculpting ion channel functional expression with engineered ubiquitin ligases

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Kanner, Scott A; Morgenstern, Travis

2017-01-01

The functional repertoire of surface ion channels is sustained by dynamic processes of trafficking, sorting, and degradation. Dysregulation of these processes underlies diverse ion channelopathies including cardiac arrhythmias and cystic fibrosis. Ubiquitination powerfully regulates multiple steps in the channel lifecycle, yet basic mechanistic understanding is confounded by promiscuity among E3 ligase/substrate interactions and ubiquitin code complexity. Here we targeted the catalytic domain of E3 ligase, CHIP, to YFP-tagged KCNQ1 ± KCNE1 subunits with a GFP-nanobody to selectively manipulate this channel complex in heterologous cells and adult rat cardiomyocytes. Engineered CHIP enhanced KCNQ1 ubiquitination, eliminated KCNQ1 surface-density, and abolished reconstituted K+ currents without affecting protein expression. A chemo-genetic variation enabling chemical control of ubiquitination revealed KCNQ1 surface-density declined with a ~ 3.5 hr t1/2 by impaired forward trafficking. The results illustrate utility of engineered E3 ligases to elucidate mechanisms underlying ubiquitin regulation of membrane proteins, and to achieve effective post-translational functional knockdown of ion channels. PMID:29256394

Fab-based inhibitors reveal ubiquitin independent functions for HIV Vif neutralization of APOBEC3 restriction factors.

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Jennifer M Binning

2018-01-01

Full Text Available The lentiviral protein Viral Infectivity Factor (Vif counteracts the antiviral effects of host APOBEC3 (A3 proteins and contributes to persistent HIV infection. Vif targets A3 restriction factors for ubiquitination and proteasomal degradation by recruiting them to a multi-protein ubiquitin E3 ligase complex. Here, we describe a degradation-independent mechanism of Vif-mediated antagonism that was revealed through detailed structure-function studies of antibody antigen-binding fragments (Fabs to the Vif complex. Two Fabs were found to inhibit Vif-mediated A3 neutralization through distinct mechanisms: shielding A3 from ubiquitin transfer and blocking Vif E3 assembly. Combined biochemical, cell biological and structural studies reveal that disruption of Vif E3 assembly inhibited A3 ubiquitination but was not sufficient to restore its packaging into viral particles and antiviral activity. These observations establish that Vif can neutralize A3 family members in a degradation-independent manner. Additionally, this work highlights the potential of Fabs as functional probes, and illuminates how Vif uses a multi-pronged approach involving both degradation dependent and independent mechanisms to suppress A3 innate immunity.

Characterization and 454 pyrosequencing of Major Histocompatibility Complex class I genes in the great tit reveal complexity in a passerine system

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Sepil Irem

2012-05-01

Full Text Available Abstract Background The critical role of Major Histocompatibility Complex (Mhc genes in disease resistance and their highly polymorphic nature make them exceptional candidates for studies investigating genetic effects on survival, mate choice and conservation. Species that harbor many Mhc loci and high allelic diversity are particularly intriguing as they are potentially under strong selection and studies of such species provide valuable information as to the mechanisms maintaining Mhc diversity. However comprehensive genotyping of complex multilocus systems has been a major challenge to date with the result that little is known about the consequences of this complexity in terms of fitness effects and disease resistance. Results In this study, we genotyped the Mhc class I exon 3 of the great tit (Parus major from two nest-box breeding populations near Oxford, UK that have been monitored for decades. Characterization of Mhc class I exon 3 was adopted and bidirectional sequencing was carried using the 454 sequencing platform. Full analysis of sequences through a stepwise variant validation procedure allowed reliable typing of more than 800 great tits based on 214,357 reads; from duplicates we estimated the repeatability of typing as 0.94. A total of 862 alleles were detected, and the presence of at least 16 functional loci was shown - the highest number characterized in a wild bird species. Finally, the functional alleles were grouped into 17 supertypes based on their antigen binding affinities. Conclusions We found extreme complexity at the Mhc class I of the great tit both in terms of allelic diversity and gene number. The presence of many functional loci was shown, together with a pseudogene family and putatively non-functional alleles; there was clear evidence that functional alleles were under strong balancing selection. This study is the first step towards an in-depth analysis of this gene complex in this species, which will help

Hepatitis C virus induces E6AP-dependent degradation of the retinoblastoma protein.

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Tsubasa Munakata

2007-09-01

Full Text Available Hepatitis C virus (HCV is a positive-strand RNA virus that frequently causes persistent infections and is uniquely associated with the development of hepatocellular carcinoma. While the mechanism(s by which the virus promotes cancer are poorly defined, previous studies indicate that the HCV RNA-dependent RNA polymerase, nonstructural protein 5B (NS5B, forms a complex with the retinoblastoma tumor suppressor protein (pRb, targeting it for degradation, activating E2F-responsive promoters, and stimulating cellular proliferation. Here, we describe the mechanism underlying pRb regulation by HCV and its relevance to HCV infection. We show that the abundance of pRb is strongly downregulated, and its normal nuclear localization altered to include a major cytoplasmic component, following infection of cultured hepatoma cells with either genotype 1a or 2a HCV. We further demonstrate that this is due to NS5B-dependent ubiquitination of pRb and its subsequent degradation via the proteasome. The NS5B-dependent ubiquitination of pRb requires the ubiquitin ligase activity of E6-associated protein (E6AP, as pRb abundance was restored by siRNA knockdown of E6AP or overexpression of a dominant-negative E6AP mutant in cells containing HCV RNA replicons. E6AP also forms a complex with pRb in an NS5B-dependent manner. These findings suggest a novel mechanism for the regulation of pRb in which the HCV NS5B protein traps pRb in the cytoplasm, and subsequently recruits E6AP to this complex in a process that leads to the ubiquitination of pRb. The disruption of pRb/E2F regulatory pathways in cells infected with HCV is likely to promote hepatocellular proliferation and chromosomal instability, factors important for the development of liver cancer.

Production of Infectious Dengue Virus in Aedes aegypti Is Dependent on the Ubiquitin Proteasome Pathway.

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Milly M Choy

2015-11-01

Full Text Available Dengue virus (DENV relies on host factors to complete its life cycle in its mosquito host for subsequent transmission to humans. DENV first establishes infection in the midgut of Aedes aegypti and spreads to various mosquito organs for lifelong infection. Curiously, studies have shown that infectious DENV titers peak and decrease thereafter in the midgut despite relatively stable viral genome levels. However, the mechanisms that regulate this decoupling of infectious virion production from viral RNA replication have never been determined. We show here that the ubiquitin proteasome pathway (UPP plays an important role in regulating infectious DENV production. Using RNA interference studies, we show in vivo that knockdown of selected UPP components reduced infectious virus production without altering viral RNA replication in the midgut. Furthermore, this decoupling effect could also be observed after RNAi knockdown in the head/thorax of the mosquito, which otherwise showed direct correlation between infectious DENV titer and viral RNA levels. The dependence on the UPP for successful DENV production is further reinforced by the observed up-regulation of key UPP molecules upon DENV infection that overcome the relatively low expression of these genes after a blood meal. Collectively, our findings indicate an important role for the UPP in regulating DENV production in the mosquito vector.

Lipidation of BmAtg8 is required for autophagic degradation of p62 bodies containing ubiquitinated proteins in the silkworm, Bombyx mori.

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Ji, Ming-Ming; Lee, Jae Man; Mon, Hiroaki; Iiyama, Kazuhiro; Tatsuke, Tsuneyuki; Morokuma, Daisuke; Hino, Masato; Yamashita, Mami; Hirata, Kazuma; Kusakabe, Takahiro

2017-10-01

p62/Sequestosome-1 (p62/SQSTM1, hereafter referred to as p62) is a major adaptor that allows ubiquitinated proteins to be degraded by autophagy, and Atg8 homologs are required for p62-mediated autophagic degradation, but their relationship is still not understood in Lepidopteran insects. Here it is clearly demonstrated that the silkworm homolog of mammalian p62, Bombyx mori p62 (Bmp62), forms p62 bodies depending on its Phox and Bem1p (PB1) and ubiquitin-associated (UBA) domains. These two domains are associated with Bmp62 binding to ubiquitinated proteins to form the p62 bodies, and the UBA domain is essential for the binding, but Bmp62 still self-associates without the PB1 or UBA domain. The p62 bodies in Bombyx cells are enclosed by BmAtg9-containing membranes and degraded via autophagy. It is revealed that the interaction between the Bmp62 AIM motif and BmAtg8 is critical for the autophagic degradation of the p62 bodies. Intriguingly, we further demonstrate that lipidation of BmAtg8 is required for the Bmp62-mediated complete degradation of p62 bodies by autophagy. Our results should be useful in future studies of the autophagic mechanism in Lepidopteran insects. Copyright © 2017 Elsevier Ltd. All rights reserved.

The study of fkbp and ubiquitin reveals interesting aspects of Artemia stress history.

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Maniatsi, Stefania; Farmaki, Theodora; Abatzopoulos, Theodore J

2015-08-01

Research on stress responses in animals has increased greatly during the last decades. Though most studies focus on the cellular and molecular bases of the stress response mechanisms, the ecological and evolutionary aspects of stress responses gain more and more interest. Here, we use species and parthenogenetic strains of the genus Artemia, an extremophile model organism, to study, for the first time, a protein well known for its chaperone activity and its involvement in stress responses. More specifically, transcription and protein accumulation of an FK506-Binding Protein (FKBP) homologue were investigated under heat and salt stresses. Additionally, the mRNA levels of ubiquitin, a heat-inducible protein related to the proteasomal pathway, were quantitated under these conditions. Biochemical and phylogenetic analyses showed that the studied FKBP orthologue is a typical representative of the family that clusters with other crustacean sequences. The expression was increased in both fkbp and ubiquitin genes after salt and heat stresses. However, our results in combination with the fact that Artemia species and parthenogenetic strains, selected for this study, exhibit different heat or salt tolerance provide useful hints about the evolutionary significance of FKBP and ubiquitin. Regarding FKBP, mRNA expression and protein accumulation seem to depend on the environmental conditions and the evolutionary history of each Artemia population while ubiquitin has a clear and more conserved role under heat shock. Copyright © 2015 Elsevier Inc. All rights reserved.

It takes two to tango: Ubiquitin and SUMO in the DNA damage response

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Bologna, Serena; Ferrari, Stefano

2013-01-01

The complexity of living cells is primarily determined by the genetic information encoded in DNA and gets fully disclosed upon translation. A major determinant of complexity is the reversible post-translational modification (PTM) of proteins, which generates variants displaying distinct biological properties such as subcellular localization, enzymatic activity and the ability to assemble in complexes. Decades of work on phosphorylation have unambiguously proven this concept. In recent years, the covalent attachment of Ubiquitin or Small Ubiquitin-like Modifiers (SUMO) to amino acid residues of target proteins has been recognized as another crucial PTM, re-directing protein fate and protein-protein interactions. This review focuses on the role of ubiquitylation and sumoylation in the control of DNA damage response proteins. To lay the ground, we begin with a description of ubiquitylation and sumoylation, providing established examples of DNA damage response elements that are controlled through these PTMs. We then examine in detail the role of PTMs in the cellular response to DNA double-strand breaks illustrating hierarchy, cross-talk, synergism or antagonism between phosphorylation, ubiquitylation and sumoylation. We conclude offering a perspective on Ubiquitin and SUMO pathways as targets in cancer therapy. PMID:23781231

Effects of exogenous ubiquitin in a polytrauma model with blunt chest trauma

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Baker, Todd A.; Romero, Jacqueline; Bach, Harold H.; Strom, Joel A.; Gamelli, Richard L.; Majetschak, Matthias

2013-01-01

Objective To determine whether treatment with the CXC chemokine receptor (CXCR) 4 agonist ubiquitin results in beneficial effects in a polytrauma model consisting of bilateral femur fractures plus blunt chest trauma (Injury Severity Score 18-25). Design Treatment study. Setting Research Laboratory. Subjects Seventeen Yorkshire pigs. Interventions Intravenous (i.v.) injection of 1.5 mg/kg ubiquitin or albumin (=control) at 60 min after polytrauma. Measurements and Main Results Anesthetized, mechanically ventilated pigs underwent polytrauma, followed by a simulated 60 min shock phase. At the end of the shock phase ubiquitin or albumin were administered and animals were resuscitated to a mean arterial blood pressure of 70 mmHg until t = 420 min. After i.v. ubiquitin, ubiquitin plasma concentrations increased sixteen-fold to 2870 ± 1015 ng/mL at t = 90 min and decreased with t1/2 = 60 min. Endogenous plasma ubiquitin increased two-fold in the albumin group with peak levels of 359 ± 210 ng/mL. Plasma levels of the cognate CXCR4 ligand stromal cell-derived factor (SDF)-1α were unchanged in both groups. Ubiquitin treatment reduced arterial lactate levels and prevented a continuous decrease in arterial oxygenation, which occurred in the albumin group during resuscitation. Wet weight to dry weight ratios of the lung contralateral from the injury, heart, spleen and jejunum were lower with ubiquitin. With ubiquitin treatment, tissue levels of IL-8, IL-10, TNFα and SDF-1α were reduced in the injured lung and of IL-8 in the contralateral lung, respectively. Conclusions Administration of exogenous ubiquitin modulates the local inflammatory response, improves resuscitation, reduces fluid shifts into tissues and preserves arterial oxygenation after blunt polytrauma with lung injury. This study further supports the notion that ubiquitin is a promising protein therapeutic and implies CXCR4 as a drug target after polytrauma. PMID:22622399

Direct Sensing and Discrimination among Ubiquitin and Ubiquitin Chains Using Solid-State Nanopores.

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Nir, Iftach; Huttner, Diana; Meller, Amit

2015-05-05

Nanopore sensing involves an electrophoretic transport of analytes through a nanoscale pore, permitting label-free sensing at the single-molecule level. However, to date, the detection of individual small proteins has been challenging, primarily due to the poor signal/noise ratio that these molecules produce during passage through the pore. Here, we show that fine adjustment of the buffer pH, close to the isoelectric point, can be used to slow down the translocation speed of the analytes, hence permitting sensing and characterization of small globular proteins. Ubiquitin (Ub) is a small protein of 8.5 kDa, which is well conserved in all eukaryotes. Ub conjugates to proteins as a posttranslational modification called ubiquitination. The immense diversity of Ub substrates, as well as the complexity of Ub modification types and the numerous physiological consequences of these modifications, make Ub and Ub chains an interesting and challenging subject of study. The ability to detect Ub and to identify Ub linkage type at the single-molecule level may provide a novel tool for investigation in the Ub field. This is especially adequate because, for most ubiquitinated substrates, Ub modifies only a few molecules in the cell at a given time. Applying our method to the detection of mono- and poly-Ub molecules, we show that we can analyze their characteristics using nanopores. Of particular importance is that two Ub dimers that are equal in molecular weight but differ in 3D structure due to their different linkage types can be readily discriminated. Thus, to our knowledge, our method offers a novel approach for analyzing proteins in unprecedented detail using solid-state nanopores. Specifically, it provides the basis for development of single-molecule sensing of differently ubiquitinated substrates with different biological significance. Finally, our study serves as a proof of concept for approaching nanopore detection of sub-10-kDa proteins and demonstrates the ability of

StUbEx PLUS-A Modified Stable Tagged Ubiquitin Exchange System for Peptide Level Purification and In-Depth Mapping of Ubiquitination Sites

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Akimov, Vyacheslav; Olsen, Louise C B; Hansen, Sten V F

2018-01-01

repair, and signal transduction. Because of its importance for numerous cellular functions, ubiquitination has become an intense topic of research in recent years, and proteomics tools have greatly facilitated the identification of many ubiquitination targets. Taking advantage of the StUbEx strategy...

The long N-terminus of the human monocarboxylate transporter 8 is a target of ubiquitin-dependent proteasomal degradation which regulates protein expression and oligomerization capacity.

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Zwanziger, Denise; Schmidt, Mathias; Fischer, Jana; Kleinau, Gunnar; Braun, Doreen; Schweizer, Ulrich; Moeller, Lars Christian; Biebermann, Heike; Fuehrer, Dagmar

2016-10-15

Monocarboxylate transporter 8 (MCT8) equilibrates thyroid hormones between the extra- and the intracellular sides. MCT8 exists either with a short or a long N-terminus, but potential functional differences between both variants are yet not known. We, therefore, generated MCT8 constructs which are different in N-terminal length: MCT8(1-613), MCT8(25-613), MCT8(49-613) and MCT8(75-613). The M75G substitution prevents translation of MCT8(75-613) and ensures expression of full-length MCT8 protein. The K56G substitution was made to prevent ubiquitinylation. Cell-surface expression, localization and proteasomal degradation were investigated using C-terminally GFP-tagged MCT8 constructs (HEK293 and MDCK1 cells) and oligomerization capacity was determined using N-terminally HA- and C-terminally FLAG-tagged MCT8 constructs (COS7 cells). MCT8(1-613)-GFP showed a lower protein expression than the shorter MCT8(75-613)-GFP protein. The proteasome inhibitor lactacystin increased MCT8(1-613)-GFP protein amount, suggesting proteasomal degradation of MCT8 with the long N-terminus. Ubiquitin conjugation of MCT8(1-613)-GFP was found by immuno-precipitation. A diminished ubiquitin conjugation caused by K56G substitution resulted in increased MCT8(1-613)-GFP protein expression. Sandwich ELISA was performed to investigate if the bands at higher molecular weight observed in Western blot analysis are due to MCT8 oligomerization, which was indeed shown. Our data imply a role of the long N-terminus of MCT8 as target of ubiquitin-dependent proteasomal degradation affecting MCT8 amount and subsequently oligomerization capacity. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Water Evaporation and Conformational Changes from Partially Solvated Ubiquitin

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Saravana Prakash Thirumuruganandham

2010-01-01

Full Text Available Using molecular dynamics simulation, we study the evaporation of water molecules off partially solvated ubiquitin. The evaporation and cooling rates are determined for a molecule at the initial temperature of 300 K. The cooling rate is found to be around 3 K/ns, and decreases with water temperature in the course of the evaporation. The conformation changes are monitored by studying a variety of intermediate partially solvated ubiquitin structures. We find that ubiquitin shrinks with decreasing hydration shell and exposes more of its hydrophilic surface area to the surrounding.

Ubiquitination of basal VEGFR2 regulates signal transduction and endothelial function.

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Smith, Gina A; Fearnley, Gareth W; Abdul-Zani, Izma; Wheatcroft, Stephen B; Tomlinson, Darren C; Harrison, Michael A; Ponnambalam, Sreenivasan

2017-10-15

Cell surface receptors can undergo recycling or proteolysis but the cellular decision-making events that sort between these pathways remain poorly defined. Vascular endothelial growth factor A (VEGF-A) and vascular endothelial growth factor receptor 2 (VEGFR2) regulate signal transduction and angiogenesis, but how signaling and proteolysis is regulated is not well understood. Here, we provide evidence that a pathway requiring the E1 ubiquitin-activating enzyme UBA1 controls basal VEGFR2 levels, hence metering plasma membrane receptor availability for the VEGF-A-regulated endothelial cell response. VEGFR2 undergoes VEGF-A-independent constitutive degradation via a UBA1-dependent ubiquitin-linked pathway. Depletion of UBA1 increased VEGFR2 recycling from endosome-to-plasma membrane and decreased proteolysis. Increased membrane receptor availability after UBA1 depletion elevated VEGF-A-stimulated activation of key signaling enzymes such as PLCγ1 and ERK1/2. Although UBA1 depletion caused an overall decrease in endothelial cell proliferation, surviving cells showed greater VEGF-A-stimulated responses such as cell migration and tubulogenesis. Our study now suggests that a ubiquitin-linked pathway regulates the balance between receptor recycling and degradation which in turn impacts on the intensity and duration of VEGF-A-stimulated signal transduction and the endothelial response. © 2017. Published by The Company of Biologists Ltd.

Genome-Wide Identification, Phylogenetic and Expression Analyses of the Ubiquitin-Conjugating Enzyme Gene Family in Maize

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Jue, Dengwei; Sang, Xuelian; Lu, Shengqiao; Dong, Chen; Zhao, Qiufang; Chen, Hongliang; Jia, Liqiang

2015-01-01

Background Ubiquitination is a post-translation modification where ubiquitin is attached to a substrate. Ubiquitin-conjugating enzymes (E2s) play a major role in the ubiquitin transfer pathway, as well as a variety of functions in plant biological processes. To date, no genome-wide characterization of this gene family has been conducted in maize (Zea mays). Methodology/Principal Findings In the present study, a total of 75 putative ZmUBC genes have been identified and located in the maize genome. Phylogenetic analysis revealed that ZmUBC proteins could be divided into 15 subfamilies, which include 13 ubiquitin-conjugating enzymes (ZmE2s) and two independent ubiquitin-conjugating enzyme variant (UEV) groups. The predicted ZmUBC genes were distributed across 10 chromosomes at different densities. In addition, analysis of exon-intron junctions and sequence motifs in each candidate gene has revealed high levels of conservation within and between phylogenetic groups. Tissue expression analysis indicated that most ZmUBC genes were expressed in at least one of the tissues, indicating that these are involved in various physiological and developmental processes in maize. Moreover, expression profile analyses of ZmUBC genes under different stress treatments (4°C, 20% PEG6000, and 200 mM NaCl) and various expression patterns indicated that these may play crucial roles in the response of plants to stress. Conclusions Genome-wide identification, chromosome organization, gene structure, evolutionary and expression analyses of ZmUBC genes have facilitated in the characterization of this gene family, as well as determined its potential involvement in growth, development, and stress responses. This study provides valuable information for better understanding the classification and putative functions of the UBC-encoding genes of maize. PMID:26606743

Altered ubiquitin causes perturbed calcium homeostasis, hyperactivation of calpain, dysregulated differentiation, and cataract.

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Liu, Ke; Lyu, Lei; Chin, David; Gao, Junyuan; Sun, Xiurong; Shang, Fu; Caceres, Andrea; Chang, Min-Lee; Rowan, Sheldon; Peng, Junmin; Mathias, Richard; Kasahara, Hideko; Jiang, Shuhong; Taylor, Allen

2015-01-27

Although the ocular lens shares many features with other tissues, it is unique in that it retains its cells throughout life, making it ideal for studies of differentiation/development. Precipitation of proteins results in lens opacification, or cataract, the major blinding disease. Lysines on ubiquitin (Ub) determine fates of Ub-protein substrates. Information regarding ubiquitin proteasome systems (UPSs), specifically of K6 in ubiquitin, is undeveloped. We expressed in the lens a mutant Ub containing a K6W substitution (K6W-Ub). Protein profiles of lenses that express wild-type ubiquitin (WT-Ub) or K6W-Ub differ by only ∼2%. Despite these quantitatively minor differences, in K6W-Ub lenses and multiple model systems we observed a fourfold Ca(2+) elevation and hyperactivation of calpain in the core of the lens, as well as calpain-associated fragmentation of critical lens proteins including Filensin, Fodrin, Vimentin, β-Crystallin, Caprin family member 2, and tudor domain containing 7. Truncations can be cataractogenic. Additionally, we observed accumulation of gap junction Connexin43, and diminished Connexin46 levels in vivo and in vitro. These findings suggest that mutation of Ub K6 alters UPS function, perturbs gap junction function, resulting in Ca(2+) elevation, hyperactivation of calpain, and associated cleavage of substrates, culminating in developmental defects and a cataractous lens. The data show previously unidentified connections between UPS and calpain-based degradative systems and advance our understanding of roles for Ub K6 in eye development. They also inform about new approaches to delay cataract and other protein precipitation diseases.

Ubiquitinated proteins enriched from tumor cells by a ubiquitin binding protein Vx3(A7) as a potent cancer vaccine.

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Aldarouish, Mohanad; Wang, Huzhan; Zhou, Meng; Hu, Hong-Ming; Wang, Li-Xin

2015-04-16

Our previous studies have demonstrated that autophagosome-enriched vaccine (named DRibbles: DRiPs-containing blebs) induce a potent anti-tumor efficacy in different murine tumor models, in which DRibble-containing ubiquitinated proteins are efficient tumor-specific antigen source for the cross-presentation after being loaded onto dendritic cells. In this study, we sought to detect whether ubiquitinated proteins enriched from tumor cells could be used directly as a novel cancer vaccine. The ubiquitin binding protein Vx3(A7) was used to isolate ubiquitinated proteins from EL4 and B16-F10 tumor cells after blocking their proteasomal degradation pathway. C57BL/6 mice were vaccinated with different doses of Ub-enriched proteins via inguinal lymph nodes or subcutaneous injection and with DRibbles, Ub-depleted proteins and whole cell lysate as comparison groups, respectively. The lymphocytes from the vaccinated mice were re-stimulated with inactivated tumor cells and the levels of IFN-γ in the supernatant were detected by ELISA. Anti-tumor efficacy of Ub-enriched proteins vaccine was evaluated by monitoring tumor growth in established tumor mice models. Graphpad Prism 5.0 was used for all statistical analysis. We found that after stimulation with inactivated tumor cells, the lymphocytes from the Ub-enriched proteins-vaccinated mice secreted high level of IFN-γ in dose dependent manner, in which the priming vaccination via inguinal lymph nodes injection induced higher IFN-γ level than that via subcutaneous injection. Moreover, the level of secreted IFN-γ in the Ub-enriched proteins group was markedly higher than that in the whole cell lysate and Ub-depleted proteins. Interestingly, the lymphocytes from mice vaccinated with Ub-enriched proteins, but not Ub-depleted proteins and whole cell lysates, isolated from EL4 or B16-F10 tumor cells also produced an obvious level of IFN-γ when stimulated alternately with inactivated B16-F10 or EL4 tumor cells. Furthermore, Ub

FANCL ubiquitinates β-catenin and enhances its nuclear function.

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Dao, Kim-Hien T; Rotelli, Michael D; Petersen, Curtis L; Kaech, Stefanie; Nelson, Whitney D; Yates, Jane E; Hanlon Newell, Amy E; Olson, Susan B; Druker, Brian J; Bagby, Grover C

2012-07-12

Bone marrow failure is a nearly universal complication of Fanconi anemia. The proteins encoded by FANC genes are involved in DNA damage responses through the formation of a multisubunit nuclear complex that facilitates the E3 ubiquitin ligase activity of FANCL. However, it is not known whether loss of E3 ubiquitin ligase activity accounts for the hematopoietic stem cell defects characteristic of Fanconi anemia. Here we provide evidence that FANCL increases the activity and expression of β-catenin, a key pluripotency factor in hematopoietic stem cells. We show that FANCL ubiquitinates β-catenin with atypical ubiquitin chain extension known to have nonproteolytic functions. Specifically, β-catenin modified with lysine-11 ubiquitin chain extension efficiently activates a lymphocyte enhancer-binding factor-T cell factor reporter. We also show that FANCL-deficient cells display diminished capacity to activate β-catenin leading to reduced transcription of Wnt-responsive targets c-Myc and Cyclin D1. Suppression of FANCL expression in normal human CD34(+) stem and progenitor cells results in fewer β-catenin active cells and inhibits expansion of multilineage progenitors. Together, these results suggest that diminished Wnt/β-catenin signaling may be an underlying molecular defect in FANCL-deficient hematopoietic stem cells leading to their accelerated loss.

Structure and catalytic regulatory function of ubiquitin specific protease 11 N-terminal and ubiquitin-like domains.

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Harper, Stephen; Gratton, Hayley E; Cornaciu, Irina; Oberer, Monika; Scott, David J; Emsley, Jonas; Dreveny, Ingrid

2014-05-13

The ubiquitin specific protease 11 (USP11) is implicated in DNA repair, viral RNA replication, and TGFβ signaling. We report the first characterization of the USP11 domain architecture and its role in regulating the enzymatic activity. USP11 consists of an N-terminal "domain present in USPs" (DUSP) and "ubiquitin-like" (UBL) domain, together referred to as DU domains, and the catalytic domain harboring a second UBL domain. Crystal structures of the DU domains show a tandem arrangement with a shortened β-hairpin at the two-domain interface and altered surface characteristics compared to the homologues USP4 and USP15. A conserved VEVY motif is a signature feature at the two-domain interface that shapes a potential protein interaction site. Small angle X-ray scattering and gel filtration experiments are consistent with the USP11DU domains and full-length USP11 being monomeric. Unexpectedly, we reveal, through kinetic assays of a series of deletion mutants, that the catalytic activity of USP11 is not regulated through intramolecular autoinhibition or activation by the N-terminal DU or UBL domains. Moreover, ubiquitin chain cleavage assays with all eight linkages reveal a preference for Lys(63)-, Lys(6)-, Lys(33)-, and Lys(11)-linked chains over Lys(27)-, Lys(29)-, and Lys(48)-linked and linear chains consistent with USP11's function in DNA repair pathways that is mediated by the protease domain. Our data support a model whereby USP11 domains outside the catalytic core domain serve as protein interaction or trafficking modules rather than a direct regulatory function of the proteolytic activity. This highlights the diversity of USPs in substrate recognition and regulation of ubiquitin deconjugation.

Giant panda genomic data provide insight into the birth-and-death process of mammalian major histocompatibility complex class II genes.

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Qiu-Hong Wan

Full Text Available To gain an understanding of the genomic structure and evolutionary history of the giant panda major histocompatibility complex (MHC genes, we determined a 636,503-bp nucleotide sequence spanning the MHC class II region. Analysis revealed that the MHC class II region from this rare species contained 26 loci (17 predicted to be expressed, of which 10 are classical class II genes (1 DRA, 2 DRB, 2 DQA, 3 DQB, 1 DYB, 1 DPA, and 2 DPB and 4 are non-classical class II genes (1 DOA, 1 DOB, 1 DMA, and 1 DMB. The presence of DYB, a gene specific to ruminants, prompted a comparison of the giant panda class II sequence with those of humans, cats, dogs, cattle, pigs, and mice. The results indicated that birth and death events within the DQ and DRB-DY regions led to major lineage differences, with absence of these regions in the cat and in humans and mice respectively. The phylogenetic trees constructed using all expressed alpha and beta genes from marsupials and placental mammals showed that: (1 because marsupials carry loci corresponding to DR, DP, DO and DM genes, those subregions most likely developed before the divergence of marsupials and placental mammals, approximately 150 million years ago (MYA; (2 conversely, the DQ and DY regions must have evolved later, but before the radiation of placental mammals (100 MYA. As a result, the typical genomic structure of MHC class II genes for the giant panda is similar to that of the other placental mammals and corresponds to BTNL2 approximately DR1 approximately DQ approximately DR2 approximately DY approximately DO_box approximately DP approximately COL11A2. Over the past 100 million years, there has been birth and death of mammalian DR, DQ, DY, and DP genes, an evolutionary process that has brought about the current species-specific genomic structure of the MHC class II region. Furthermore, facing certain similar pathogens, mammals have adopted intra-subregion (DR and DQ and inter-subregion (between DQ and DP

Roles of mono-ubiquitinated Smad4 in the formation of Smad transcriptional complexes

International Nuclear Information System (INIS)

Wang Bei; Suzuki, Hiroyuki; Kato, Mitsuyasu

2008-01-01

TGF-β activates receptor-regulated Smad (R-Smad) through phosphorylation by type I receptors. Activated R-Smad binds to Smad4 and the complex translocates into the nucleus and stimulates the transcription of target genes through association with co-activators including p300. It is not clear, however, how activated Smad complexes are removed from target genes. In this study, we show that TGF-β enhances the mono-ubiquitination of Smad4. Smad4 mono-ubiquitination was promoted by p300 and suppressed by the c-Ski co-repressor. Smad4 mono-ubiquitination disrupted the interaction with Smad2 in the presence of constitutively active TGF-β type I receptor. Furthermore, mono-ubiquitinated Smad4 was not found in DNA-binding Smad complexes. A Smad4-Ubiquitin fusion protein, which mimics mono-ubiquitinated Smad4, enhanced localization to the cytoplasm. These results suggest that mono-ubiquitination of Smad4 occurs in the transcriptional activator complex and facilitates the turnover of Smad complexes at target genes

Break-induced ATR and Ddb1-Cul4(Cdt)² ubiquitin ligase-dependent nucleotide synthesis promotes homologous recombination repair in fission yeast

DEFF Research Database (Denmark)

Moss, Jennifer; Tinline-Purvis, Helen; Walker, Carol A

2010-01-01

Nucleotide synthesis is a universal response to DNA damage, but how this response facilitates DNA repair and cell survival is unclear. Here we establish a role for DNA damage-induced nucleotide synthesis in homologous recombination (HR) repair in fission yeast. Using a genetic screen, we found...... the Ddb1-Cul4(Cdt)² ubiquitin ligase complex and ribonucleotide reductase (RNR) to be required for HR repair of a DNA double-strand break (DSB). The Ddb1-Cul4(Cdt)² ubiquitin ligase complex is required for degradation of Spd1, an inhibitor of RNR in fission yeast. Accordingly, deleting spd1(+) suppressed...

Mechanism for the selective conjugation of ubiquitin to phytochrome

Energy Technology Data Exchange (ETDEWEB)

Vierstra, R.D.

1990-01-01

The goal of this project is to understand at the molecular level how phytochrome functions and how intracellular proteins are degraded. Phytochrome is marked for degradation by covalent attachment of ubiquitin. Ubiquitin-phytochrome conjugates (UbP) were characterized with respect to formation kinetics, subcellular localization and site of ubiquitin attachment. UbP appears to be a general phenomenon during phytochrome degradation in a variety of species. UbP was isolated from oat seedlings and characterized. Residues 747-830 of phytochrome have been identified as a possible attachment site for ubiquitin. By placing the gene for etiolated phytochrome in tobacco we have created a transgenic system for over expressing phytochrome. The effects of this over expression are described, and it appears that tobacco degrades this foreign protein through formation of UbP. We have created a series of site-directed mutants of the oat phytochrome gene, and are in the process of characterizing them to determine sequence requirements for ubiquination. 8 refs., 1 fig. (MHB)

Histocompatibility Testing for Organ Transplantation Purposes in Albania: A Single Center Experience

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Erkena Shyti

2014-06-01

Full Text Available Background: Histocompatibility testing (HT which includes donor-recipient human leukocyte antigen (HLA matching, cross-match testing (XMT and anti-HLA antibody searching are crucial examinations in solid organ transplantation aiming to avoid the hyperacute graft rejection and also to predict the immunological outcome of the graft. Aims: The aim of this study was to analyse the tissue typing data collected at the Laboratory of Immunology and Histocompatibility of the University Hospital Center of Tirana, Albania, in order to define those actions that should be taken for improvements in the situation of kidney transplantation in Albania. Design: Descriptive study. Methods: The donor/recipient cross-match testing was performed through a standard complement-dependent cytotoxicity (CDC assay using separated donor T and B cells that were tested in parallel with the recipient serum sample. All recipient sera were screened for anti-Class I and anti-Class II HLA antibodies using a bead based Luminex anti-HLA antibody screening test. In the case of detected positivity, an allele-specific anti-HLA antibody determination was conducted with the respective Luminex anti-Class I and Class II HLA antibody determination kits. Results: A total of 174 recipients and 202 donors were typed for the purpose of living donor kidney transplantation at our laboratory between January 2006 and December 2012. The mean age and female gender proportion of patients were 34.9 years and 34.5%, respectively, and 48.0 years and 65.3% for the donors, respectively. Here, 25.9% of the patients reported a positive complement-dependent cytotoxicity cross-match test and/or a positive anti-HLA antibody testing result. Eighteen patients that were negative for the complement-dependent cytotoxicity cross-match test were positive for anti-HLA antibodies. Conclusion: The predominant causes of end-stage renal disease (ESRD in our patient population are chronic pyelonephritis and

Genetic variation in the extended major histocompatibility complex and susceptibility to childhood acute lymphoblastic leukemia: a review of the evidence

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Kevin Y Urayama

2013-12-01

Full Text Available The enduring suspicion that infections and immunologic response may play a role in the etiology of childhood leukemia, particularly acute lymphoblastic leukemia (ALL, is now supported, albeit still indirectly, by numerous epidemiological studies. The cumulative evidence includes, for example, descriptive observations of a peculiar peak incidence at age 2-5 years for ALL in economically developed countries, clustering of cases in situations of population mixing associated with unusual patterns of personal contacts, associations with various proxy measures for immune modulatory exposures early in life, and genetic susceptibility conferred by variation in genes involved in the immune system. In this review, our focus is the extended major histocompatibility complex (xMHC, an approximately 7.6 megabase region that is well-known for its high density of expressed genes, extensive polymorphisms exhibiting complex linkage disequilibrium patterns, and its disproportionately large number of immune-related genes, including human leukocyte antigen (HLA. First discovered through the role they play in transplant rejection, the classical HLA class I (HLA-A, -B, and -C and class II (HLA-DR, HLA-DQ, and HLA-DP molecules reside at the epicenter of the immune response pathways and are now the targets of many disease susceptibility studies, including those for childhood leukemia. The genes encoding the HLA molecules are only a minority of the over 250 expressed genes in the xMHC, and a growing number of studies are beginning to evaluate other loci through targeted investigations or utilizing a mapping approach with a comprehensive screen of the entire region. Here, we review the current epidemiologic evidence available to date regarding genetic variation contained within this highly unique region of the genome and its relationship with childhood ALL risk.

Biochemical function of typical and variant Arabidopsis thaliana U-box E3 ubiquitin-protein ligases.

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Wiborg, Jakob; O'Shea, Charlotte; Skriver, Karen

2008-08-01

The variance of the U-box domain in 64 Arabidopsis thaliana (thale cress) E3s (ubiquitin-protein ligases) was used to examine the interactions between E3s and E2s (ubiquitin-conjugating enzymes). E2s and E3s are components of the ubiquitin protein degradation pathway. Seven U-box proteins were analysed for their ability to ubiquitinate proteins in vitro in co-operation with different E2s. All U-box domains exhibited ubiquitination activity and interacted productively with UBC4/5-type E2s. Three and four of the U-box domains mediated ubiquitin addition in the presence of UBC13 and UBC7 E2s respectively, but no productive interaction was observed with the UBC15 E2 tested. The activity of AtPUB54 [Arabidopsis thaliana (thale cress) plant U-box 54 protein] was dependent on Trp(266) in the E2-binding cleft, and the E2 selectivity was changed by substitution of this position. The function of the distant U-box protein, AtPUB49, representing a large family of eukaryotic proteins containing a U-box linked to a cyclophilin-like peptidyl-prolyl cis-trans isomerase domain, was characterized biochemically. AtPUB49 functioned both as a prolyl isomerase and a chaperone by catalysing cis-trans isomerization of peptidyl-prolyl bonds and dissolving protein aggregates. In conclusion, both typical and atypical Arabidopsis U-box proteins were active E3s. The overlap in the E3/E2 selectivity suggests that in vivo specificity is not determined only by the E3-E2 interactions, but also by other parameters, e.g. co-existence or interactions with additional domains. The biochemical functions of AtPUB49 suggest that the protein can be involved in folding or degradation of protein substrates. Similar functions can also be retained within a protein complex with separate chaperone and U-box proteins.

The cell biology of T-dependent B cell activation

DEFF Research Database (Denmark)

Owens, T; Zeine, R

1989-01-01

The requirement that CD4+ helper T cells recognize antigen in association with class II Major Histocompatibility Complex (MHC) encoded molecules constrains T cells to activation through intercellular interaction. The cell biology of the interactions between CD4+ T cells and antigen-presenting cells...... includes multipoint intermolecular interactions that probably involve aggregation of both polymorphic and monomorphic T cell surface molecules. Such aggregations have been shown in vitro to markedly enhance and, in some cases, induce T cell activation. The production of T-derived lymphokines that have been...... implicated in B cell activation is dependent on the T cell receptor for antigen and its associated CD3 signalling complex. T-dependent help for B cell activation is therefore similarly MHC-restricted and involves T-B intercellular interaction. Recent reports that describe antigen-independent B cell...

Characterization of major histocompatibility complex class I, and class II DRB loci of captive and wild Indian leopards (Panthera pardus fusca).

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Parmar, Drashti R; Mitra, Siuli; Bhadouriya, Snehalata; Rao, Tirupathi; Kunteepuram, Vaishnavi; Gaur, Ajay

2017-12-01

The major histocompatibility complex (MHC), in vertebrate animals, is a multi-genic protein complex that encodes various receptors. During a disease, MHC interacts with the antigen and triggers a cascade of adaptive immune responses to overcome a disease outbreak. The MHC is very important region from immunological point of view, but it is poorly characterized among Indian leopards. During this investigation, we examined genetic diversity for MHC class I (MHC-I) and MHC class II-DRB (MHC-II) among wild and captive Indian leopards. This study estimated a pool of 9 and 17 alleles for MHC-I and MHC-II, respectively. The wild group of individuals showed higher nucleotide diversity and amino acid polymorphism compared to the captive group. A phylogenetic comparison with other felids revealed a clustering in MHC-I and interspersed presence in MHC-II sequences. A test for selection also revealed a deviation from neutrality at MHC-II DRB loci and higher non-synonymous substitution rate (dN) among the individuals from wild group. Further, the wild individuals showed higher dN for both MHC I and II genes compared to the group that was bred under captive conditions. These findings suggest the role of micro-evolutionary forces, such as pathogen-mediated selection, to cause MHC variations among the two groups of Indian leopards, because the two groups have been bred in two different environments for a substantial period of time. Since, MHC diversity is often linked with the quality of immunological health; the results obtained from this study fill the gap of knowledge on disease predisposition among wild and captive Indian leopards.

Bone marrow transplantation across major histocompatibility barriers in mice. II. T cell requirement for engraftment in total lymphoid irradiation-conditioned recipients

International Nuclear Information System (INIS)

Vallera, D.A.; Soderling, C.C.; Carlson, G.J.; Kersey, J.H.

1982-01-01

Studies were undertaken to examine the role of T lymphocytes in engraftment of bone marrow (BM) in animals conditioned with total lymphoid irradiation (TLI) prior to transplantation across major histocompatibility barriers. Donor BM (added as a source of lymphohematopoietic stem cells) and spleen cells (added as a source of graft-versus-host disease (GVHD)-causing cells) were pretreated in vitro with monoclonal anti-Thy-1.2 plus complement (C). T cell-depleted grafts were then give to allogeneic mice conditioned with 900 rad of single dose TLI plus cyclophosphamide (CY). These mice did not engraft. Even in the absence of added spleen cells, elimination of the small T cell population from donor BM grafts prevented engraftment compared with animals that received the same conditioning regimen and untreated donor cells. These control animals demonstrated uniform evidence of engraftment about 1 month after transplantation. Similar findings were reported when recipients were conditioned with fractionated 17 x 200-rad TLI. In TLI plus CY-conditional recipients, we have also observed that increasing the donation of treated bone marrow cells still did not result in significant engraftment. Furthermore, graft failure in mice receiving normal dosages of anti-Thy-1.2 plus C-treated donor cells was not a strain-restricted phenomenon. Moreover, removal of bone marrow T cells with monoclonal anti-Lyt-1 plus complement also resulted in graft failure in TLI-conditioned recipients. In contrast to TLI conditioning, when Thy-1.2 plus C-treated donor cells were given to recipients conditioned with total body irradiation (TBI), a high percentage of engraftment was demonstrated by an H-2 microcytotoxicity assay. Plausible mechanisms for there findings are discussed

Modulation of the major histocompatibility complex by neural stem cell-derived neurotrophic factors used for regenerative therapy in a rat model of stroke

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Sun Chongran

2010-08-01

Full Text Available Abstract Background The relationship between functional improvements in ischemic rats given a neural stem cell (NSC transplant and the modulation of the class I major histocompatibility complex (MHC mediated by NSC-derived neurotrophins was investigated. Methods The levels of gene expression of nerve growth factor (NGF, brain-derived neurotropic factor (BDNF and neurotrophin-3 (NT-3 were assayed from cultures of cortical NSC from Sprague-Dawley rat E16 embryos. The levels of translated NGF in spent culture media from NSC cultures and the cerebral spinal fluid (CSF of rats with and without NGF injection or NSC transplant were also measured. Results We found a significant increase of NGF, BDNF and NT-3 transcripts and NGF proteins in both the NSC cultures and the CSF of the rats. The immunochemical staining for MHC in brain sections and the enzyme-linked immunosorbent assay of CSF were carried out in sham-operated rats and rats with surgically induced focal cerebral ischemia. These groups were further divided into animals that did and did not receive NGF administration or NSC transplant into the cisterna magna. Our results show an up-regulation of class I MHC in the ischemic rats with NGF and NSC administration. The extent of caspase-III immunoreactivity was comparable among three arms in the ischemic rats. Conclusion Readouts of somatosensory evoked potential and the trap channel test illustrated improvements in the neurological function of ischemic rats treated with NGF administration and NSC transplant.

Ubiquitination and sumoylation of the HTLV-2 Tax-2B protein regulate its NF-κB activity: a comparative study with the HTLV-1 Tax-1 protein

Science.gov (United States)

2012-01-01

Background Retroviruses HTLV-1 and HTLV-2 have homologous genomic structures but differ significantly in pathogenicity. HTLV-1 is associated with Adult T cell Leukemia (ATL), whereas infection by HTLV-2 has no association with neoplasia. Transformation of T lymphocytes by HTLV-1 is linked to the capacity of its oncoprotein Tax-1 to alter cell survival and cell cycle control mechanisms. Among these functions, Tax-1-mediated activation of cellular gene expression via the NF-κB pathway depends on Tax-1 post-translational modifications by ubiquitination and sumoylation. The Tax-2 protein of HTLV-2B (Tax-2B) is also modified by ubiquitination and sumoylation and activates the NF-κB pathway to a level similar to that of Tax-1. The present study aims to understand whether ubiquitination and sumoylation modifications are involved in Tax-2B-mediated activation of the NF-κB pathway. Results The comparison of Tax-1 and Tax-2B lysine to arginine substitution mutants revealed conserved patterns and levels of ubiquitination with notable difference in the lysine usage for sumoylation. Neither Tax-1 nor Tax-2B ubiquitination and sumoylation deficient mutants could activate the NF-κB pathway and fusion of ubiquitin or SUMO-1 to the C-terminus of the ubiquitination and sumoylation deficient Tax-2B mutant strikingly restored transcriptional activity. In addition, ubiquitinated forms of Tax-2B colocalized with RelA and IKKγ in prominent cytoplasmic structures associated with the Golgi apparatus, whereas colocalization of Tax-2B with the RelA subunit of NF-κB and the transcriptional coactivator p300 in punctate nuclear structures was dependent on Tax-2B sumoylation, as previously observed for Tax-1. Conclusions Both Tax-1 and Tax-2 activate the NF-κB pathway via similar mechanisms involving ubiquitination and sumoylation. Therefore, the different transforming potential of HTLV-1 and HTLV-2 is unlikely to be related to different modes of activation of the canonical NF-κB pathway

Ubiquitination is absolutely required for the degradation of hypoxia-inducible factor - 1 alpha protein in hypoxic conditions

International Nuclear Information System (INIS)

Wang, Ronghai; Zhang, Ping; Li, Jinhang; Guan, Hongzai; Shi, Guangjun

2016-01-01

The hypoxia-inducible factor (HIF) is recognized as the master regulator of hypoxia response. HIF-α subunits expression are tightly regulated. In this study, our data show that ts20 cells still expressed detectable E1 protein even at 39.5° C for 12 h, and complete depletion of E1 protein expression at 39.5° C by siRNA enhanced HIF-1α and P53 protein expression. Further inhibition of E1 at 39.5 °C by siRNA, or E1 inhibitor Ube1-41 completely blocked HIF-1α degradation. Moreover, immunoprecipitations of co-transfection of HA-ubiquitin and FLAG–HIF–1α plasmids directly confirmed the involvement of ubiquitin in the hypoxic degradation of HIF-1α. Additionally, hypoxic HIF-1 α degradation is independent of HAF, RACK1, sumoylation or nuclear/cytoplasmic localization. Taken together, our data suggest that constitutive HIF-1α protein degradation in hypoxia is absolutely ubiquitination-dependent, and unidentified E3 ligase may exist for this degradation pathway. - Highlights: • HIF-1α protein is constitutively degraded in hypoxic conditions. • Requirement of ubiquitination for HIF-1α degradation in hypoxia. • Hypoxic HIF-1α degradation is independent of HAF, RACK1, sumoylation or nuclear/cytoplasmic localization.

Ubiquitination is absolutely required for the degradation of hypoxia-inducible factor - 1 alpha protein in hypoxic conditions

Energy Technology Data Exchange (ETDEWEB)

Wang, Ronghai [Department of Urology, Linzi District People' s Hospital, Zibo, 255400 (China); Zhang, Ping, E-mail: zpskx001@163.com [Department of Gynecology, Qingdao Municipal Hospital, Qingdao, 266011 (China); Li, Jinhang [Department of Gynecology, Qingdao Municipal Hospital, Qingdao, 266011 (China); Guan, Hongzai [Laboratory Department, School of Medicine, Qingdao University, Qingdao, 266071 (China); Shi, Guangjun, E-mail: qdmhshigj@yahoo.com [Department of Hepatobiliary Surgery, Qingdao Municipal Hospital, Qingdao, 266071 (China)

2016-01-29

The hypoxia-inducible factor (HIF) is recognized as the master regulator of hypoxia response. HIF-α subunits expression are tightly regulated. In this study, our data show that ts20 cells still expressed detectable E1 protein even at 39.5° C for 12 h, and complete depletion of E1 protein expression at 39.5° C by siRNA enhanced HIF-1α and P53 protein expression. Further inhibition of E1 at 39.5 °C by siRNA, or E1 inhibitor Ube1-41 completely blocked HIF-1α degradation. Moreover, immunoprecipitations of co-transfection of HA-ubiquitin and FLAG–HIF–1α plasmids directly confirmed the involvement of ubiquitin in the hypoxic degradation of HIF-1α. Additionally, hypoxic HIF-1 α degradation is independent of HAF, RACK1, sumoylation or nuclear/cytoplasmic localization. Taken together, our data suggest that constitutive HIF-1α protein degradation in hypoxia is absolutely ubiquitination-dependent, and unidentified E3 ligase may exist for this degradation pathway. - Highlights: • HIF-1α protein is constitutively degraded in hypoxic conditions. • Requirement of ubiquitination for HIF-1α degradation in hypoxia. • Hypoxic HIF-1α degradation is independent of HAF, RACK1, sumoylation or nuclear/cytoplasmic localization.

Ubiquitin-SUMO Circuitry Controls Activated Fanconi Anemia ID Complex Dosage in Response to DNA Damage

DEFF Research Database (Denmark)

Gibbs-Seymour, Ian; Oka, Yasuyoshi; Rajendra, Eeson

2015-01-01

We show that central components of the Fanconi anemia (FA) DNA repair pathway, the tumor suppressor proteins FANCI and FANCD2 (the ID complex), are SUMOylated in response to replication fork stalling. The ID complex is SUMOylated in a manner that depends on the ATR kinase, the FA ubiquitin ligase...

Qualitative ubiquitome unveils the potential significances of protein lysine ubiquitination in hyphal growth of Aspergillus nidulans.

Science.gov (United States)

Chu, Xin-Ling; Feng, Ming-Guang; Ying, Sheng-Hua

2016-02-01

Protein ubiquitination is an evolutionarily conserved post-translational modification process in eukaryotes, and it plays an important role in many biological processes. Aspergillus nidulans, a model filamentous fungus, contributes to our understanding of cellular physiology, metabolism and genetics, but its ubiquitination is not completely revealed. In this study, the ubiquitination sites in the proteome of A. nidulans were identified using a highly sensitive mass spectrometry combined with immuno-affinity enrichment of the ubiquitinated peptides. The 4816 ubiquitination sites were identified in 1913 ubiquitinated proteins, accounting for 18.1% of total proteins in A. nidulans. Bioinformatic analysis suggested that the ubiquitinated proteins associated with a number of biological functions and displayed various sub-cellular localisations. Meanwhile, seven motifs were revealed from the ubiquitinated peptides, and significantly over-presented in the different pathways. Comparison of the enriched functional catalogues indicated that the ubiquitination functions divergently during growth of A. nidulans and Saccharomyces cerevisiae. Additionally, the proteins in A. nidulans-specific sub-category (cell growth/morphogenesis) were subjected to the protein interaction analysis which demonstrated that ubiquitination is involved in the comprehensive protein interactions. This study presents a first proteomic view of ubiquitination in the filamentous fungus, and provides an initial framework for exploring the physiological roles of ubiquitination in A. nidulans.

Aβ-Induced Synaptic Alterations Require the E3 Ubiquitin Ligase Nedd4-1.

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Rodrigues, Elizabeth M; Scudder, Samantha L; Goo, Marisa S; Patrick, Gentry N

2016-02-03

Alzheimer's disease (AD) is a neurodegenerative disease in which patients experience progressive cognitive decline. A wealth of evidence suggests that this cognitive impairment results from synaptic dysfunction in affected brain regions caused by cleavage of amyloid precursor protein into the pathogenic peptide amyloid-β (Aβ). Specifically, it has been shown that Aβ decreases surface AMPARs, dendritic spine density, and synaptic strength, and also alters synaptic plasticity. The precise molecular mechanisms by which this occurs remain unclear. Here we demonstrate a role for ubiquitination in Aβ-induced synaptic dysfunction in cultured rat neurons. We find that Aβ promotes the ubiquitination of AMPARs, as well as the redistribution and recruitment of Nedd4-1, a HECT E3 ubiquitin ligase we previously demonstrated to target AMPARs for ubiquitination and degradation. Strikingly, we show that Nedd4-1 is required for Aβ-induced reductions in surface AMPARs, synaptic strength, and dendritic spine density. Our findings, therefore, indicate an important role for Nedd4-1 and ubiquitin in the synaptic alterations induced by Aβ. Synaptic changes in Alzheimer's disease (AD) include surface AMPAR loss, which can weaken synapses. In a cell culture model of AD, we found that AMPAR loss correlates with increased AMPAR ubiquitination. In addition, the ubiquitin ligase Nedd4-1, known to ubiquitinate AMPARs, is recruited to synapses in response to Aβ. Strikingly, reducing Nedd4-1 levels in this model prevented surface AMPAR loss and synaptic weakening. These findings suggest that, in AD, Nedd4-1 may ubiquitinate AMPARs to promote their internalization and weaken synaptic strength, similar to what occurs in Nedd4-1's established role in homeostatic synaptic scaling. This is the first demonstration of Aβ-mediated control of a ubiquitin ligase to regulate surface AMPAR expression. Copyright © 2016 the authors 0270-6474/16/361590-06$15.00/0.

Giant panda BAC library construction and assembly of a 650-kb contig spanning major histocompatibility complex class II region

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Pan Hui-Juan

2007-09-01

Full Text Available Abstract Background Giant panda is rare and endangered species endemic to China. The low rates of reproductive success and infectious disease resistance have severely hampered the development of captive and wild populations of the giant panda. The major histocompatibility complex (MHC plays important roles in immune response and reproductive system such as mate choice and mother-fetus bio-compatibility. It is thus essential to understand genetic details of the giant panda MHC. Construction of a bacterial artificial chromosome (BAC library will provide a new tool for panda genome physical mapping and thus facilitate understanding of panda MHC genes. Results A giant panda BAC library consisting of 205,800 clones has been constructed. The average insert size was calculated to be 97 kb based on the examination of 174 randomly selected clones, indicating that the giant panda library contained 6.8-fold genome equivalents. Screening of the library with 16 giant panda PCR primer pairs revealed 6.4 positive clones per locus, in good agreement with an expected 6.8-fold genomic coverage of the library. Based on this BAC library, we constructed a contig map of the giant panda MHC class II region from BTNL2 to DAXX spanning about 650 kb by a three-step method: (1 PCR-based screening of the BAC library with primers from homologous MHC class II gene loci, end sequences and BAC clone shotgun sequences, (2 DNA sequencing validation of positive clones, and (3 restriction digest fingerprinting verification of inter-clone overlapping. Conclusion The identifications of genes and genomic regions of interest are greatly favored by the availability of this giant panda BAC library. The giant panda BAC library thus provides a useful platform for physical mapping, genome sequencing or complex analysis of targeted genomic regions. The 650 kb sequence-ready BAC contig map of the giant panda MHC class II region from BTNL2 to DAXX, verified by the three-step method, offers a

Regulation of HTLV-1 Tax Stability, Cellular Trafficking and NF-κB Activation by the Ubiquitin-Proteasome Pathway

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Lavorgna, Alfonso; Harhaj, Edward William

2014-01-01

Human T-cell leukemia virus type 1 (HTLV-1) is a complex retrovirus that infects CD4+ T cells and causes adult T-cell leukemia/lymphoma (ATLL) in 3%–5% of infected individuals after a long latent period. HTLV-1 Tax is a trans-activating protein that regulates viral gene expression and also modulates cellular signaling pathways to enhance T-cell proliferation and cell survival. The Tax oncoprotein promotes T-cell transformation, in part via constitutive activation of the NF-κB transcription factor; however, the underlying mechanisms remain unknown. Ubiquitination is a type of post-translational modification that occurs in a three-step enzymatic cascade mediated by E1, E2 and E3 enzymes and regulates protein stability as well as signal transduction, protein trafficking and the DNA damage response. Emerging studies indicate that Tax hijacks the ubiquitin machinery to activate ubiquitin-dependent kinases and downstream NF-κB signaling. Tax interacts with the E2 conjugating enzyme Ubc13 and is conjugated on C-terminal lysine residues with lysine 63-linked polyubiquitin chains. Tax K63-linked polyubiquitination may serve as a platform for signaling complexes since this modification is critical for interactions with NEMO and IKK. In addition to NF-κB signaling, mono- and polyubiquitination of Tax also regulate its subcellular trafficking and stability. Here, we review recent advances in the diverse roles of ubiquitin in Tax function and how Tax usurps the ubiquitin-proteasome pathway to promote oncogenesis. PMID:25341660

Preparation of ubiquitin-conjugated proteins using an insect cell-free protein synthesis system.

Science.gov (United States)

Suzuki, Takashi; Ezure, Toru; Ando, Eiji; Nishimura, Osamu; Utsumi, Toshihiko; Tsunasawa, Susumu

2010-01-01

Ubiquitination is one of the most significant posttranslational modifications (PTMs). To evaluate the ability of an insect cell-free protein synthesis system to carry out ubiquitin (Ub) conjugation to in vitro translated proteins, poly-Ub chain formation was studied in an insect cell-free protein synthesis system. Poly-Ub was generated in the presence of Ub aldehyde (UA), a de-ubiquitinating enzyme inhibitor. In vitro ubiquitination of the p53 tumor suppressor protein was also analyzed, and p53 was poly-ubiquitinated when Ub, UA, and Mdm2, an E3 Ub ligase (E3) for p53, were added to the in vitro reaction mixture. These results suggest that the insect cell-free protein synthesis system contains enzymatic activities capable of carrying out ubiquitination. CBB-detectable ubiquitinated p53 was easily purified from the insect cell-free protein synthesis system, allowing analysis of the Ub-conjugated proteins by mass spectrometry (MS). Lys 305 of p53 was identified as one of the Ub acceptor sites using this strategy. Thus, we conclude that the insect cell-free protein synthesis system is a powerful tool for studying various PTMs of eukaryotic proteins including ubiqutination presented here.

Structural changes induced by L50P and I61T single mutations of ubiquitin affect cell cycle progression while impairing its regulatory and degradative functions in Saccharomyces cerevisiae.

Science.gov (United States)

Doshi, Ankita; Sharma, Mrinal; Prabha, C Ratna

2017-06-01

Posttranslational conjugation of ubiquitin to proteins either regulates their function directly or concentration through ubiquitination dependent degradation. High degree of conservation of ubiquitin's sequence implies structural and functional importance of the conserved residues. Ubiquitin gene of Saccharomyces cerevisiae was evolved in vitro by us to study the significance of conserved residues. Present study investigates the structural changes in the protein resulting from the single mutations UbS20F, UbA46S, UbL50P, UbI61T and their functional consequences in the SUB60 strain of S. cerevisiae. Expression of UbL50P and UbI61T decreased Cdc28 protein kinase, enhanced Fus3 levels, caused dosage dependent lethality and at sublethal level produced drastic effects on stress tolerance, protein sorting, protein degradation by ubiquitin fusion degradation pathway and by lysosomes. UbS20F and UbA46S produced insignificant effects over the cells. All four mutations of ubiquitin were incorporated into polyubiquitin. However, polyubiquitination with K63 linkage decreased significantly in cells expressing UbL50P and UbI61T. Structural studies on UbL50P and UbI61T revealed distorted structure with greatly reduced α-helical and elevated β-sheet contents, while UbS20F and UbA46S show mild structural alterations. Our results on functional efficacy of ubiquitin in relation to structural integrity may be useful for designing inhibitors to investigate and modulate eukaryotic cellular dynamics. Copyright © 2017 Elsevier B.V. All rights reserved.

UbSRD: The Ubiquitin Structural Relational Database.

Science.gov (United States)

Harrison, Joseph S; Jacobs, Tim M; Houlihan, Kevin; Van Doorslaer, Koenraad; Kuhlman, Brian

2016-02-22

The structurally defined ubiquitin-like homology fold (UBL) can engage in several unique protein-protein interactions and many of these complexes have been characterized with high-resolution techniques. Using Rosetta's structural classification tools, we have created the Ubiquitin Structural Relational Database (UbSRD), an SQL database of features for all 509 UBL-containing structures in the PDB, allowing users to browse these structures by protein-protein interaction and providing a platform for quantitative analysis of structural features. We used UbSRD to define the recognition features of ubiquitin (UBQ) and SUMO observed in the PDB and the orientation of the UBQ tail while interacting with certain types of proteins. While some of the interaction surfaces on UBQ and SUMO overlap, each molecule has distinct features that aid in molecular discrimination. Additionally, we find that the UBQ tail is malleable and can adopt a variety of conformations upon binding. UbSRD is accessible as an online resource at rosettadesign.med.unc.edu/ubsrd. Copyright © 2015 Elsevier Ltd. All rights reserved.

Molecular and structural insight into lysine selection on substrate and ubiquitin lysine 48 by the ubiquitin-conjugating enzyme Cdc34

DEFF Research Database (Denmark)

Suryadinata, Randy; Holien, Jessica K; Yang, George

2013-01-01

, the mechanisms of lysine selection are not clearly understood. The positioning of lysine(s) toward the E2/E3 active site and residues proximal to lysines are critical in their selection. We investigated determinants of lysine specificity of the ubiquitin-conjugating enzyme Cdc34, toward substrate and Ub lysines....... Evaluation of the relative importance of different residues positioned -2, -1, +1 and +2 toward ubiquitination of its substrate, Sic1, on lysine 50 showed that charged residues in the -1 and -2 positions negatively impact on ubiquitination. Modeling suggests that charged residues at these positions alter...... the native salt-bridge interactions in Ub and Cdc34, resulting in misplacement of Sic1 lysine 50 in the Cdc34 catalytic cleft. During polyubiquitination, Cdc34 showed a strong preference for Ub lysine 48 (K48), with lower activity towards lysine 11 (K11) and lysine 63 (K63). Mutating the -2, -1, +1 and +2...

The Prader-Willi syndrome proteins MAGEL2 and necdin regulate leptin receptor cell surface abundance through ubiquitination pathways.

Science.gov (United States)

Wijesuriya, Tishani Methsala; De Ceuninck, Leentje; Masschaele, Delphine; Sanderson, Matthea R; Carias, Karin Vanessa; Tavernier, Jan; Wevrick, Rachel

2017-11-01

In Prader-Willi syndrome (PWS), obesity is caused by the disruption of appetite-controlling pathways in the brain. Two PWS candidate genes encode MAGEL2 and necdin, related melanoma antigen proteins that assemble into ubiquitination complexes. Mice lacking Magel2 are obese and lack leptin sensitivity in hypothalamic pro-opiomelanocortin neurons, suggesting dysregulation of leptin receptor (LepR) activity. Hypothalamus from Magel2-null mice had less LepR and altered levels of ubiquitin pathway proteins that regulate LepR processing (Rnf41, Usp8, and Stam1). MAGEL2 increased the cell surface abundance of LepR and decreased their degradation. LepR interacts with necdin, which interacts with MAGEL2, which complexes with RNF41 and USP8. Mutations in the MAGE homology domain of MAGEL2 suppress RNF41 stabilization and prevent the MAGEL2-mediated increase of cell surface LepR. Thus, MAGEL2 and necdin together control LepR sorting and degradation through a dynamic ubiquitin-dependent pathway. Loss of MAGEL2 and necdin may uncouple LepR from ubiquitination pathways, providing a cellular mechanism for obesity in PWS. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

E1AF degradation by a ubiquitin-proteasome pathway

International Nuclear Information System (INIS)

Takahashi, Akiko; Higashino, Fumihiro; Aoyagi, Mariko; Yoshida, Koichi; Itoh, Miyuki; Kobayashi, Masanobu; Totsuka, Yasunori; Kohgo, Takao; Shindoh, Masanobu

2005-01-01

E1AF is a member of the ETS family of transcription factors. In mammary tumors, overexpression of E1AF is associated with tumorigenesis, but E1AF protein has hardly been detected and its degradation mechanism is not yet clear. Here we show that E1AF protein is stabilized by treatment with the 26S protease inhibitor MG132. We found that E1AF was modified by ubiquitin through the C-terminal region and ubiquitinated E1AF aggregated in nuclear dots, and that the inhibition of proteasome-activated transcription from E1AF target promoters. These results suggest that E1AF is degraded via the ubiquitin-proteasome pathway, which has some effect on E1AF function

Brief review of the chicken Major Histocompatibility Complex: the genes, their distribution on chromosome 16, and their contributions to disease resistance

Science.gov (United States)

Miller, Marcia M.; Taylor, Robert L.

2016-01-01

Nearly all genes presently mapped to chicken chromosome 16 (GGA 16) have either a demonstrated role in immune responses or are considered to serve in immunity by reason of sequence homology with immune system genes defined in other species. The genes are best described in regional units. Among these, the best known is the polymorphic major histocompatibility complex-B (MHC-B) region containing genes for classical peptide antigen presentation. Nearby MHC-B is a small region containing two CD1 genes, which encode molecules known to bind lipid antigens and which will likely be found in chickens to present lipids to specialized T cells, as occurs with CD1 molecules in other species. Another region is the MHC-Y region, separated from MHC-B by an intervening region of tandem repeats. Like MHC-B, MHC-Y is polymorphic. It contains specialized class I and class II genes and c-type lectin-like genes. Yet another region, separated from MHC-Y by the single nucleolar organizing region (NOR) in the chicken genome, contains olfactory receptor genes and scavenger receptor genes, which are also thought to contribute to immunity. The structure, distribution, linkages and patterns of polymorphism in these regions, suggest GGA 16 evolves as a microchromosome devoted to immune defense. Many GGA 16 genes are polymorphic and polygenic. At the moment most disease associations are at the haplotype level. Roles of individual MHC genes in disease resistance are documented in only a very few instances. Provided suitable experimental stocks persist, the availability of increasingly detailed maps of GGA 16 genes combined with new means for detecting genetic variability will lead to investigations defining the contributions of individual loci and more applications for immunogenetics in breeding healthy poultry. PMID:26740135

A small-molecule inhibitor of the ubiquitin activating enzyme for cancer treatment.

Science.gov (United States)

Hyer, Marc L; Milhollen, Michael A; Ciavarri, Jeff; Fleming, Paul; Traore, Tary; Sappal, Darshan; Huck, Jessica; Shi, Judy; Gavin, James; Brownell, Jim; Yang, Yu; Stringer, Bradley; Griffin, Robert; Bruzzese, Frank; Soucy, Teresa; Duffy, Jennifer; Rabino, Claudia; Riceberg, Jessica; Hoar, Kara; Lublinsky, Anya; Menon, Saurabh; Sintchak, Michael; Bump, Nancy; Pulukuri, Sai M; Langston, Steve; Tirrell, Stephen; Kuranda, Mike; Veiby, Petter; Newcomb, John; Li, Ping; Wu, Jing Tao; Powe, Josh; Dick, Lawrence R; Greenspan, Paul; Galvin, Katherine; Manfredi, Mark; Claiborne, Chris; Amidon, Benjamin S; Bence, Neil F

2018-02-01

The ubiquitin-proteasome system (UPS) comprises a network of enzymes that is responsible for maintaining cellular protein homeostasis. The therapeutic potential of this pathway has been validated by the clinical successes of a number of UPS modulators, including proteasome inhibitors and immunomodulatory imide drugs (IMiDs). Here we identified TAK-243 (formerly known as MLN7243) as a potent, mechanism-based small-molecule inhibitor of the ubiquitin activating enzyme (UAE), the primary mammalian E1 enzyme that regulates the ubiquitin conjugation cascade. TAK-243 treatment caused depletion of cellular ubiquitin conjugates, resulting in disruption of signaling events, induction of proteotoxic stress, and impairment of cell cycle progression and DNA damage repair pathways. TAK-243 treatment caused death of cancer cells and, in primary human xenograft studies, demonstrated antitumor activity at tolerated doses. Due to its specificity and potency, TAK-243 allows for interrogation of ubiquitin biology and for assessment of UAE inhibition as a new approach for cancer treatment.

The interaction of beta 2-microglobulin (beta 2m) with mouse class I major histocompatibility antigens and its ability to support peptide binding. A comparison of human and mouse beta 2m

DEFF Research Database (Denmark)

Pedersen, L O; Stryhn, A; Holter, T L

1995-01-01

of class I molecules are involved in peptide binding, whereas most of class I molecules are involved in beta 2m binding. We propose that mouse beta 2m interacts with the minor peptide binding (i.e. the "empty") fraction with a lower affinity than human beta 2m does, whereas mouse and human beta 2m interact......The function of major histocompatibility complex (MHC) class I molecules is to sample peptides derived from intracellular proteins and to present these peptides to CD8+ cytotoxic T lymphocytes. In this paper, biochemical assays addressing MHC class I binding of both peptide and beta 2-microglobulin...... (beta 2m) have been used to examine the assembly of the trimolecular MHC class I/beta 2m/peptide complex. Recombinant human beta 2m and mouse beta 2ma have been generated to compare the binding of the two beta 2m to mouse class I. It is frequently assumed that human beta 2m binds to mouse class I heavy...

Ubiquitination is absolutely required for the degradation of hypoxia-inducible factor--1 alpha protein in hypoxic conditions.

Science.gov (United States)

Wang, Ronghai; Zhang, Ping; Li, Jinhang; Guan, Hongzai; Shi, Guangjun

2016-01-29

The hypoxia-inducible factor (HIF) is recognized as the master regulator of hypoxia response. HIF-α subunits expression are tightly regulated. In this study, our data show that ts20 cells still expressed detectable E1 protein even at 39.5° C for 12 h, and complete depletion of E1 protein expression at 39.5° C by siRNA enhanced HIF-1α and P53 protein expression. Further inhibition of E1 at 39.5 °C by siRNA, or E1 inhibitor Ube1-41 completely blocked HIF-1α degradation. Moreover, immunoprecipitations of co-transfection of HA-ubiquitin and FLAG-HIF-1α plasmids directly confirmed the involvement of ubiquitin in the hypoxic degradation of HIF-1α. Additionally, hypoxic HIF-1 α degradation is independent of HAF, RACK1, sumoylation or nuclear/cytoplasmic localization. Taken together, our data suggest that constitutive HIF-1α protein degradation in hypoxia is absolutely ubiquitination-dependent, and unidentified E3 ligase may exist for this degradation pathway. Copyright © 2016 Elsevier Inc. All rights reserved.

Associative link of clinical manifestations of the secondary syphilis of skin and mucosa with histocompatibility antigens Class I

Directory of Open Access Journals (Sweden)

S. V. Koshkin

2017-01-01

Full Text Available Sixty patients with different clinical symptoms of secondary syphilis (ulcer chancres, pustular syphilis, hypertrophic papules, widespread leukoderma and alopecia were examined in order to study the distribution pattern of histocompatibility antigens of the first class in patients with secondary syphilis of the skin and mucous membranes. As a result of the study, the presence of an associative relationship between the distribution pattern of histocompatibility antigens of the first class and various clinical manifestations in patients with secondary syphilis was established.

VEGFR2 Trafficking, Signaling and Proteolysis is Regulated by the Ubiquitin Isopeptidase USP8.

Science.gov (United States)

Smith, Gina A; Fearnley, Gareth W; Abdul-Zani, Izma; Wheatcroft, Stephen B; Tomlinson, Darren C; Harrison, Michael A; Ponnambalam, Sreenivasan

2016-01-01

Vascular endothelial growth factor A (VEGF-A) regulates many aspects of vascular function. VEGF-A binding to vascular endothelial growth factor receptor 2 (VEGFR2) stimulates endothelial signal transduction and regulates multiple cellular responses. Activated VEGFR2 undergoes ubiquitination but the enzymes that regulate this post-translational modification are unclear. In this study, the de-ubiquitinating enzyme, USP8, is shown to regulate VEGFR2 trafficking, de-ubiquitination, proteolysis and signal transduction. USP8-depleted endothelial cells displayed altered VEGFR2 ubiquitination and production of a unique VEGFR2 extracellular domain proteolytic fragment caused by VEGFR2 accumulation in the endosome-lysosome system. In addition, perturbed VEGFR2 trafficking impaired VEGF-A-stimulated signal transduction in USP8-depleted cells. Thus, regulation of VEGFR2 ubiquitination and de-ubiquitination has important consequences for the endothelial cell response and vascular physiology. © 2015 The Authors. Traffic published by John Wiley & Sons Ltd.

The major genetic determinants of HIV-1 control affect HLA class I peptide presentation

NARCIS (Netherlands)

Pereyra, Florencia; Jia, Xiaoming; McLaren, Paul J.; Telenti, Amalio; de Bakker, Paul I. W.; Walker, Bruce D.; Ripke, Stephan; Brumme, Chanson J.; Pulit, Sara L.; Carrington, Mary; Kadie, Carl M.; Carlson, Jonathan M.; Heckerman, David; Graham, Robert R.; Plenge, Robert M.; Deeks, Steven G.; Gianniny, Lauren; Crawford, Gabriel; Sullivan, Jordan; Gonzalez, Elena; Davies, Leela; Camargo, Amy; Moore, Jamie M.; Beattie, Nicole; Gupta, Supriya; Crenshaw, Andrew; Burtt, Noël P.; Guiducci, Candace; Gupta, Namrata; Gao, Xiaojiang; Qi, Ying; Yuki, Yuko; Piechocka-Trocha, Alicja; Cutrell, Emily; Rosenberg, Rachel; Moss, Kristin L.; Lemay, Paul; O'Leary, Jessica; Schaefer, Todd; Verma, Pranshu; Toth, Ildiko; Block, Brian; Baker, Brett; Rothchild, Alissa; Lian, Jeffrey; Proudfoot, Jacqueline; Alvino, Donna Marie L.; Vine, Seanna; Addo, Marylyn M.; Allen, Todd M.; Altfeld, Marcus; Henn, Matthew R.; Le Gall, Sylvie; Streeck, Hendrik; Haas, David W.; Kuritzkes, Daniel R.; Robbins, Gregory K.; Shafer, Robert W.; Gulick, Roy M.; Shikuma, Cecilia M.; Haubrich, Richard; Riddler, Sharon; Sax, Paul E.; Daar, Eric S.; Ribaudo, Heather J.; Agan, Brian; Agarwal, Shanu; Ahern, Richard L.; Allen, Brady L.; Altidor, Sherly; Altschuler, Eric L.; Ambardar, Sujata; Anastos, Kathryn; Anderson, Ben; Anderson, Val; Andrady, Ushan; Antoniskis, Diana; Bangsberg, David; Barbaro, Daniel; Barrie, William; Bartczak, J.; Barton, Simon; Basden, Patricia; Basgoz, Nesli; Bazner, Suzane; Bellos, Nicholaos C.; Benson, Anne M.; Berger, Judith; Bernard, Nicole F.; Bernard, Annette M.; Birch, Christopher; Bodner, Stanley J.; Bolan, Robert K.; Boudreaux, Emilie T.; Bradley, Meg; Braun, James F.; Brndjar, Jon E.; Brown, Stephen J.; Brown, Katherine; Brown, Sheldon T.; Burack, Jedidiah; Bush, Larry M.; Cafaro, Virginia; Campbell, Omobolaji; Campbell, John; Carlson, Robert H.; Carmichael, J. Kevin; Casey, Kathleen K.; Cavacuiti, Chris; Celestin, Gregory; Chambers, Steven T.; Chez, Nancy; Chirch, Lisa M.; Cimoch, Paul J.; Cohen, Daniel; Cohn, Lillian E.; Conway, Brian; Cooper, David A.; Cornelson, Brian; Cox, David T.; Cristofano, Michael V.; Cuchural, George; Czartoski, Julie L.; Dahman, Joseph M.; Daly, Jennifer S.; Davis, Benjamin T.; Davis, Kristine; Davod, Sheila M.; DeJesus, Edwin; Dietz, Craig A.; Dunham, Eleanor; Dunn, Michael E.; Ellerin, Todd B.; Eron, Joseph J.; Fangman, John J. W.; Farel, Claire E.; Ferlazzo, Helen; Fidler, Sarah; Fleenor-Ford, Anita; Frankel, Renee; Freedberg, Kenneth A.; French, Neel K.; Fuchs, Jonathan D.; Fuller, Jon D.; Gaberman, Jonna; Gallant, Joel E.; Gandhi, Rajesh T.; Garcia, Efrain; Garmon, Donald; Gathe, Joseph C.; Gaultier, Cyril R.; Gebre, Wondwoosen; Gilman, Frank D.; Gilson, Ian; Goepfert, Paul A.; Gottlieb, Michael S.; Goulston, Claudia; Groger, Richard K.; Gurley, T. Douglas; Haber, Stuart; Hardwicke, Robin; Hardy, W. David; Harrigan, P. Richard; Hawkins, Trevor N.; Heath, Sonya; Hecht, Frederick M.; Henry, W. Keith; Hladek, Melissa; Hoffman, Robert P.; Horton, James M.; Hsu, Ricky K.; Huhn, Gregory D.; Hunt, Peter; Hupert, Mark J.; Illeman, Mark L.; Jaeger, Hans; Jellinger, Robert M.; John, Mina; Johnson, Jennifer A.; Johnson, Kristin L.; Johnson, Heather; Johnson, Kay; Joly, Jennifer; Jordan, Wilbert C.; Kauffman, Carol A.; Khanlou, Homayoon; Killian, Robert K.; Kim, Arthur Y.; Kim, David D.; Kinder, Clifford A.; Kirchner, Jeffrey T.; Kogelman, Laura; Kojic, Erna Milunka; Korthuis, P. Todd; Kurisu, Wayne; Kwon, Douglas S.; LaMar, Melissa; Lampiris, Harry; Lanzafame, Massimiliano; Lederman, Michael M.; Lee, David M.; Lee, Jean M. L.; Lee, Marah J.; Lee, Edward T. Y.; Lemoine, Janice; Levy, Jay A.; Llibre, Josep M.; Liguori, Michael A.; Little, Susan J.; Liu, Anne Y.; Lopez, Alvaro J.; Loutfy, Mono R.; Loy, Dawn; Mohammed, Debbie Y.; Man, Alan; Mansour, Michael K.; Marconi, Vincent C.; Markowitz, Martin; Marques, Rui; Martin, Jeffrey N.; Martin, Harold L.; Mayer, Kenneth Hugh; McElrath, M. Juliana; McGhee, Theresa A.; McGovern, Barbara H.; McGowan, Katherine; McIntyre, Dawn; Mcleod, Gavin X.; Menezes, Prema; Mesa, Greg; Metroka, Craig E.; Meyer-Olson, Dirk; Miller, Andy O.; Montgomery, Kate; Mounzer, Karam C.; Nagami, Ellen H.; Nagin, Iris; Nahass, Ronald G.; Nelson, Margret O.; Nielsen, Craig; Norene, David L.; O'Connor, David H.; Ojikutu, Bisola O.; Okulicz, Jason; Oladehin, Olakunle O.; Oldfield, Edward C.; Olender, Susan A.; Ostrowski, Mario; Owen, William F.; Pae, Eunice; Parsonnet, Jeffrey; Pavlatos, Andrew M.; Perlmutter, Aaron M.; Pierce, Michael N.; Pincus, Jonathan M.; Pisani, Leandro; Price, Lawrence Jay; Proia, Laurie; Prokesch, Richard C.; Pujet, Heather Calderon; Ramgopal, Moti; Rathod, Almas; Rausch, Michael; Ravishankar, J.; Rhame, Frank S.; Richards, Constance Shamuyarira; Richman, Douglas D.; Rodes, Berta; Rodriguez, Milagros; Rose, Richard C.; Rosenberg, Eric S.; Rosenthal, Daniel; Ross, Polly E.; Rubin, David S.; Rumbaugh, Elease; Saenz, Luis; Salvaggio, Michelle R.; Sanchez, William C.; Sanjana, Veeraf M.; Santiago, Steven; Schmidt, Wolfgang; Schuitemaker, Hanneke; Sestak, Philip M.; Shalit, Peter; Shay, William; Shirvani, Vivian N.; Silebi, Vanessa I.; Sizemore, James M.; Skolnik, Paul R.; Sokol-Anderson, Marcia; Sosman, James M.; Stabile, Paul; Stapleton, Jack T.; Starrett, Sheree; Stein, Francine; Stellbrink, Hans-Jurgen; Sterman, F. Lisa; Stone, Valerie E.; Stone, David R.; Tambussi, Giuseppe; Taplitz, Randy A.; Tedaldi, Ellen M.; Theisen, William; Torres, Richard; Tosiello, Lorraine; Tremblay, Cecile; Tribble, Marc A.; Trinh, Phuong D.; Tsao, Alice; Ueda, Peggy; Vaccaro, Anthony; Valadas, Emilia; Vanig, Thanes J.; Vecino, Isabel; Vega, Vilma M.; Veikley, Wenoah; Wade, Barbara H.; Walworth, Charles; Wanidworanun, Chingchai; Ward, Douglas J.; Warner, Daniel A.; Weber, Robert D.; Webster, Duncan; Weis, Steve; Wheeler, David A.; White, David J.; Wilkins, Ed; Winston, Alan; Wlodaver, Clifford G.; van't Wout, Angelique; Wright, David P.; Yang, Otto O.; Yurdin, David L.; Zabukovic, Brandon W.; Zachary, Kimon C.; Zeeman, Beth; Zhao, Meng

2010-01-01

Infectious and inflammatory diseases have repeatedly shown strong genetic associations within the major histocompatibility complex (MHC); however, the basis for these associations remains elusive. To define host genetic effects on the outcome of a chronic viral infection, we performed genome-wide

Residues 240-250 in the C-terminus of the Pirh2 protein complement the function of the RING domain in self-ubiquitination of the Pirh2 protein.

Directory of Open Access Journals (Sweden)

Rami Abou Zeinab

Full Text Available Pirh2 is a p53 inducible gene that encodes a RING-H2 domain and is proposed to be a main regulator of p53 protein, thus fine tuning the DNA damage response. Pirh2 interacts physically with p53 and promotes its MDM2-independent ubiquitination and subsequent degradation as well as participates in an auto-regulatory feedback loop that controls p53 function. Pirh2 also self-ubiquitinates. Interestingly, Pirh2 is overexpressed in a wide range of human tumors. In this study, we investigated the domains and residues essential for Pirh2 self-ubiquitination. Deletions were made in each of the three major domains of Pirh2: the N-terminal domain (NTD, Ring domain (RING, and C-terminal domain (CTD. The effects of these deletions on Pirh2 self-ubiquitination were then assessed using in vitro ubiquitination assays. Our results demonstrate that the RING domain is essential, but not sufficient, for Pirh2 self-ubiquitination and that residues 240-250 of the C-terminal domain are also essential. Our results demonstrate that Pirh2 mediated p53 polyubiquitination occurs mainly through the K48 residue of ubiquitin in vitro. Our data further our understanding of the mechanism of Pirh2 self-ubiquitination and may help identify valuable therapeutic targets that play roles in reducing the effects of the overexpression of Pirh2, thus maximizing p53's response to DNA damage.

Expression of bovine non-classical major histocompatibility complex class I proteins in mouse P815 and human K562 cells.

Science.gov (United States)

Parasar, Parveen; Wilhelm, Amanda; Rutigliano, Heloisa M; Thomas, Aaron J; Teng, Lihong; Shi, Bi; Davis, William C; Suarez, Carlos E; New, Daniel D; White, Kenneth L; Davies, Christopher J

2016-08-01

Major histocompatibility complex class I (MHC-I) proteins can be expressed as cell surface or secreted proteins. To investigate whether bovine non-classical MHC-I proteins are expressed as cell surface or secreted proteins, and to assess the reactivity pattern of monoclonal antibodies with non-classical MHC-I isoforms, we expressed the MHC proteins in murine P815 and human K562 (MHC-I deficient) cells. Following antibiotic selection, stably transfected cell lines were stained with H1A or W6/32 antibodies to detect expression of the MHC-I proteins by flow cytometry. Two non-classical proteins (BoLA-NC1*00501 and BoLA-NC3*00101) were expressed on the cell surface in both cell lines. Surprisingly, the BoLA-NC4*00201 protein was expressed on the cell membrane of human K562 but not mouse P815 cells. Two non-classical proteins (BoLA-NC1*00401, which lacks a transmembrane domain, and BoLA-NC2*00102) did not exhibit cell surface expression. Nevertheless, Western blot analyses demonstrated expression of the MHC-I heavy chain in all transfected cell lines. Ammonium-sulfate precipitation of proteins from culture supernatants showed that BoLA-NC1*00401 was secreted and that all surface expressed proteins where shed from the cell membrane by the transfected cells. Interestingly, the surface expressed MHC-I proteins were present in culture supernatants at a much higher concentration than BoLA-NC1*00401. This comprehensive study shows that bovine non-classical MHC-I proteins BoLA-NC1*00501, BoLA-NC3*00101, and BoLA-NC4*00201 are expressed as surface isoforms with the latter reaching the cell membrane only in K562 cells. Furthermore, it demonstrated that BoLA-NC1*00401 is a secreted isoform and that significant quantities of membrane associated MHC-I proteins can be shed from the cell membrane. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

The impact of sex-role reversal on the diversity of the major histocompatibility complex: Insights from the seahorse (Hippocampus abdominalis

Directory of Open Access Journals (Sweden)

Wilson Anthony B

2011-05-01

Full Text Available Abstract Background Both natural and sexual selection are thought to influence genetic diversity, but the study of the relative importance of these two factors on ecologically-relevant traits has traditionally focused on species with conventional sex-roles, with male-male competition and female-based mate choice. With its high variability and significance in both immune function and olfactory-mediated mate choice, the major histocompatibility complex (MHC/MH is an ideal system in which to evaluate the relative contributions of these two selective forces to genetic diversity. Intrasexual competition and mate choice are both reversed in sex-role reversed species, and sex-related differences in the detection and use of MH-odor cues are expected to influence the intensity of sexual selection in such species. The seahorse, Hippocampus abdominalis, has an exceptionally highly developed form of male parental care, with female-female competition and male mate choice. Results Here, we demonstrate that the sex-role reversed seahorse has a single MH class II beta-chain gene and that the diversity of the seahorse MHIIβ locus and its pattern of variation are comparable to those detected in species with conventional sex roles. Despite the presence of only a single gene copy, intralocus MHIIβ allelic diversity in this species exceeds that observed in species with multiple copies of this locus. The MHIIβ locus of the seahorse exhibits a novel expression domain in the male brood pouch. Conclusions The high variation found at the seahorse MHIIβ gene indicates that sex-role reversed species are capable of maintaining the high MHC diversity typical in most vertebrates. Whether such species have evolved the capacity to use MH-odor cues during mate choice is presently being investigated using mate choice experiments. If this possibility can be rejected, such systems would offer an exceptional opportunity to study the effects of natural selection in isolation

The impact of sex-role reversal on the diversity of the major histocompatibility complex: insights from the seahorse (Hippocampus abdominalis).

Science.gov (United States)

Bahr, Angela; Wilson, Anthony B

2011-05-10

Both natural and sexual selection are thought to influence genetic diversity, but the study of the relative importance of these two factors on ecologically-relevant traits has traditionally focused on species with conventional sex-roles, with male-male competition and female-based mate choice. With its high variability and significance in both immune function and olfactory-mediated mate choice, the major histocompatibility complex (MHC/MH) is an ideal system in which to evaluate the relative contributions of these two selective forces to genetic diversity. Intrasexual competition and mate choice are both reversed in sex-role reversed species, and sex-related differences in the detection and use of MH-odor cues are expected to influence the intensity of sexual selection in such species. The seahorse, Hippocampus abdominalis, has an exceptionally highly developed form of male parental care, with female-female competition and male mate choice. Here, we demonstrate that the sex-role reversed seahorse has a single MH class II beta-chain gene and that the diversity of the seahorse MHIIβ locus and its pattern of variation are comparable to those detected in species with conventional sex roles. Despite the presence of only a single gene copy, intralocus MHIIβ allelic diversity in this species exceeds that observed in species with multiple copies of this locus. The MHIIβ locus of the seahorse exhibits a novel expression domain in the male brood pouch. The high variation found at the seahorse MHIIβ gene indicates that sex-role reversed species are capable of maintaining the high MHC diversity typical in most vertebrates.Whether such species have evolved the capacity to use MH-odor cues during mate choice is presently being investigated using mate choice experiments. If this possibility can be rejected, such systems would offer an exceptional opportunity to study the effects of natural selection in isolation, providing powerful comparative models for understanding the

Ubiquitin-specific Protease 11 (USP11) Deubiquitinates Hybrid Small Ubiquitin-like Modifier (SUMO)-Ubiquitin Chains to Counteract RING Finger Protein 4 (RNF4)

DEFF Research Database (Denmark)

Hendriks, Ivo A; Schimmel, Joost; Eifler, Karolin

2015-01-01

of RNF4 as a counterbalancing factor. In response to DNA damage induced by methyl methanesulfonate, USP11 could counteract RNF4 to inhibit the dissolution of nuclear bodies. Thus, we provide novel insight into cross-talk between ubiquitin and SUMO and uncover USP11 and RNF4 as a balanced SUMO...

Lys48 ubiquitination during the intraerythrocytic cycle of the rodent malaria parasite, Plasmodium chabaudi.

Science.gov (United States)

González-López, Lorena; Carballar-Lejarazú, Rebeca; Arrevillaga Boni, Gerardo; Cortés-Martínez, Leticia; Cázares-Raga, Febe Elena; Trujillo-Ocampo, Abel; Rodríguez, Mario H; James, Anthony A; Hernández-Hernández, Fidel de la Cruz

2017-01-01

Ubiquitination tags proteins for different functions within the cell. One of the most abundant and studied ubiquitin modification is the Lys48 polyubiquitin chain that modifies proteins for their destruction by proteasome. In Plasmodium is proposed that post-translational regulation is fundamental for parasite development during its complex life-cycle; thus, the objective of this work was to analyze the ubiquitination during Plasmodium chabaudi intraerythrocytic stages. Ubiquitinated proteins were detected during intraerythrocytic stages of Plasmodium chabaudi by immunofluorescent microscopy, bidimensional electrophoresis (2-DE) combined with immunoblotting and mass spectrometry. All the studied stages presented protein ubiquitination and Lys48 polyubiquitination with more abundance during the schizont stage. Three ubiquitinated proteins were identified for rings, five for trophozoites and twenty for schizonts. Only proteins detected with a specific anti- Lys48 polyubiquitin antibody were selected for Mass Spectrometry analysis and two of these identified proteins were selected in order to detect the specific amino acid residues where ubiquitin is placed. Ubiquitinated proteins during the ring and trophozoite stages were related with the invasion process and in schizont proteins were related with nucleic acid metabolism, glycolysis and protein biosynthesis. Most of the ubiquitin detection was during the schizont stage and the Lys48 polyubiquitination during this stage was related to proteins that are expected to be abundant during the trophozoite stage. The evidence that these Lys48 polyubiquitinated proteins are tagged for destruction by the proteasome complex suggests that this type of post-translational modification is important in the regulation of protein abundance during the life-cycle and may also contribute to the parasite cell-cycle progression.

Dietary flavonoids bind to mono-ubiquitinated annexin A1 in nuclei, and inhibit chemical induced mutagenesis

Energy Technology Data Exchange (ETDEWEB)

Hirata, Fusao, E-mail: fhirata@wayne.edu; Harada, Takasuke; Corcoran, George B.; Hirata, Aiko

2014-01-15

Highlight: • Nuclear mono-ubiquitinated annexin A1 is involved in DNA damage induced mutagenesis. • Dietary flavonoids bind to and inhibit purified mono-ubiquitinated annexin A1 helicase. • Dietary flavonoids show anti-mutagenic action. • Annexin A1 may serve as a putative target of cancer chemoprevention by flavonoids. - Abstract: In order to investigate the mechanisms of anti-mutagenic action by dietary flavonoids, we investigated if they inhibit mutation of the thymidine kinase (tk) gene in L5178Ytk(±) lymphoma cells. Silibinin, quercetin and genistein suppressed mutation of the tk gene induced in L5178Ytk(±) lymphoma cells by methyl methanesulfonate (MMS) and As{sup 3+}. Flavone and flavonol were less effective. To establish that mutation of the tk gene in L5178Ytk(±) lymphoma cells by MMS and As{sup 3+} is mediated through mono-ubiquitinated annexin A1, L5178Ytk(±) lymphoma cells were treated with annexin A1 anti-sense oligonucleotide. The treatment reduced mRNA as well as protein levels of annexin A1, and suppressed mutation of the tk gene. Nuclear extracts from L5178Ytk(±) lymphoma cells catalyzed translesion DNA synthesis with an oligonucleotide template containing 8-oxo-guanosine in an annexin A1 dependent manner. This translesion DNA synthesis was inhibited by the anti-mutagenic flavonoids, silibinin, quercetin and genistein, in a concentration dependent manner, but only slightly by flavone and flavonol. Because these observations implicate involvement of annexin A1 in mutagenesis, we examined if flavonoids suppress nuclear annexin A1 helicase activity. Silibinin, quercetin and genistein inhibited ssDNA binding, DNA chain annealing and DNA unwinding activities of purified nuclear mono-ubiquitinated annexin A1. Flavone and flavonol were ineffective. The apparent direct binding of anti-mutagenic flavonoids to the annexin A1 molecule was supported by fluorescence quenching. Taken together, these findings illustrate that nuclear annexin A1 may be

Dietary flavonoids bind to mono-ubiquitinated annexin A1 in nuclei, and inhibit chemical induced mutagenesis

International Nuclear Information System (INIS)

Hirata, Fusao; Harada, Takasuke; Corcoran, George B.; Hirata, Aiko

2014-01-01

Highlight: • Nuclear mono-ubiquitinated annexin A1 is involved in DNA damage induced mutagenesis. • Dietary flavonoids bind to and inhibit purified mono-ubiquitinated annexin A1 helicase. • Dietary flavonoids show anti-mutagenic action. • Annexin A1 may serve as a putative target of cancer chemoprevention by flavonoids. - Abstract: In order to investigate the mechanisms of anti-mutagenic action by dietary flavonoids, we investigated if they inhibit mutation of the thymidine kinase (tk) gene in L5178Ytk(±) lymphoma cells. Silibinin, quercetin and genistein suppressed mutation of the tk gene induced in L5178Ytk(±) lymphoma cells by methyl methanesulfonate (MMS) and As 3+ . Flavone and flavonol were less effective. To establish that mutation of the tk gene in L5178Ytk(±) lymphoma cells by MMS and As 3+ is mediated through mono-ubiquitinated annexin A1, L5178Ytk(±) lymphoma cells were treated with annexin A1 anti-sense oligonucleotide. The treatment reduced mRNA as well as protein levels of annexin A1, and suppressed mutation of the tk gene. Nuclear extracts from L5178Ytk(±) lymphoma cells catalyzed translesion DNA synthesis with an oligonucleotide template containing 8-oxo-guanosine in an annexin A1 dependent manner. This translesion DNA synthesis was inhibited by the anti-mutagenic flavonoids, silibinin, quercetin and genistein, in a concentration dependent manner, but only slightly by flavone and flavonol. Because these observations implicate involvement of annexin A1 in mutagenesis, we examined if flavonoids suppress nuclear annexin A1 helicase activity. Silibinin, quercetin and genistein inhibited ssDNA binding, DNA chain annealing and DNA unwinding activities of purified nuclear mono-ubiquitinated annexin A1. Flavone and flavonol were ineffective. The apparent direct binding of anti-mutagenic flavonoids to the annexin A1 molecule was supported by fluorescence quenching. Taken together, these findings illustrate that nuclear annexin A1 may be a novel

Hyperthermia enhances mapatumumab-induced apoptotic death through ubiquitin-mediated degradation of cellular FLIP(long) in human colon cancer cells.

Science.gov (United States)

Song, X; Kim, S-Y; Zhou, Z; Lagasse, E; Kwon, Y T; Lee, Y J

2013-04-04

Colorectal cancer is the third leading cause of cancer-related mortality in the world; the main cause of death of colorectal cancer is hepatic metastases, which can be treated with hyperthermia using isolated hepatic perfusion (IHP). In this study, we report that mild hyperthermia potently reduced cellular FLIP(long), (c-FLIP(L)), a major regulator of the death receptor (DR) pathway of apoptosis, thereby enhancing humanized anti-DR4 antibody mapatumumab (Mapa)-mediated mitochondria-independent apoptosis. We observed that overexpression of c-FLIP(L) in CX-1 cells abrogated the synergistic effect of Mapa and hyperthermia, whereas silencing of c-FLIP in CX-1 cells enhanced Mapa-induced apoptosis. Hyperthermia altered c-FLIP(L) protein stability without concomitant reductions in FLIP mRNA. Ubiquitination of c-FLIP(L) was increased by hyperthermia, and proteasome inhibitor MG132 prevented heat-induced downregulation of c-FLIP(L). These results suggest the involvement of the ubiquitin-proteasome system in this process. We also found lysine residue 195 (K195) to be essential for c-FLIP(L) ubiquitination and proteolysis, as mutant c-FLIP(L) lysine 195 arginine (arginine replacing lysine) was left virtually un-ubiquitinated and was refractory to hyperthermia-triggered degradation, and thus partially blocked the synergistic effect of Mapa and hyperthermia. Our observations reveal that hyperthermia transiently reduced c-FLIP(L) by proteolysis linked to K195 ubiquitination, which contributed to the synergistic effect between Mapa and hyperthermia. This study supports the application of hyperthermia combined with other regimens to treat colorectal hepatic metastases.

Hydrophobic Collapse of Ubiquitin Generates Rapid Protein-Water Motions.

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Wirtz, Hanna; Schäfer, Sarah; Hoberg, Claudius; Reid, Korey M; Leitner, David M; Havenith, Martina

2018-06-04

We report time-resolved measurements of the coupled protein-water modes of solvated ubiquitin during protein folding. Kinetic terahertz absorption (KITA) spectroscopy serves as a label-free technique for monitoring large scale conformational changes and folding of proteins subsequent to a sudden T-jump. We report here KITA measurements at an unprecedented time resolution of 500 ns, a resolution 2 orders of magnitude better than those of any previous KITA measurements, which reveal the coupled ubiquitin-solvent dynamics even in the initial phase of hydrophobic collapse. Complementary equilibrium experiments and molecular simulations of ubiquitin solutions are performed to clarify non-equilibrium contributions and reveal the molecular picture upon a change in structure, respectively. On the basis of our results, we propose that in the case of ubiquitin a rapid (<500 ns) initial phase of the hydrophobic collapse from the elongated protein to a molten globule structure precedes secondary structure formation. We find that these very first steps, including large-amplitude changes within the unfolded manifold, are accompanied by a rapid (<500 ns) pronounced change of the coupled protein-solvent response. The KITA response upon secondary structure formation exhibits an opposite sign, which indicates a distinct effect on the solvent-exposed surface.

Proinflammatory cytokines downregulate connexin 43-gap junctions via the ubiquitin-proteasome system in rat spinal astrocytes.

Science.gov (United States)

Zhang, Fang Fang; Morioka, Norimitsu; Kitamura, Tomoya; Hisaoka-Nakashima, Kazue; Nakata, Yoshihiro

2015-09-04

Astrocytic gap junctions formed by connexin 43 (Cx43) are crucial for intercellular communication between spinal cord astrocytes. Various neurological disorders are associated with dysfunctional Cx43-gap junctions. However, the mechanism modulating Cx43-gap junctions in spinal astrocytes under pathological conditions is not entirely clear. A previous study showed that treatment of spinal astrocytes in culture with pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) decreased both Cx43 expression and gap junction intercellular communication (GJIC) via a c-jun N-terminal kinase (JNK)-dependent pathway. The current study further elaborates the intracellular mechanism that decreases Cx43 under an inflammatory condition. Cycloheximide chase analysis revealed that TNF-α (10 ng/ml) alone or in combination with IFN-γ (5 ng/ml) accelerated the degradation of Cx43 protein in cultured spinal astrocytes. The reduction of both Cx43 expression and GJIC induced by a mixture of TNF-α and IFN-γ were blocked by pretreatment with proteasome inhibitors MG132 (0.5 μM) and epoxomicin (25 nM), a mixture of TNF-α and IFN-γ significantly increased proteasome activity and Cx43 ubiquitination. In addition, TNF-α and IFN-γ-induced activation of ubiquitin-proteasome systems was prevented by SP600125, a JNK inhibitor. Together, these results indicate that a JNK-dependent ubiquitin-proteasome system is induced under an inflammatory condition that disrupts astrocytic gap junction expression and function, leading to astrocytic dysfunction and the maintenance of the neuroinflammatory state. Copyright © 2015 Elsevier Inc. All rights reserved.

Major histocompatibility complex (MHC) class III genetics in two Amerindian tribes from southern Brazil: the Kaingang and the Guarani.

Science.gov (United States)

Weg-Remers, S; Brenden, M; Schwarz, E; Witzel, K; Schneider, P M; Guerra, L K; Rehfeldt, I R; Lima, M T; Hartmann, D; Petzl-Erler, M L; de Messias, I J; Mauff, G

1997-10-01

Population genetic studies of the major histocompatibility complex (MHC) class III region, comprising C2, BF and C4 phenotypes, and molecular genetic data are rarely available for populations other than Caucasoids. We have investigated three Amerindian populations from Southern Brazil: 131 Kaingang from Ivaí (KIV), 111 Kaingang (KRC) and 100 Guarani (GRC) from Rio das Cobras. Extended MHC haplotypes were derived after standard C2, BF, C4 phenotyping and restriction fragment length polymorphism (RFLP) analysis with TaqI, together with HLA data published previously by segregation analysis. C2 and BF frequencies corresponded to other Amerindian populations. C4B*Q0 frequency was high in the GRC (0.429) but low in the Kaingang. Unusual C4 alleles were found, viz. C4A*58, A*55 and C4B*22 (presumably non-Amerindian) and aberrant C4A*3 of Amerindian origin occurring with a frequency of 0.223 in the GRC. C4A*3 bands of homo- and heterozygous individuals carrying this variant were Rodgers 1 positive and Chido 1,3 positive, showed a C4A specific lysis type and a C4A like alpha-chain. Polymerase chain reaction studies and sequencing showed that this is based on a C4A*3 duplication with a regular C4A*3 and a partially converted C4A*0304 carrying the C4B specific epitopes Ch 6 and Ch 1,3. Associations of class III haplotypes with particular RFLP patterns were similar to those reported for Caucasoids. The previously described association between combined C4A and CYP21P deletions and the 6.4 kb TaqI fragment was not seen in these Amerindians. This fragment occurred within a regular two locus gene structure in the Kaingang, representing a "short" gene at C4 locus I. C4 and CYP21 duplications were frequently observed. The distribution of extended MHC haplotypes provides evidence for a close relationship between the KIV and KRC and a larger genetic distance between the two Kaingang groups and the GRC.

When ubiquitin meets NF-κB: a trove for anti-cancer drug development.

Science.gov (United States)

Wu, Zhao-Hui; Shi, Yuling

2013-01-01

During the last two decades, the studies on ubiquitination in regulating transcription factor NF-κB activation have elucidated the expanding role of ubiquitination in modulating cellular events by non-proteolytic mechanisms, as well as by proteasomal degradation. The significance of ubiquitination has also been recognized in regulating gene transcription, epigenetic modifications, kinase activation, DNA repair and subcellular translocation. This progress has been translated into novel strategies for developing anti-cancer therapeutics, exemplified by the success of the first FDA-approved proteasome inhibitor drug Bortezomib. Here we discuss the current understanding of the ubiquitin-proteasome system and how it is involved in regulating NF-κB signaling pathways in response to a variety of stimuli. We also focus on the recent progress of anti-cancer drug development targeting various steps of ubiquitination process, and the potential of these drugs in cancer treatment as related to their impact on NF-κB activation.

The ubiquitin-proteasome system and autophagy are defective in the taurine-deficient heart.

Science.gov (United States)

Jong, Chian Ju; Ito, Takashi; Schaffer, Stephen W

2015-12-01

Taurine depletion leads to impaired mitochondrial function, as characterized by reduced ATP production and elevated superoxide generation. These defects can fundamentally alter cardiomyocyte function and if left unchanged can result in cell death. To protect against these stresses, cardiomyocytes possess quality control processes, such as the ubiquitin-proteasome system (UPS) and autophagy, which can rejuvenate cells through the degradation of damaged proteins and organelles. Hence, the present study tested the hypothesis that reactive oxygen species generated by damaged mitochondria initiates UPS and autophagy in the taurine-deficient heart. Using transgenic mice lacking the taurine transporter (TauTKO) as a model of taurine deficiency, it was shown that the levels of ubiquitinated protein were elevated, an effect associated with a decrease in ATP-dependent 26S β5 proteasome activity. Treating the TauTKO mouse with the mitochondria-specific antioxidant, mitoTEMPO, largely abolished the increase in ubiquitinated protein content. The TauTKO heart was also associated with impaired autophagy, characterized by an increase in the initiator, Beclin-1, and autophagosome content, but a defect in the generation of active autophagolysosomes. Although mitoTEMPO treatment only restores the oxidative balance within the mitochondria, it appeared to completely disrupt the crosstalk between the damaged mitochondria and the quality control processes. Thus, mitochondrial oxidative stress is the main trigger initiating the quality control systems in the taurine-deficient heart. We conclude that the activation of the UPS and autophagy is another fundamental function of mitochondria.

Fine-tuning the ubiquitin code at DNA double-strand breaks: deubiquitinating enzymes at work

Directory of Open Access Journals (Sweden)

Elisabetta eCitterio

2015-09-01

Full Text Available Ubiquitination is a reversible protein modification broadly implicated in cellular functions. Signaling processes mediated by ubiquitin are crucial for the cellular response to DNA double-strand breaks (DSBs, one of the most dangerous types of DNA lesions. In particular, the DSB response critically relies on active ubiquitination by the RNF8 and RNF168 ubiquitin ligases at the chromatin, which is essential for proper DSB signaling and repair. How this pathway is fine-tuned and what the functional consequences are of its deregulation for genome integrity and tissue homeostasis are subject of intense investigation. One important regulatory mechanism is by reversal of substrate ubiquitination through the activity of specific deubiquitinating enzymes (DUBs, as supported by the implication of a growing number of DUBs in DNA damage response (DDR processes. Here, we discuss the current knowledge of how ubiquitin-mediated signaling at DSBs is controlled by deubiquitinating enzymes, with main focus on DUBs targeting histone H2A and on their recent implication in stem cell biology and cancer.

An ethanolic extract of Artemisia dracunculus L. regulates gene expression of ubiquitin-proteasome system enzymes in skeletal muscle: potential role in the treatment of sarcopenic obesity.

Science.gov (United States)

Kirk-Ballard, Heather; Kilroy, Gail; Day, Britton C; Wang, Zhong Q; Ribnicky, David M; Cefalu, William T; Floyd, Z Elizabeth

2014-01-01

Obesity is linked to insulin resistance, a primary component of metabolic syndrome and type 2 diabetes. The problem of obesity-related insulin resistance is compounded when age-related skeletal muscle loss, called sarcopenia, occurs with obesity. Skeletal muscle loss results from elevated levels of protein degradation and prevention of obesity-related sarcopenic muscle loss will depend on strategies that target pathways involved in protein degradation. An extract from Artemisia dracunculus, termed PMI 5011, improves insulin signaling and increases skeletal muscle myofiber size in a rodent model of obesity-related insulin resistance. The aim of this study was to examine the effect of PMI 5011 on the ubiquitin-proteasome system, a central regulator of muscle protein degradation. Gastrocnemius and vastus lateralis skeletal muscle was obtained from KK-A(y) obese diabetic mice fed a control or 1% (w/w) PMI 5011-supplemented diet. Regulation of genes encoding enzymes of the ubiquitin-proteasome system was determined using real-time quantitative reverse transcriptase polymerase chain reaction. Although MuRF-1 ubiquitin ligase gene expression is consistently down-regulated in skeletal muscle, atrogin-1, Fbxo40, and Traf6 expression is differentially regulated by PMI 5011. Genes encoding other enzymes of the ubiquitin-proteasome system ranging from ubiquitin to ubiquitin-specific proteases are also regulated by PMI 5011. Additionally, expression of the gene encoding the microtubule-associated protein-1 light chain 3 (LC3), a ubiquitin-like protein pivotal to autophagy-mediated protein degradation, is down-regulated by PMI 5011 in the vastus lateralis. PMI 5011 alters the gene expression of ubiquitin-proteasome system enzymes that are essential regulators of skeletal muscle mass. This suggests that PMI 5011 has therapeutic potential in the treatment of obesity-linked sarcopenia by regulating ubiquitin-proteasome-mediated protein degradation. Copyright © 2014 Elsevier Inc

Cellular Cholesterol Regulates Ubiquitination and Degradation of the Cholesterol Export Proteins ABCA1 and ABCG1*

Science.gov (United States)

Hsieh, Victar; Kim, Mi-Jurng; Gelissen, Ingrid C.; Brown, Andrew J.; Sandoval, Cecilia; Hallab, Jeannette C.; Kockx, Maaike; Traini, Mathew; Jessup, Wendy; Kritharides, Leonard

2014-01-01

The objective of this study was to examine the influence of cholesterol in post-translational control of ABCA1 and ABCG1 protein expression. Using CHO cell lines stably expressing human ABCA1 or ABCG1, we observed that the abundance of these proteins is increased by cell cholesterol loading. The response to increased cholesterol is rapid, is independent of transcription, and appears to be specific for these membrane proteins. The effect is mediated through cholesterol-dependent inhibition of transporter protein degradation. Cell cholesterol loading similarly regulates degradation of endogenously expressed ABCA1 and ABCG1 in human THP-1 macrophages. Turnover of ABCA1 and ABCG1 is strongly inhibited by proteasomal inhibitors and is unresponsive to inhibitors of lysosomal proteolysis. Furthermore, cell cholesterol loading inhibits ubiquitination of ABCA1 and ABCG1. Our findings provide evidence for a rapid, cholesterol-dependent, post-translational control of ABCA1 and ABCG1 protein levels, mediated through a specific and sterol-sensitive mechanism for suppression of transporter protein ubiquitination, which in turn decreases proteasomal degradation. This provides a mechanism for acute fine-tuning of cholesterol transporter activity in response to fluctuations in cell cholesterol levels, in addition to the longer term cholesterol-dependent transcriptional regulation of these genes. PMID:24500716

The ubiquitin proteasome system in glia and its role in neurodegenerative diseases

NARCIS (Netherlands)

Jansen, Anne H. P.; Reits, Eric A. J.; Hol, Elly M.

2014-01-01

The ubiquitin proteasome system (UPS) is crucial for intracellular protein homeostasis and for degradation of aberrant and damaged proteins. The accumulation of ubiquitinated proteins is a hallmark of many neurodegenerative diseases, including amyotrophic lateral sclerosis, Alzheimer's, Parkinson's,

Ubiquitin regulates GGA3-mediated degradation of BACE1.

Science.gov (United States)

Kang, Eugene L; Cameron, Andrew N; Piazza, Fabrizio; Walker, Kendall R; Tesco, Giuseppina

2010-07-30

BACE1 (beta-site amyloid precursor protein-cleaving enzyme 1) is a membrane-tethered member of the aspartyl proteases, essential for the production of beta-amyloid, a toxic peptide that accumulates in the brain of subjects affected by Alzheimer disease. The BACE1 C-terminal fragment contains a DXXLL motif that has been shown to bind the VHS (VPS27, Hrs, and STAM) domain of GGA1-3 (Golgi-localized gamma-ear-containing ARF-binding proteins). GGAs are trafficking molecules involved in the transport of proteins containing the DXXLL signal from the Golgi complex to endosomes. Moreover, GGAs bind ubiquitin and traffic synthetic and endosomal ubiquitinated cargoes to lysosomes. We have previously shown that depletion of GGA3 results in increased BACE1 levels and activity because of impaired lysosomal degradation. Here, we report that the accumulation of BACE1 is rescued by the ectopic expression of GGA3 in H4 neuroglioma cells depleted of GGA3. Accordingly, the overexpression of GGA3 reduces the levels of BACE1 and beta-amyloid. We then established that mutations in the GGA3 VPS27, Hrs, and STAM domain (N91A) or in BACE1 di-leucine motif (L499A/L500A), able to abrogate their binding, did not affect the ability of ectopically expressed GGA3 to rescue BACE1 accumulation in cells depleted of GGA3. Instead, we found that BACE1 is ubiquitinated at lysine 501 and is mainly monoubiquitinated and Lys-63-linked polyubiquitinated. Finally, a GGA3 mutant with reduced ability to bind ubiquitin (GGA3L276A) was unable to regulate BACE1 levels both in rescue and overexpression experiments. These findings indicate that levels of GGA3 tightly and inversely regulate BACE1 levels via interaction with ubiquitin sorting machinery.

Ubiquitin Regulates GGA3-mediated Degradation of BACE1*

Science.gov (United States)

Kang, Eugene L.; Cameron, Andrew N.; Piazza, Fabrizio; Walker, Kendall R.; Tesco, Giuseppina

2010-01-01

BACE1 (β-site amyloid precursor protein-cleaving enzyme 1) is a membrane-tethered member of the aspartyl proteases, essential for the production of β-amyloid, a toxic peptide that accumulates in the brain of subjects affected by Alzheimer disease. The BACE1 C-terminal fragment contains a DXXLL motif that has been shown to bind the VHS (VPS27, Hrs, and STAM) domain of GGA1–3 (Golgi-localized γ-ear-containing ARF-binding proteins). GGAs are trafficking molecules involved in the transport of proteins containing the DXXLL signal from the Golgi complex to endosomes. Moreover, GGAs bind ubiquitin and traffic synthetic and endosomal ubiquitinated cargoes to lysosomes. We have previously shown that depletion of GGA3 results in increased BACE1 levels and activity because of impaired lysosomal degradation. Here, we report that the accumulation of BACE1 is rescued by the ectopic expression of GGA3 in H4 neuroglioma cells depleted of GGA3. Accordingly, the overexpression of GGA3 reduces the levels of BACE1 and β-amyloid. We then established that mutations in the GGA3 VPS27, Hrs, and STAM domain (N91A) or in BACE1 di-leucine motif (L499A/L500A), able to abrogate their binding, did not affect the ability of ectopically expressed GGA3 to rescue BACE1 accumulation in cells depleted of GGA3. Instead, we found that BACE1 is ubiquitinated at lysine 501 and is mainly monoubiquitinated and Lys-63-linked polyubiquitinated. Finally, a GGA3 mutant with reduced ability to bind ubiquitin (GGA3L276A) was unable to regulate BACE1 levels both in rescue and overexpression experiments. These findings indicate that levels of GGA3 tightly and inversely regulate BACE1 levels via interaction with ubiquitin sorting machinery. PMID:20484053

Genotyping of major histocompatibility complex Class II DRB gene in Rohilkhandi goats by polymerase chain reaction-restriction fragment length polymorphism and DNA sequencing

Directory of Open Access Journals (Sweden)

Kush Shrivastava

2015-10-01

Full Text Available Aim: To study the major histocompatibility complex (MHC Class II DRB1 gene polymorphism in Rohilkhandi goat using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP and nucleotide sequencing techniques. Materials and Methods: DNA was isolated from 127 Rohilkhandi goats maintained at sheep and goat farm, Indian Veterinary Research Institute, Izatnagar, Bareilly. A 284 bp fragment of exon 2 of DRB1 gene was amplified and digested using BsaI and TaqI restriction enzymes. Population genetic parameters were calculated using Popgene v 1.32 and SAS 9.0. The genotypes were then sequenced using Sanger dideoxy chain termination method and were compared with related breeds/species using MEGA 6.0 and Megalign (DNASTAR software. Results: TaqI locus showed three and BsaI locus showed two genotypes. Both the loci were found to be in Hardy–Weinberg equilibrium (HWE, however, population genetic parameters suggest that heterozygosity is still maintained in the population at both loci. Percent diversity and divergence matrix, as well as phylogenetic analysis revealed that the MHC Class II DRB1 gene of Rohilkhandi goats was found to be in close cluster with Garole and Scottish blackface sheep breeds as compared to other goat breeds included in the sequence comparison. Conclusion: The PCR-RFLP patterns showed population to be in HWE and absence of one genotype at one locus (BsaI, both the loci showed excess of one or the other homozygote genotype, however, effective number of alleles showed that allelic diversity is present in the population. Sequence comparison of DRB1 gene of Rohilkhandi goat with other sheep and goat breed assigned Rohilkhandi goat in divergence with Jamanupari and Angora goats.

The APC/C Ubiquitin Ligase: From Cell Biology to Tumorigenesis

Energy Technology Data Exchange (ETDEWEB)

Penas, Clara; Ramachandran, Vimal [John P. Hussman Institute for Human Genomics, University of Miami Miller School of Medicine, Miami, FL (United States); Ayad, Nagi George, E-mail: nayad@med.miami.edu [John P. Hussman Institute for Human Genomics, University of Miami Miller School of Medicine, Miami, FL (United States); Department of Psychiatry and Behavioral Sciences, University of Miami Miller School of Medicine, Miami, FL (United States)

2012-01-09

The ubiquitin proteasome system (UPS) is required for normal cell proliferation, vertebrate development, and cancer cell transformation. The UPS consists of multiple proteins that work in concert to target a protein for degradation via the 26S proteasome. Chains of an 8.5-kDa protein called ubiquitin are attached to substrates, thus allowing recognition by the 26S proteasome. Enzymes called ubiquitin ligases or E3s mediate specific attachment to substrates. Although there are over 600 different ubiquitin ligases, the Skp1–Cullin–F-box (SCF) complexes and the anaphase promoting complex/cyclosome (APC/C) are the most studied. SCF involvement in cancer has been known for some time while APC/C’s cancer role has recently emerged. In this review we will discuss the importance of APC/C to normal cell proliferation and development, underscoring its possible contribution to transformation. We will also examine the hypothesis that modulating a specific interaction of the APC/C may be therapeutically attractive in specific cancer subtypes. Finally, given that the APC/C pathway is relatively new as a cancer target, therapeutic interventions affecting APC/C activity may be beneficial in cancers that are resistant to classical chemotherapy.

The APC/C Ubiquitin Ligase: From Cell Biology to Tumorigenesis

International Nuclear Information System (INIS)

Penas, Clara; Ramachandran, Vimal; Ayad, Nagi George

2012-01-01

The ubiquitin proteasome system (UPS) is required for normal cell proliferation, vertebrate development, and cancer cell transformation. The UPS consists of multiple proteins that work in concert to target a protein for degradation via the 26S proteasome. Chains of an 8.5-kDa protein called ubiquitin are attached to substrates, thus allowing recognition by the 26S proteasome. Enzymes called ubiquitin ligases or E3s mediate specific attachment to substrates. Although there are over 600 different ubiquitin ligases, the Skp1–Cullin–F-box (SCF) complexes and the anaphase promoting complex/cyclosome (APC/C) are the most studied. SCF involvement in cancer has been known for some time while APC/C’s cancer role has recently emerged. In this review we will discuss the importance of APC/C to normal cell proliferation and development, underscoring its possible contribution to transformation. We will also examine the hypothesis that modulating a specific interaction of the APC/C may be therapeutically attractive in specific cancer subtypes. Finally, given that the APC/C pathway is relatively new as a cancer target, therapeutic interventions affecting APC/C activity may be beneficial in cancers that are resistant to classical chemotherapy.

A central role for ubiquitination within a circadian clock protein modification code

Directory of Open Access Journals (Sweden)

Katarina eStojkovic

2014-08-01

Full Text Available Circadian rhythms, endogenous cycles of about 24 h in physiology, are generated by a master clock located in the suprachiasmatic nucleus of the hypothalamus and other clocks located in the brain and peripheral tissues. Circadian disruption is known to increase the incidence of various illnesses, such as mental disorders, metabolic syndrome and cancer. At the molecular level, periodicity is established by a set of clock genes via autoregulatory translation-transcription feedback loops. This clock mechanism is regulated by post-translational modifications such as phosphorylation and ubiquitination, which set the pace of the clock. Ubiquitination in particular has been found to regulate the stability of core clock components, but also other clock protein functions. Mutation of genes encoding ubiquitin ligases can cause either elongation or shortening of the endogenous circadian period. Recent research has also started to uncover roles for deubiquitination in the molecular clockwork. Here we review the role of the ubiquitin pathway in regulating the circadian clock and we propose that ubiquitination is a key element in a clock protein modification code that orchestrates clock mechanisms and circadian behavior over the daily cycle.

circRNA Mediates Silica-Induced Macrophage Activation Via HECTD1/ZC3H12A-Dependent Ubiquitination

Science.gov (United States)

Zhou, Zewei; Jiang, Rong; Yang, Xiyue; Guo, Huifang; Fang, Shencun; Zhang, Yingming; Cheng, Yusi; Wang, Jing; Yao, Honghong; Chao, Jie

2018-01-01

Rationale: Phagocytosis of silicon dioxide (SiO2) into lung cells causes an inflammatory cascade that results in fibroblast proliferation and migration, followed by fibrosis. Circular RNAs (circRNAs) are a subclass of non-coding RNAs detected within mammalian cells; however, researchers have not determined whether circRNAs are involved in the pathophysiological process of silicosis. The upstream molecular mechanisms and functional effects on cell apoptosis, proliferation and migration were investigated to elucidate the role of circRNAs in SiO2-induced inflammation in pulmonary macrophages. Methods: Primary cultures of alveolar macrophages from healthy donors and patients as well as the RAW264.7 macrophage cell line were used to explore the functions of circHECTD1 (HECT domain E3 ubiquitin protein ligase 1) in macrophage activation. Results: The results of the experiments indicated that 1) SiO2 concomitantly decreased circHECTD1 levels and increased HECTD1 protein expression; 2) circHECTD1 and HECTD1 were involved in SiO2-induced macrophage activation via ubiquitination; and 3) SiO2-activated macrophages promoted fibroblast proliferation and migration via the circHECTD1/HECTD1 pathway. Tissue samples from silicosis patients confirmed the upregulation of HECTD1. Conclusions: Our study elucidated a link between SiO2-induced macrophage activation and the circHECTD1/HECTD1 pathway, thereby providing new insight into the potential use of HECTD1 in the development of novel therapeutic strategies for treating silicosis. PMID:29290828

circRNA Mediates Silica-Induced Macrophage Activation Via HECTD1/ZC3H12A-Dependent Ubiquitination.

Science.gov (United States)

Zhou, Zewei; Jiang, Rong; Yang, Xiyue; Guo, Huifang; Fang, Shencun; Zhang, Yingming; Cheng, Yusi; Wang, Jing; Yao, Honghong; Chao, Jie

2018-01-01

Rationale: Phagocytosis of silicon dioxide (SiO 2 ) into lung cells causes an inflammatory cascade that results in fibroblast proliferation and migration, followed by fibrosis. Circular RNAs (circRNAs) are a subclass of non-coding RNAs detected within mammalian cells; however, researchers have not determined whether circRNAs are involved in the pathophysiological process of silicosis. The upstream molecular mechanisms and functional effects on cell apoptosis, proliferation and migration were investigated to elucidate the role of circRNAs in SiO 2 -induced inflammation in pulmonary macrophages. Methods: Primary cultures of alveolar macrophages from healthy donors and patients as well as the RAW264.7 macrophage cell line were used to explore the functions of circHECTD1 (HECT domain E3 ubiquitin protein ligase 1) in macrophage activation. Results: The results of the experiments indicated that 1) SiO 2 concomitantly decreased circHECTD1 levels and increased HECTD1 protein expression; 2) circHECTD1 and HECTD1 were involved in SiO 2 -induced macrophage activation via ubiquitination; and 3) SiO 2 -activated macrophages promoted fibroblast proliferation and migration via the circHECTD1/HECTD1 pathway. Tissue samples from silicosis patients confirmed the upregulation of HECTD1. Conclusions: Our study elucidated a link between SiO 2 -induced macrophage activation and the circHECTD1/HECTD1 pathway, thereby providing new insight into the potential use of HECTD1 in the development of novel therapeutic strategies for treating silicosis.

Biochemical function of typical and variant Arabidopsis thaliana U-box E3 ubiquitin-protein ligases

DEFF Research Database (Denmark)

Wiborg, Jakob; O'Shea, Charlotte; Skriver, Karen

2008-01-01

of the distant U-box protein, AtPUB49, representing a large family of eukaryotic proteins containing a U-box linked to a cyclophilin-like peptidyl-prolyl cis-trans isomerase domain, was characterized biochemically. AtPUB49 functioned both as a prolyl isomerase and a chaperone by catalysing cis......The variance of the U-box domain in 64 Arabidopsis thaliana (thale cress) E3s (ubiquitin-protein ligases) was used to examine the interactions between E3s and E2s (ubiquitin-conjugating enzymes). E2s and E3s are components of the ubiquitin protein degradation pathway. Seven U-box proteins were...... analysed for their ability to ubiquitinate proteins in vitro in co-operation with different E2s. All U-box domains exhibited ubiquitination activity and interacted productively with UBC4/5-type E2s. Three and four of the U-box domains mediated ubiquitin addition in the presence of UBC13 and UBC7 E2s...

The E3 ubiquitin ligases β-TrCP and FBXW7 cooperatively mediates GSK3-dependent Mcl-1 degradation induced by the Akt inhibitor API-1, resulting in apoptosis.

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Ren, Hui; Koo, Junghui; Guan, Baoxiang; Yue, Ping; Deng, Xingming; Chen, Mingwei; Khuri, Fadlo R; Sun, Shi-Yong

2013-11-22

The novel Akt inhibitor, API-1, induces apoptosis through undefined mechanisms. The current study focuses on revealing the mechanisms by which API-1 induces apoptosis. API-1 rapidly and potently reduced the levels of Mcl-1 primarily in API-1-senstive lung cancer cell lines. Ectopic expression of Mcl-1 protected cells from induction of apoptosis by API-1. API-1 treatment decreased the half-life of Mcl-1, whereas inhibition of the proteasome with MG132 rescued Mcl-1 reduction induced by API-1. API-1 decreased Mcl-1 levels accompanied with a rapid increase in Mcl-1 phosphorylation (S159/T163). Moreover, inhibition of GSK3 inhibited Mcl-1 phosphorylation and reduction induced by API-1 and antagonized the effect of API-1 on induction of apoptosis. Knockdown of either FBXW7 or β-TrCP alone, both of which are E3 ubiquitin ligases involved in Mcl-1 degradation, only partially rescued Mcl-1 reduction induced by API-1. However, double knockdown of both E3 ubiquitin ligases enhanced the rescue of API-1-induced Mcl-1 reduction. API-1 induces GSK3-dependent, β-TrCP- and FBXW7-mediated Mcl-1 degradation, resulting in induction of apoptosis.

Brucella infection inhibits macrophages apoptosis via Nedd4-dependent degradation of calpain2.

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Cui, Guimei; Wei, Pan; Zhao, Yuxi; Guan, Zhenhong; Yang, Li; Sun, Wanchun; Wang, Shuangxi; Peng, Qisheng

2014-11-07

The calcium-dependent protease calpain2 is involved in macrophages apoptosis. Brucella infection-induced up-regulation of intracellular calcium level is an essential factor for the intracellular survival of Brucella within macrophages. Here, we hypothesize that calcium-dependent E3 ubiquitin ligase Nedd4 ubiquitinates calpain2 and inhibits Brucella infection-induced macrophage apoptosis via degradation of calpain2.Our results reveal that Brucella infection induces increases in Nedd4 activity in an intracellular calcium dependent manner. Furthermore, Brucella infection-induced degradation of calpain2 is mediated by Nedd4 ubiquitination of calpain2. Brucella infection-induced calpain2 degradation inhibited macrophages apoptosis. Treatment of Brucella infected macrophages with calcium chelator BAPTA or Nedd4 knock-down decreased Nedd4 activity, prevented calpain2 degradation, and resulted in macrophages apoptosis. Copyright © 2014 Elsevier B.V. All rights reserved.

What do we really know about the ubiquitin-proteasome pathway in muscle atrophy?

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Jagoe, R. T.; Goldberg, A. L.

2001-01-01

Studies of many different rodent models of muscle wasting have indicated that accelerated proteolysis via the ubiquitin-proteasome pathway is the principal cause of muscle atrophy induced by fasting, cancer cachexia, metabolic acidosis, denervation, disuse, diabetes, sepsis, burns, hyperthyroidism and excess glucocorticoids. However, our understanding about how muscle proteins are degraded, and how the ubiquitin-proteasome pathway is activated in muscle under these conditions, is still very limited. The identities of the important ubiquitin-protein ligases in skeletal muscle, and the ways in which they recognize substrates are still largely unknown. Recent in-vitro studies have suggested that one set of ubquitination enzymes, E2(14K) and E3(alpha), which are responsible for the 'N-end rule' system of ubiquitination, plays an important role in muscle, especially in catabolic states. However, their functional significance in degrading different muscle proteins is still unclear. This review focuses on the many gaps in our understanding of the functioning of the ubiquitin-proteasome pathway in muscle atrophy, and highlights the strengths and limitations of the different experimental approaches used in such studies.

Spermidine mediates degradation of ornithine decarboxylase by a non-lysosomal, ubiquitin-independent mechanism

International Nuclear Information System (INIS)

Glass, J.R.; Gerner, E.W.

1987-01-01

The mechanism of spermidine-induced ornithine decarboxylase (OCD, E.C. 4.1.1.17) inactivation was investigated using Chinese hamster ovary (CHO) cells, maintained in serum-free medium, which display a stabilization of ODC owing to the lack of accumulation of putrescine and spermidine. Treatment of cells with 10 μM exogenous spermidine leads to rapid decay of ODC activity accompanied by a parallel decrease in enzyme protein. Analysis of the decay of [ 35 S]methionine-labeled ODC and separation by two-dimensional electrophoresis revealed no detectable modification in ODC structure during enhanced degradation. Spermidine-mediated inactivation of ODC occurred in a temperature-dependent manner exhibiting pseudo-first-order kinetics over a temperature range of 22-37 0 C. In cultures treated continuously, an initial lag was observed after treatment with spermidine, followed by a rapid decline in activity as an apparent critical concentration of intracellular spermidine was achieved. Treating cells at 22 0 C for 3 hours with 10 μ M spermidine, followed by removal of exogenous polyamine, and then shifting to varying temperatures, resulted in rates of ODC inactivation identical with that determined with a continuous treatment. Arrhenius analysis showed that polyamine mediated inactivation of ODC occurred with an activation energy of approximately 16 kcal/mol. Treatment of cells with lysosomotrophic agents had no effect of ODC degradation. ODC turnover was not dependent on ubiquitin-dependent proteolysis. These data support the hypothesis that spermidine regulates ODC degradation via a mechanism requiring new protein synthesis, and that this occurs via a non-lysosomal, ubiquitin-independent pathway

A quantitative and qualitative comparison of illumina MiSeq and 454 amplicon sequencing for genotyping the highly polymorphic major histocompatibility complex (MHC) in a non-model species.

Science.gov (United States)

Razali, Haslina; O'Connor, Emily; Drews, Anna; Burke, Terry; Westerdahl, Helena

2017-07-28

High-throughput sequencing enables high-resolution genotyping of extremely duplicated genes. 454 amplicon sequencing (454) has become the standard technique for genotyping the major histocompatibility complex (MHC) genes in non-model organisms. However, illumina MiSeq amplicon sequencing (MiSeq), which offers a much higher read depth, is now superseding 454. The aim of this study was to quantitatively and qualitatively evaluate the performance of MiSeq in relation to 454 for genotyping MHC class I alleles using a house sparrow (Passer domesticus) dataset with pedigree information. House sparrows provide a good study system for this comparison as their MHC class I genes have been studied previously and, consequently, we had prior expectations concerning the number of alleles per individual. We found that 454 and MiSeq performed equally well in genotyping amplicons with low diversity, i.e. amplicons from individuals that had fewer than 6 alleles. Although there was a higher rate of failure in the 454 dataset in resolving amplicons with higher diversity (6-9 alleles), the same genotypes were identified by both 454 and MiSeq in 98% of cases. We conclude that low diversity amplicons are equally well genotyped using either 454 or MiSeq, but the higher coverage afforded by MiSeq can lead to this approach outperforming 454 in amplicons with higher diversity.

Ubiquitin-aldehyde: a general inhibitor of ubiquitin-recycling processes

International Nuclear Information System (INIS)

Hershko, A.; Rose, I.A.

1987-01-01

The generation and characterization of ubiquitin (Ub)-aldehyde, a potent inhibitor of Ub-C-terminal hydrolase, has previously been reported. The authors examine the action of this compound on the Ub-mediated proteolytic pathway using the system derived from rabbit reticulocytes. Addition of Ub-aldehyde was found to strongly inhibit breakdown of added 125 I-labeled lysozyme, but inhibition was overcome by increasing concentrations of Ub. The following evidence shows the effect of Ub-aldehyde on protein breakdown to be indirectly caused by its interference with the recycling of Ub, leading to exhaustion of the supply of free Ub: (i) Ub-aldehyde markedly increased the accumulation of Ub-protein conjugates coincident with a much decreased rate of conjugate breakdown; (ii) release of Ub from isolated Ub-protein conjugates in the absence of ATP (and therefore not coupled to protein degradation) is markedly inhibited by Ub-aldehyde. On the other hand, the ATP-dependent degradation of the protein moiety of Ub conjugates, which is an integral part of the proteolytic process, is not inhibited by this agent; (iii) direct measurement of levels of free Ub showed a rapid disappearance caused by the inhibitor. The Ub is found to be distributed in derivatives of a wide range of molecular weight classes. It thus seems that Ub-aldehyde, previously demonstrated to inhibit the hydrolysis of Ub conjugates of small molecules, also inhibits the activity of a series of enzymes that regenerate free Ub from adducts with proteins and intermediates in protein breakdown

HIV Controllers Exhibit Enhanced Frequencies of Major Histocompatibility Complex Class II Tetramer+ Gag-Specific CD4+ T Cells in Chronic Clade C HIV-1 Infection.

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Laher, Faatima; Ranasinghe, Srinika; Porichis, Filippos; Mewalal, Nikoshia; Pretorius, Karyn; Ismail, Nasreen; Buus, Søren; Stryhn, Anette; Carrington, Mary; Walker, Bruce D; Ndung'u, Thumbi; Ndhlovu, Zaza M

2017-04-01

Immune control of viral infections is heavily dependent on helper CD4 + T cell function. However, the understanding of the contribution of HIV-specific CD4 + T cell responses to immune protection against HIV-1, particularly in clade C infection, remains incomplete. Recently, major histocompatibility complex (MHC) class II tetramers have emerged as a powerful tool for interrogating antigen-specific CD4 + T cells without relying on effector functions. Here, we defined the MHC class II alleles for immunodominant Gag CD4 + T cell epitopes in clade C virus infection, constructed MHC class II tetramers, and then used these to define the magnitude, function, and relation to the viral load of HIV-specific CD4 + T cell responses in a cohort of untreated HIV clade C-infected persons. We observed significantly higher frequencies of MHC class II tetramer-positive CD4 + T cells in HIV controllers than progressors ( P = 0.0001), and these expanded Gag-specific CD4 + T cells in HIV controllers showed higher levels of expression of the cytolytic proteins granzymes A and B. Importantly, targeting of the immunodominant Gag41 peptide in the context of HLA class II DRB1*1101 was associated with HIV control ( r = -0.5, P = 0.02). These data identify an association between HIV-specific CD4 + T cell targeting of immunodominant Gag epitopes and immune control, particularly the contribution of a single class II MHC-peptide complex to the immune response against HIV-1 infection. Furthermore, these results highlight the advantage of the use of class II tetramers in evaluating HIV-specific CD4 + T cell responses in natural infections. IMPORTANCE Increasing evidence suggests that virus-specific CD4 + T cells contribute to the immune-mediated control of clade B HIV-1 infection, yet there remains a relative paucity of data regarding the role of HIV-specific CD4 + T cells in shaping adaptive immune responses in individuals infected with clade C, which is responsible for the majority of HIV

The APC/C Ubiquitin Ligase: From Cell Biology to Tumorigenesis

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Penas, Clara; Ramachandran, Vimal; Ayad, Nagi George

2011-01-01

The ubiquitin proteasome system (UPS) is required for normal cell proliferation, vertebrate development, and cancer cell transformation. The UPS consists of multiple proteins that work in concert to target a protein for degradation via the 26S proteasome. Chains of an 8.5-kDa protein called ubiquitin are attached to substrates, thus allowing recognition by the 26S proteasome. Enzymes called ubiquitin ligases or E3s mediate specific attachment to substrates. Although there are over 600 different ubiquitin ligases, the Skp1–Cullin–F-box (SCF) complexes and the anaphase promoting complex/cyclosome (APC/C) are the most studied. SCF involvement in cancer has been known for some time while APC/C’s cancer role has recently emerged. In this review we will discuss the importance of APC/C to normal cell proliferation and development, underscoring its possible contribution to transformation. We will also examine the hypothesis that modulating a specific interaction of the APC/C may be therapeutically attractive in specific cancer subtypes. Finally, given that the APC/C pathway is relatively new as a cancer target, therapeutic interventions affecting APC/C activity may be beneficial in cancers that are resistant to classical chemotherapy. PMID:22655255

The Crystal Structure and Conformations of an Unbranched Mixed Tri-Ubiquitin Chain Containing K48 and K63 Linkages.

Science.gov (United States)

Padala, Prasanth; Soudah, Nadine; Giladi, Moshe; Haitin, Yoni; Isupov, Michail N; Wiener, Reuven

2017-12-08

The ability of ubiquitin to function in a wide range of cellular processes is ascribed to its capacity to cause a diverse spectrum of modifications. While a target protein can be modified with monoubiquitin, it can also be modified with ubiquitin chains. The latter include seven types of homotypic chains as well as mixed ubiquitin chains. In a mixed chain, not all the isopeptide bonds are restricted to a specific lysine of ubiquitin, resulting in a chain possessing more than one type of linkage. While structural characterization of homotypic chains has been well elucidated, less is known about mixed chains. Here we present the crystal structure of a mixed tri-ubiquitin chain at 3.1-Å resolution. In the structure, the proximal ubiquitin is connected to the middle ubiquitin via K48 and these two ubiquitins adopt a compact structure as observed in K48 di-ubiquitin. The middle ubiquitin links to the distal ubiquitin via its K63 and these ubiquitins adopt two conformations, suggesting a flexible structure. Using small-angle X-ray scattering, we unexpectedly found differences between the conformational ensembles of the above tri-ubiquitin chains and chains possessing the same linkages but in the reverse order. In addition, cleavage of the K48 linkage by DUB is faster if this linkage is at the distal end. Taken together, our results suggest that in mixed chains, not only the type of the linkages but also their sequence determine the structural and functional properties of the chain. Copyright © 2017 Elsevier Ltd. All rights reserved.

Direct observation of a single nanoparticle-ubiquitin corona formation

Science.gov (United States)

Ding, Feng; Radic, Slaven; Chen, Ran; Chen, Pengyu; Geitner, Nicholas K.; Brown, Jared M.; Ke, Pu Chun

2013-09-01

The advancement of nanomedicine and the increasing applications of nanoparticles in consumer products have led to administered biological exposure and unintentional environmental accumulation of nanoparticles, causing concerns over the biocompatibility and sustainability of nanotechnology. Upon entering physiological environments, nanoparticles readily assume the form of a nanoparticle-protein corona that dictates their biological identity. Consequently, understanding the structure and dynamics of a nanoparticle-protein corona is essential for predicting the fate, transport, and toxicity of nanomaterials in living systems and for enabling the vast applications of nanomedicine. Here we combined multiscale molecular dynamics simulations and complementary experiments to characterize the silver nanoparticle-ubiquitin corona formation. Notably, ubiquitins competed with citrates for the nanoparticle surface, governed by specific electrostatic interactions. Under a high protein/nanoparticle stoichiometry, ubiquitins formed a multi-layer corona on the particle surface. The binding exhibited an unusual stretched-exponential behavior, suggesting a rich binding kinetics. Furthermore, the binding destabilized the α-helices while increasing the β-sheet content of the proteins. This study revealed the atomic and molecular details of the structural and dynamic characteristics of nanoparticle-protein corona formation.The advancement of nanomedicine and the increasing applications of nanoparticles in consumer products have led to administered biological exposure and unintentional environmental accumulation of nanoparticles, causing concerns over the biocompatibility and sustainability of nanotechnology. Upon entering physiological environments, nanoparticles readily assume the form of a nanoparticle-protein corona that dictates their biological identity. Consequently, understanding the structure and dynamics of a nanoparticle-protein corona is essential for predicting the fate

Ubiquitin C-Terminal Hydrolase L1 in Tumorigenesis

Directory of Open Access Journals (Sweden)

Jennifer Hurst-Kennedy

2012-01-01

Full Text Available Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1, aka PGP9.5 is an abundant, neuronal deubiquitinating enzyme that has also been suggested to possess E3 ubiquitin-protein ligase activity and/or stabilize ubiquitin monomers in vivo. Recent evidence implicates dysregulation of UCH-L1 in the pathogenesis and progression of human cancers. Although typically only expressed in neurons, high levels of UCH-L1 have been found in many nonneuronal tumors, including breast, colorectal, and pancreatic carcinomas. UCH-L1 has also been implicated in the regulation of metastasis and cell growth during the progression of nonsmall cell lung carcinoma, colorectal cancer, and lymphoma. Together these studies suggest UCH-L1 has a potent oncogenic role and drives tumor development. Conversely, others have observed promoter methylation-mediated silencing of UCH-L1 in certain tumor subtypes, suggesting a potential tumor suppressor role for UCH-L1. In this paper, we provide an overview of the evidence supporting the involvement of UCH-L1 in tumor development and discuss the potential mechanisms of action of UCH-L1 in oncogenesis.

The Endosome-associated Deubiquitinating Enzyme USP8 Regulates BACE1 Enzyme Ubiquitination and Degradation.

Science.gov (United States)

Yeates, Eniola Funmilayo Aduke; Tesco, Giuseppina

2016-07-22

The β-site amyloid precursor protein-cleaving enzyme (BACE1) is the rate-limiting enzyme in the production of amyloid-β, the toxic peptide that accumulates in the brain of subjects affected by Alzheimer disease. Our previous studies have shown that BACE1 is degraded via the lysosomal pathway and that that depletion of the trafficking molecule Golgi-localized γ-ear-containing ARF-binding protein 3 (GGA3) results in increased BACE1 levels and activity because of impaired lysosomal degradation. We also determined that GGA3 regulation of BACE1 levels requires its ability to bind ubiquitin. Accordingly, we reported that BACE1 is ubiquitinated at lysine 501 and that lack of ubiquitination at lysine 501 produces BACE1 stabilization. Ubiquitin conjugation is a reversible process mediated by deubiquitinating enzymes. The ubiquitin-specific peptidase 8 (USP8), an endosome-associated deubiquitinating enzyme, regulates the ubiquitination, trafficking, and lysosomal degradation of several plasma membrane proteins. Here, we report that RNAi-mediated depletion of USP8 reduced levels of both ectopically expressed and endogenous BACE1 in H4 human neuroglioma cells. Moreover, USP8 depletion increased BACE1 ubiquitination, promoted BACE1 accumulation in the early endosomes and late endosomes/lysosomes, and decreased levels of BACE1 in the recycling endosomes. We also found that decreased BACE1 protein levels were accompanied by a decrease in BACE1-mediated amyloid precursor protein cleavage and amyloid-β levels. Our findings demonstrate that USP8 plays a key role in the trafficking and degradation of BACE1 by deubiquitinating lysine 501. These studies suggest that therapies able to accelerate BACE1 degradation (e.g. by increasing BACE1 ubiquitination) may represent a potential treatment for Alzheimer disease. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

The Endosome-associated Deubiquitinating Enzyme USP8 Regulates BACE1 Enzyme Ubiquitination and Degradation*

Science.gov (United States)

Yeates, Eniola Funmilayo Aduke; Tesco, Giuseppina

2016-01-01

The β-site amyloid precursor protein-cleaving enzyme (BACE1) is the rate-limiting enzyme in the production of amyloid-β, the toxic peptide that accumulates in the brain of subjects affected by Alzheimer disease. Our previous studies have shown that BACE1 is degraded via the lysosomal pathway and that that depletion of the trafficking molecule Golgi-localized γ-ear-containing ARF-binding protein 3 (GGA3) results in increased BACE1 levels and activity because of impaired lysosomal degradation. We also determined that GGA3 regulation of BACE1 levels requires its ability to bind ubiquitin. Accordingly, we reported that BACE1 is ubiquitinated at lysine 501 and that lack of ubiquitination at lysine 501 produces BACE1 stabilization. Ubiquitin conjugation is a reversible process mediated by deubiquitinating enzymes. The ubiquitin-specific peptidase 8 (USP8), an endosome-associated deubiquitinating enzyme, regulates the ubiquitination, trafficking, and lysosomal degradation of several plasma membrane proteins. Here, we report that RNAi-mediated depletion of USP8 reduced levels of both ectopically expressed and endogenous BACE1 in H4 human neuroglioma cells. Moreover, USP8 depletion increased BACE1 ubiquitination, promoted BACE1 accumulation in the early endosomes and late endosomes/lysosomes, and decreased levels of BACE1 in the recycling endosomes. We also found that decreased BACE1 protein levels were accompanied by a decrease in BACE1-mediated amyloid precursor protein cleavage and amyloid-β levels. Our findings demonstrate that USP8 plays a key role in the trafficking and degradation of BACE1 by deubiquitinating lysine 501. These studies suggest that therapies able to accelerate BACE1 degradation (e.g. by increasing BACE1 ubiquitination) may represent a potential treatment for Alzheimer disease. PMID:27302062

Major histocompatibility complex class I molecule expression is normal on peripheral blood lymphocytes from patients with insulin-dependent diabetes mellitus.

OpenAIRE

Hao, W; Gladstone, P; Engardt, S; Greenbaum, C; Palmer, J P

1996-01-01

Recent work from one laboratory has shown, in both nonobese diabetic mice and humans, an association between insulin-dependent diabetes mellitus (IDDM) and quantitative difference in MHC class I molecule expression. This reported decrease in MHC class I molecule expression is very controversial in the nonobese diabetic mouse model of IDDM, but to our knowledge, it has not been evaluated by another group in human IDDM. To evaluate this question, we studied 30 patients with IDDM and 30 age- and...

Atomic structure of the APC/C and its mechanism of protein ubiquitination

Science.gov (United States)

Yang, Jing; McLaughlin, Stephen H.; Barford, David

2015-01-01

The anaphase-promoting complex (APC/C) is a multimeric RING E3 ubiquitin ligase that controls chromosome segregation and mitotic exit. Its regulation by coactivator subunits, phosphorylation, the mitotic checkpoint complex, and interphase inhibitor Emi1 ensures the correct order and timing of distinct cell cycle transitions. Here, we used cryo-electron microscopy to determine atomic structures of APC/C-coactivator complexes with either Emi1 or a UbcH10-ubiquitin conjugate. These structures define the architecture of all APC/C subunits, the position of the catalytic module, and explain how Emi1 mediates inhibition of the two E2s UbcH10 and Ube2S. Definition of Cdh1 interactions with the APC/C indicates how they are antagonized by Cdh1 phosphorylation. The structure of the APC/C with UbcH10-ubiquitin reveals insights into the initiating ubiquitination reaction. Our results provide a quantitative framework for the design of experiments to further investigate APC/C functions in vivo. PMID:26083744

Chaperones, but not oxidized proteins, are ubiquitinated after oxidative stress

DEFF Research Database (Denmark)

Kästle, Marc; Reeg, Sandra; Rogowska-Wrzesinska, Adelina

2012-01-01

of these proteins by MALDI tandem mass spectrometry (MALDI MS/MS). As a result we obtained 24 different proteins which can be categorized into the following groups: chaperones, energy metabolism, cytoskeleton/intermediate filaments, and protein translation/ribosome biogenesis. The special set of identified......, ubiquitinated proteins confirm the thesis that ubiquitination upon oxidative stress is no random process to degrade the mass of oxidized proteins, but concerns a special group of functional proteins....

Comparative analysis of minor histocompatibility antigens genotyping methods

Directory of Open Access Journals (Sweden)

A. S. Vdovin

2016-01-01

Full Text Available The wide range of techniques could be employed to find mismatches in minor histocompatibility antigens between transplant recipients and their donors. In the current study we compared three genotyping methods based on polymerase chain reaction (PCR for four minor antigens. Three of the tested methods: allele-specific PCR, restriction fragment length polymorphism and real-time PCR with TaqMan probes demonstrated 100% reliability when compared to Sanger sequencing for all of the studied polymorphisms. High resolution melting analysis was unsuitable for genotyping of one of the tested minor antigens (HA-1 as it has linked synonymous polymorphism. Obtained data could be used to select the strategy for large-scale clinical genotyping.

DMPD: Ubiquitin: tool and target for intracellular NF-kappaB inhibitors. [Dynamic Macrophage Pathway CSML Database

Lifescience Database Archive (English)

Full Text Available 16982211 Ubiquitin: tool and target for intracellular NF-kappaB inhibitors. Wullaer...vg) (.html) (.csml) Show Ubiquitin: tool and target for intracellular NF-kappaB inhibitors. PubmedID 1698221...1 Title Ubiquitin: tool and target for intracellular NF-kappaB inhibitors. Author

Typing of human fetal organs for the histocompatibility antigens A, B and DR.

Science.gov (United States)

Tuch, B E; Doran, T J; Messel, N; Turtle, J R

1985-01-01

In the transplantation of human fetal pancreatic explants into diabetic man, the importance of matching the histocompatibility antigens of donor and recipient to decrease the chances of rejection is unknown. Before this question can be answered human fetuses must be tissue typed. We have shown that lymphocytes harvested from fetal liver, thymus, bone marrow and spleen can be successfully HLA DR typed in 64% and A and B typed in 57% of 58 fetuses aged 15 wk or more. Typing should ideally be carried out on unseparated T and B cells. Best results were achieved if all four of the above organs were available and more than one million viable cells were able to be harvested for typing. Whilst the DR antigens could be typed from all tissues, the A and B antigens could be typed, with few exceptions only from thymus, spleen and bone marrow. The efficacy of matching the histocompatibility antigens of recipient and donor fetuses, especially the DR antigens can now be tested in the human diabetic being transplanted with pancreatic explants.

Bacterial Effectors and Their Functions in the Ubiquitin-Proteasome System: Insight from the Modes of Substrate Recognition

Directory of Open Access Journals (Sweden)

Minsoo Kim

2014-08-01

Full Text Available Protein ubiquitination plays indispensable roles in the regulation of cell homeostasis and pathogenesis of neoplastic, infectious, and neurodegenerative diseases. Given the importance of this modification, it is to be expected that several pathogenic bacteria have developed the ability to utilize the host ubiquitin system for their own benefit. Modulation of the host ubiquitin system by bacterial effector proteins inhibits innate immune responses and hijacks central signaling pathways. Bacterial effectors mimic enzymes of the host ubiquitin system, but may or may not be structurally similar to the mammalian enzymes. Other effectors bind and modify components of the host ubiquitin system, and some are themselves subject to ubiquitination. This review will describe recent findings, based on structural analyses, regarding how pathogens use post-translational modifications of proteins to establish an infection.

Bacterial effectors and their functions in the ubiquitin-proteasome system: insight from the modes of substrate recognition.

Science.gov (United States)

Kim, Minsoo; Otsubo, Ryota; Morikawa, Hanako; Nishide, Akira; Takagi, Kenji; Sasakawa, Chihiro; Mizushima, Tsunehiro

2014-08-18

Protein ubiquitination plays indispensable roles in the regulation of cell homeostasis and pathogenesis of neoplastic, infectious, and neurodegenerative diseases. Given the importance of this modification, it is to be expected that several pathogenic bacteria have developed the ability to utilize the host ubiquitin system for their own benefit. Modulation of the host ubiquitin system by bacterial effector proteins inhibits innate immune responses and hijacks central signaling pathways. Bacterial effectors mimic enzymes of the host ubiquitin system, but may or may not be structurally similar to the mammalian enzymes. Other effectors bind and modify components of the host ubiquitin system, and some are themselves subject to ubiquitination. This review will describe recent findings, based on structural analyses, regarding how pathogens use post-translational modifications of proteins to establish an infection.

Targeted ubiquitination of CDT1 by the DDB1-CUL4A-ROC1 ligase in response to DNA damage.

Science.gov (United States)

Hu, Jian; McCall, Chad M; Ohta, Tomohiko; Xiong, Yue

2004-10-01

Cullins assemble a potentially large number of ubiquitin ligases by binding to the RING protein ROC1 to catalyse polyubiquitination, as well as binding to various specificity factors to recruit substrates. The Cul4A gene is amplified in human breast and liver cancers, and loss-of-function of Cul4 results in the accumulation of the replication licensing factor CDT1 in Caenorhabditis elegans embryos and ultraviolet (UV)-irradiated human cells. Here, we report that human UV-damaged DNA-binding protein DDB1 associates stoichiometrically with CUL4A in vivo, and binds to an amino-terminal region in CUL4A in a manner analogous to SKP1, SOCS and BTB binding to CUL1, CUL2 and CUL3, respectively. As with SKP1-CUL1, the DDB1-CUL4A association is negatively regulated by the cullin-associated and neddylation-dissociated protein, CAND1. Recombinant DDB1 and CDT1 bind directly to each other in vitro, and ectopically expressed DDB1 bridges CDT1 to CUL4A in vivo. Silencing DDB1 prevented UV-induced rapid CDT1 degradation in vivo and CUL4A-mediated CDT1 ubiquitination in vitro. We suggest that DDB1 targets CDT1 for ubiquitination by a CUL4A-dependent ubiquitin ligase, CDL4A(DDB1), in response to UV irradiation.

Molecular characterization and functional analysis of ubiquitin extension genes from the potato cyst nematode Globodera rostochiensis

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Ubiquitin is a highly conserved 76-amino acid protein found in every eukaryotic cell. It has been proposed that ubiquitin has many cellular functions including DNA repair, transcription regulation, regulation of cell cycle and apoptosis. We identified two ubiquitin extension genes (Gr-Ubi1 and Gr-Ub...

Signals in hepatitis A virus P3 region proteins recognized by the ubiquitin-mediated proteolytic system

International Nuclear Information System (INIS)

Losick, Vicki P.; Schlax, Peter E.; Emmons, Rebecca A.; Lawson, T. Glen

2003-01-01

The hepatitis A virus 3C protease and 3D RNA polymerase are present in low concentrations in infected cells. The 3C protease was previously shown to be rapidly degraded by the ubiquitin/26S proteasome system and we present evidence here that the 3D polymerase is also subject to ubiquitination-mediated proteolysis. Our results show that the sequence 32 LGVKDDWLLV 41 in the 3C protease serves as a protein destruction signal recognized by the ubiquitin-protein ligase E3α and that the destruction signal for the RNA polymerase does not require the carboxyl-terminal 137 amino acids. Both the viral 3ABCD polyprotein and the 3CD diprotein were also found to be substrates for ubiquitin-mediated proteolysis. Attempts to determine if the 3C protease or the 3D polymerase destruction signals trigger the ubiquitination and degradation of these precursors yielded evidence suggesting, but not unequivocally proving, that the recognition of the 3D polymerase by the ubiquitin system is responsible

PCNA mono-ubiquitination and activation of translesion DNA polymerases by DNA polymerase {alpha}.

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Suzuki, Motoshi; Niimi, Atsuko; Limsirichaikul, Siripan; Tomida, Shuta; Miao Huang, Qin; Izuta, Shunji; Usukura, Jiro; Itoh, Yasutomo; Hishida, Takashi; Akashi, Tomohiro; Nakagawa, Yoshiyuki; Kikuchi, Akihiko; Pavlov, Youri; Murate, Takashi; Takahashi, Takashi

2009-07-01

Translesion DNA synthesis (TLS) involves PCNA mono-ubiquitination and TLS DNA polymerases (pols). Recent evidence has shown that the mono-ubiquitination is induced not only by DNA damage but also by other factors that induce stalling of the DNA replication fork. We studied the effect of spontaneous DNA replication errors on PCNA mono-ubiquitination and TLS induction. In the pol1L868F strain, which expressed an error-prone pol alpha, PCNA was spontaneously mono-ubiquitinated. Pol alpha L868F had a rate-limiting step at the extension from mismatched primer termini. Electron microscopic observation showed the accumulation of a single-stranded region at the DNA replication fork in yeast cells. For pol alpha errors, pol zeta participated in a generation of +1 frameshifts. Furthermore, in the pol1L868F strain, UV-induced mutations were lower than in the wild-type and a pol delta mutant strain (pol3-5DV), and deletion of the RAD30 gene (pol eta) suppressed this defect. These data suggest that nucleotide misincorporation by pol alpha induces exposure of single-stranded DNA, PCNA mono-ubiquitination and activates TLS pols.

Toward the prediction of class I and II mouse major histocompatibility complex-peptide-binding affinity: in silico bioinformatic step-by-step guide using quantitative structure-activity relationships.

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Hattotuwagama, Channa K; Doytchinova, Irini A; Flower, Darren R

2007-01-01

Quantitative structure-activity relationship (QSAR) analysis is a cornerstone of modern informatics. Predictive computational models of peptide-major histocompatibility complex (MHC)-binding affinity based on QSAR technology have now become important components of modern computational immunovaccinology. Historically, such approaches have been built around semiqualitative, classification methods, but these are now giving way to quantitative regression methods. We review three methods--a 2D-QSAR additive-partial least squares (PLS) and a 3D-QSAR comparative molecular similarity index analysis (CoMSIA) method--which can identify the sequence dependence of peptide-binding specificity for various class I MHC alleles from the reported binding affinities (IC50) of peptide sets. The third method is an iterative self-consistent (ISC) PLS-based additive method, which is a recently developed extension to the additive method for the affinity prediction of class II peptides. The QSAR methods presented here have established themselves as immunoinformatic techniques complementary to existing methodology, useful in the quantitative prediction of binding affinity: current methods for the in silico identification of T-cell epitopes (which form the basis of many vaccines, diagnostics, and reagents) rely on the accurate computational prediction of peptide-MHC affinity. We have reviewed various human and mouse class I and class II allele models. Studied alleles comprise HLA-A*0101, HLA-A*0201, HLA-A*0202, HLA-A*0203, HLA-A*0206, HLA-A*0301, HLA-A*1101, HLA-A*3101, HLA-A*6801, HLA-A*6802, HLA-B*3501, H2-K(k), H2-K(b), H2-D(b) HLA-DRB1*0101, HLA-DRB1*0401, HLA-DRB1*0701, I-A(b), I-A(d), I-A(k), I-A(S), I-E(d), and I-E(k). In this chapter we show a step-by-step guide into predicting the reliability and the resulting models to represent an advance on existing methods. The peptides used in this study are available from the AntiJen database (http://www.jenner.ac.uk/AntiJen). The PLS method

Insulin alleviates degradation of skeletal muscle protein by inhibiting the ubiquitin-proteasome system in septic rats.

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Chen, Qiyi; Li, Ning; Zhu, Weiming; Li, Weiqin; Tang, Shaoqiu; Yu, Wenkui; Gao, Tao; Zhang, Juanjuan; Li, Jieshou

2011-06-03

Hypercatabolism is common under septic conditions. Skeletal muscle is the main target organ for hypercatabolism, and this phenomenon is a vital factor in the deterioration of recovery in septic patients. In skeletal muscle, activation of the ubiquitin-proteasome system plays an important role in hypercatabolism under septic status. Insulin is a vital anticatabolic hormone and previous evidence suggests that insulin administration inhibits various steps in the ubiquitin-proteasome system. However, whether insulin can alleviate the degradation of skeletal muscle protein by inhibiting the ubiquitin-proteasome system under septic condition is unclear. This paper confirmed that mRNA and protein levels of the ubiquitin-proteasome system were upregulated and molecular markers of skeletal muscle proteolysis (tyrosine and 3-methylhistidine) simultaneously increased in the skeletal muscle of septic rats. Septic rats were infused with insulin at a constant rate of 2.4 mU.kg-1.min-1 for 8 hours. Concentrations of mRNA and proteins of the ubiquitin-proteasome system and molecular markers of skeletal muscle proteolysis were mildly affected. When the insulin infusion dose increased to 4.8 mU.kg-1.min-1, mRNA for ubiquitin, E2-14 KDa, and the C2 subunit were all sharply downregulated. At the same time, the levels of ubiquitinated proteins, E2-14KDa, and the C2 subunit protein were significantly reduced. Tyrosine and 3-methylhistidine decreased significantly. We concluded that the ubiquitin-proteasome system is important skeletal muscle hypercatabolism in septic rats. Infusion of insulin can reverse the detrimental metabolism of skeletal muscle by inhibiting the ubiquitin-proteasome system, and the effect is proportional to the insulin infusion dose.

Ube2V2 Is a Rosetta Stone Bridging Redox and Ubiquitin Codes, Coordinating DNA Damage Responses.

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Zhao, Yi; Long, Marcus J C; Wang, Yiran; Zhang, Sheng; Aye, Yimon

2018-02-28

Posttranslational modifications (PTMs) are the lingua franca of cellular communication. Most PTMs are enzyme-orchestrated. However, the reemergence of electrophilic drugs has ushered mining of unconventional/non-enzyme-catalyzed electrophile-signaling pathways. Despite the latest impetus toward harnessing kinetically and functionally privileged cysteines for electrophilic drug design, identifying these sensors remains challenging. Herein, we designed "G-REX"-a technique that allows controlled release of reactive electrophiles in vivo. Mitigating toxicity/off-target effects associated with uncontrolled bolus exposure, G-REX tagged first-responding innate cysteines that bind electrophiles under true k cat / K m conditions. G-REX identified two allosteric ubiquitin-conjugating proteins-Ube2V1/Ube2V2-sharing a novel privileged-sensor-cysteine. This non-enzyme-catalyzed-PTM triggered responses specific to each protein. Thus, G-REX is an unbiased method to identify novel functional cysteines. Contrasting conventional active-site/off-active-site cysteine-modifications that regulate target activity, modification of Ube2V2 allosterically hyperactivated its enzymatically active binding-partner Ube2N, promoting K63-linked client ubiquitination and stimulating H2AX-dependent DNA damage response. This work establishes Ube2V2 as a Rosetta-stone bridging redox and ubiquitin codes to guard genome integrity.

Experimental hyperthyroidism in rats increases the expression of the ubiquitin ligases atrogin-1 and MuRF1 and stimulates multiple proteolytic pathways in skeletal muscle.

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O'Neal, Patrick; Alamdari, Nima; Smith, Ira; Poylin, Vitaliy; Menconi, Michael; Hasselgren, Per-Olof

2009-11-01

Muscle wasting is commonly seen in patients with hyperthyroidism and is mainly caused by stimulated muscle proteolysis. Loss of muscle mass in several catabolic conditions is associated with increased expression of the muscle-specific ubiquitin ligases atrogin-1 and MuRF1 but it is not known if atrogin-1 and MuRF1 are upregulated in hyperthyroidism. In addition, it is not known if thyroid hormone increases the activity of proteolytic mechanisms other than the ubiquitin-proteasome pathway. We tested the hypotheses that experimental hyperthyroidism in rats, induced by daily intraperitoneal injections of 100 microg/100 g body weight of triiodothyronine (T3), upregulates the expression of atrogin-1 and MuRF1 in skeletal muscle and stimulates lysosomal, including cathepsin L, calpain-, and caspase-3-dependent protein breakdown in addition to proteasome-dependent protein breakdown. Treatment of rats with T3 for 3 days resulted in an approximately twofold increase in atrogin-1 and MuRF1 mRNA levels. The same treatment increased proteasome-, cathepsin L-, and calpain-dependent proteolytic rates by approximately 40% but did not influence caspase-3-dependent proteolysis. The expression of atrogin-1 and MuRF1 remained elevated during a more prolonged period (7 days) of T3 treatment. The results provide support for a role of the ubiquitin-proteasome pathway in muscle wasting during hyperthyroidism and suggest that other proteolytic pathways as well may be activated in the hyperthyroid state. (c) 2009 Wiley-Liss, Inc.

The Major Histocompatibility Complex Class II Transactivator CIITA Inhibits the Persistent Activation of NF-κB by the Human T Cell Lymphotropic Virus Type 1 Tax-1 Oncoprotein.

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Forlani, Greta; Abdallah, Rawan; Accolla, Roberto S; Tosi, Giovanna

2016-01-20

Human T cell lymphotropic virus type 1 (HTLV-1) Tax-1, a key protein in HTLV-1-induced T cell transformation, deregulates diverse cell signaling pathways. Among them, the NF-κB pathway is constitutively activated by Tax-1, which binds to NF-κB proteins and activates the IκB kinase (IKK). Upon phosphorylation-dependent IκB degradation, NF-κB migrates into the nucleus, mediating Tax-1-stimulated gene expression. We show that the transcriptional regulator of major histocompatibility complex class II genes CIITA (class II transactivator), endogenously or ectopically expressed in different cells, inhibits the activation of the canonical NF-κB pathway by Tax-1 and map the region that mediates this effect. CIITA affects the subcellular localization of Tax-1, which is mostly retained in the cytoplasm, and this correlates with impaired migration of RelA into the nucleus. Cytoplasmic and nuclear mutant forms of CIITA reveal that CIITA exploits different strategies to suppress Tax-1-mediated NF-κB activation in both subcellular compartments. CIITA interacts with Tax-1 without preventing Tax-1 binding to both IKKγ and RelA. Nevertheless, CIITA affects Tax-1-induced IKK activity, causing retention of the inactive p50/RelA/IκB complex in the cytoplasm. Nuclear CIITA associates with Tax-1/RelA in nuclear bodies, blocking Tax-1-dependent activation of NF-κB-responsive genes. Thus, CIITA inhibits cytoplasmic and nuclear steps of Tax-1-mediated NF-κB activation. These results, together with our previous finding that CIITA acts as a restriction factor inhibiting Tax-1-promoted HTLV-1 gene expression and replication, indicate that CIITA is a versatile molecule that might also counteract Tax-1 transforming activity. Unveiling the molecular basis of CIITA-mediated inhibition of Tax-1 functions may be important in defining new strategies to control HTLV-1 spreading and oncogenic potential. HTLV-1 is the causative agent of human adult T cell leukemia-lymphoma (ATLL). The viral

Viral Mimicry to Usurp Ubiquitin and SUMO Host Pathways

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Peter Wimmer

2015-08-01

Full Text Available Posttranslational modifications (PTMs of proteins include enzymatic changes by covalent addition of cellular regulatory determinants such as ubiquitin (Ub and small ubiquitin-like modifier (SUMO moieties. These modifications are widely used by eukaryotic cells to control the functional repertoire of proteins. Over the last decade, it became apparent that the repertoire of ubiquitiylation and SUMOylation regulating various biological functions is not restricted to eukaryotic cells, but is also a feature of human virus families, used to extensively exploit complex host-cell networks and homeostasis. Intriguingly, besides binding to host SUMO/Ub control proteins and interfering with the respective enzymatic cascade, many viral proteins mimic key regulatory factors to usurp this host machinery and promote efficient viral outcomes. Advanced detection methods and functional studies of ubiquitiylation and SUMOylation during virus-host interplay have revealed that human viruses have evolved a large arsenal of strategies to exploit these specific PTM processes. In this review, we highlight the known viral analogs orchestrating ubiquitin and SUMO conjugation events to subvert and utilize basic enzymatic pathways.

The role of the ubiquitin proteasome system in the memory process.

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Lip, Philomena Z Y; Demasi, Marilene; Bonatto, Diego

2017-01-01

Quite intuitive is the notion that memory formation and consolidation is orchestrated by protein synthesis because of the synaptic plasticity necessary for those processes. Nevertheless, recent advances have begun accumulating evidences of a high requirement for protein degradation on the molecular mechanisms of the memory process in the mammalian brain. Because degradation determines protein half-life, degradation has been increasingly recognized as an important intracellular regulatory mechanism. The proteasome is the main player in the degradation of intracellular proteins. Proteasomal substrates are mainly degraded after a post-translational modification by a poly-ubiquitin chain. Latter process, namely poly-ubiquitination, is highly regulated at the step of the ubiquitin molecule transferring to the protein substrate mediated by a set of proteins whose genes represent almost 2% of the human genome. Understanding the role of polyubiquitin-mediated protein degradation has challenging researchers in many fields of investigation as a new source of targets for therapeutic intervention, e.g. E3 ligases that transfer ubiquitin moieties to the substrate. The goal of present work was to uncover mechanisms underlying memory processes regarding the role of the ubiquitin-proteasome system (UPS). For that purpose, preceded of a short review on UPS and memory processes a top-down systems biology approach was applied to establish central proteins involved in memory formation and consolidation highlighting their cross-talking with the UPS. According to that approach, the pattern of expression of several elements of the UPS were found overexpressed in regions of the brain involved in processing cortical inputs. Copyright © 2016 Elsevier Ltd. All rights reserved.

The Host E3-Ubiquitin Ligase TRIM6 Ubiquitinates the Ebola Virus VP35 Protein and Promotes Virus Replication.

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Bharaj, Preeti; Atkins, Colm; Luthra, Priya; Giraldo, Maria Isabel; Dawes, Brian E; Miorin, Lisa; Johnson, Jeffrey R; Krogan, Nevan J; Basler, Christopher F; Freiberg, Alexander N; Rajsbaum, Ricardo

2017-09-15

Ebola virus (EBOV), a member of the Filoviridae family, is a highly pathogenic virus that causes severe hemorrhagic fever in humans and is responsible for epidemics throughout sub-Saharan, central, and West Africa. The EBOV genome encodes VP35, an important viral protein involved in virus replication by acting as an essential cofactor of the viral polymerase as well as a potent antagonist of the host antiviral type I interferon (IFN-I) system. By using mass spectrometry analysis and coimmunoprecipitation assays, we show here that VP35 is ubiquitinated on lysine 309 (K309), a residue located on its IFN antagonist domain. We also found that VP35 interacts with TRIM6, a member of the E3-ubiquitin ligase tripartite motif (TRIM) family. We recently reported that TRIM6 promotes the synthesis of unanchored K48-linked polyubiquitin chains, which are not covalently attached to any protein, to induce efficient antiviral IFN-I-mediated responses. Consistent with this notion, VP35 also associated noncovalently with polyubiquitin chains and inhibited TRIM6-mediated IFN-I induction. Intriguingly, we also found that TRIM6 enhances EBOV polymerase activity in a minigenome assay and TRIM6 knockout cells have reduced replication of infectious EBOV, suggesting that VP35 hijacks TRIM6 to promote EBOV replication through ubiquitination. Our work provides evidence that TRIM6 is an important host cellular factor that promotes EBOV replication, and future studies will focus on whether TRIM6 could be targeted for therapeutic intervention against EBOV infection. IMPORTANCE EBOV belongs to a family of highly pathogenic viruses that cause severe hemorrhagic fever in humans and other mammals with high mortality rates (40 to 90%). Because of its high pathogenicity and lack of licensed antivirals and vaccines, EBOV is listed as a tier 1 select-agent risk group 4 pathogen. An important mechanism for the severity of EBOV infection is its suppression of innate immune responses. The EBOV VP35

Strategies for future histocompatible stem cell therapy

DEFF Research Database (Denmark)

Nehlin, Jan; Barington, Torben

2009-01-01

Stem cell therapy based on the safe and unlimited self-renewal of human pluripotent stem cells is envisioned for future use in tissue or organ replacement after injury or disease. A gradual decline of regenerative capacity has been documented among the adult stem cell population in some body organs...... during the aging process. Recent progress in human somatic cell nuclear transfer and inducible pluripotent stem cell technologies has shown that patient-derived nuclei or somatic cells can be reprogrammed in vitro to become pluripotent stem cells, from which the three germ layer lineages can be generated......, genetically identical to the recipient. Once differentiation protocols and culture conditions can be defined and optimized, patient-histocompatible pluripotent stem cells could be directed towards virtually every cell type in the human body. Harnessing this capability to enrich for given cells within...

Construction and functional characterization of double and triple mutants of parallel beta-bulge of ubiquitin.

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Sharma, Mrinal; Prabha, C Ratna

2011-12-01

Ubiquitin, a small eukaryotic protein serving as a post-translational modification on many important proteins, plays central role in cellular homeostasis and cell cycle regulation. Ubiquitin features two beta-bulges, the second beta-bulge, located at the C-terminal region of the protein along with type II turn, holds 3 residues Glu64(1), Ser65(2) and Gln2(X). Percent frequency of occurrence of such a sequence in parallel beta-bulge is very low. However, the sequence and structure have been conserved in ubiquitin through out the evolution. Present study involves replacement of residues in unusual beta-bulge of ubiquitin by introducing mutations in combination through site directed mutagenesis, generating double and triple mutants and their functional characterization. Mutant ubiquitins cloned in yeast expression vector YEp96 tested for growth profile, viability assay and heat stress complementation study have revealed significant decrease in growth rate, loss of viability and non-complementation of heat sensitive phenotype with UbE64G-S65D and UbQ2N-E64G-S65D mutations. However, UbQ2N-S65D did not show any negative effects in the above assays. Present results show that, replacement of residues in beta-bulge of ubiquitin exerts severe effects on growth and viability in Saccharomyces cerevisiae due to functional failure of the mutant ubiquitins UbE64G-S65D and UbQ2N-E64G-S65D.

Ubiquitinated CD36 sustains insulin-stimulated Akt activation by stabilizing insulin receptor substrate 1 in myotubes.

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Sun, Shishuo; Tan, Pengcheng; Huang, Xiaoheng; Zhang, Wei; Kong, Chen; Ren, Fangfang; Su, Xiong

2018-02-16

Both the magnitude and duration of insulin signaling are important in executing its cellular functions. Insulin-induced degradation of insulin receptor substrate 1 (IRS1) represents a key negative feedback loop that restricts insulin signaling. Moreover, high concentrations of fatty acids (FAs) and glucose involved in the etiology of obesity-associated insulin resistance also contribute to the regulation of IRS1 degradation. The scavenger receptor CD36 binds many lipid ligands, and its contribution to insulin resistance has been extensively studied, but the exact regulation of insulin sensitivity by CD36 is highly controversial. Herein, we found that CD36 knockdown in C2C12 myotubes accelerated insulin-stimulated Akt activation, but the activated signaling was sustained for a much shorter period of time as compared with WT cells, leading to exacerbated insulin-induced insulin resistance. This was likely due to enhanced insulin-induced IRS1 degradation after CD36 knockdown. Overexpression of WT CD36, but not a ubiquitination-defective CD36 mutant, delayed IRS1 degradation. We also found that CD36 functioned through ubiquitination-dependent binding to IRS1 and inhibiting its interaction with cullin 7, a key component of the multisubunit cullin-RING E3 ubiquitin ligase complex. Moreover, dissociation of the Src family kinase Fyn from CD36 by free FAs or Fyn knockdown/inhibition accelerated insulin-induced IRS1 degradation, likely due to disrupted IRS1 interaction with CD36 and thus enhanced binding to cullin 7. In summary, we identified a CD36-dependent FA-sensing pathway that plays an important role in negative feedback regulation of insulin activation and may open up strategies for preventing or managing type 2 diabetes mellitus. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Arabidopsis SH3P2 is an ubiquitin-binding protein that functions together with ESCRT-I and the deubiquitylating enzyme AMSH3.

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Nagel, Marie-Kristin; Kalinowska, Kamila; Vogel, Karin; Reynolds, Gregory D; Wu, Zhixiang; Anzenberger, Franziska; Ichikawa, Mie; Tsutsumi, Chie; Sato, Masa H; Kuster, Bernhard; Bednarek, Sebastian Y; Isono, Erika

2017-08-22

Clathrin-mediated endocytosis of plasma membrane proteins is an essential regulatory process that controls plasma membrane protein abundance and is therefore important for many signaling pathways, such as hormone signaling and biotic and abiotic stress responses. On endosomal sorting, plasma membrane proteins maybe recycled or targeted for vacuolar degradation, which is dependent on ubiquitin modification of the cargos and is driven by the endosomal sorting complexes required for transport (ESCRTs). Components of the ESCRT machinery are highly conserved among eukaryotes, but homologs of ESCRT-0 that are responsible for recognition and concentration of ubiquitylated proteins are absent in plants. Recently several ubiquitin-binding proteins have been identified that serve in place of ESCRT-0; however, their function in ubiquitin recognition and endosomal trafficking is not well understood yet. In this study, we identified Src homology-3 (SH3) domain-containing protein 2 (SH3P2) as a ubiquitin- and ESCRT-I-binding protein that functions in intracellular trafficking. SH3P2 colocalized with clathrin light chain-labeled punctate structures and interacted with clathrin heavy chain in planta , indicating a role for SH3P2 in clathrin-mediated endocytosis. Furthermore, SH3P2 cofractionates with clathrin-coated vesicles (CCVs), suggesting that it associates with CCVs in planta Mutants of SH3P2 and VPS23 genetically interact, suggesting that they could function in the same pathway. Based on these results, we suggest a role of SH3P2 as an ubiquitin-binding protein that binds and transfers ubiquitylated proteins to the ESCRT machinery.

Definitive evidence for Ufd2-catalyzed elongation of the ubiquitin chain through Lys48 linkage

International Nuclear Information System (INIS)

Saeki, Yasushi; Tayama, Yoko; Toh-e, Akio; Yokosawa, Hideyoshi

2004-01-01

Saccharomyces cerevisiae Ufd2 is a ubiquitin chain elongation factor in the ubiquitin fusion degradation (UFD) pathway and functions in stress tolerance. A recent study has suggested that the mammalian Ufd2 homologue UFD2a catalyzes formation of Lys27- and Lys33-linked polyubiquitin chains rather than the Lys48-linked chain, but the linkage type of the polyubiquitin chain formed by yeast Ufd2 remains unclear. To determine the property of Ufd2, we reconstituted the UFD pathway using purified enzymes from yeast. Direct determination of the ubiquitin chain linkage type in polyubiquitinated UFD substrates by MALDI-TOF mass spectrometry revealed that Ufd2 catalyzes elongation of the ubiquitin chain through Lys48 linkage

Chain Assembly and Disassembly Processes Differently Affect the Conformational Space of Ubiquitin Chains.

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Kniss, Andreas; Schuetz, Denise; Kazemi, Sina; Pluska, Lukas; Spindler, Philipp E; Rogov, Vladimir V; Husnjak, Koraljka; Dikic, Ivan; Güntert, Peter; Sommer, Thomas; Prisner, Thomas F; Dötsch, Volker

2018-02-06

Ubiquitination is the most versatile posttranslational modification. The information is encoded by linkage type as well as chain length, which are translated by ubiquitin binding domains into specific signaling events. Chain topology determines the conformational space of a ubiquitin chain and adds an additional regulatory layer to this ubiquitin code. In particular, processes that modify chain length will be affected by chain conformations as they require access to the elongation or cleavage sites. We investigated conformational distributions in the context of chain elongation and disassembly using pulsed electron-electron double resonance spectroscopy in combination with molecular modeling. Analysis of the conformational space of diubiquitin revealed conformational selection or remodeling as mechanisms for chain recognition during elongation or hydrolysis, respectively. Chain elongation to tetraubiquitin increases the sampled conformational space, suggesting that a high intrinsic flexibility of K48-linked chains may contribute to efficient proteasomal degradation. Copyright © 2017 Elsevier Ltd. All rights reserved.

Phosphorylation by PINK1 releases the UBL domain and initializes the conformational opening of the E3 ubiquitin ligase Parkin.

Directory of Open Access Journals (Sweden)

Thomas R Caulfield

2014-11-01

Full Text Available Loss-of-function mutations in PINK1 or PARKIN are the most common causes of autosomal recessive Parkinson's disease. Both gene products, the Ser/Thr kinase PINK1 and the E3 Ubiquitin ligase Parkin, functionally cooperate in a mitochondrial quality control pathway. Upon stress, PINK1 activates Parkin and enables its translocation to and ubiquitination of damaged mitochondria to facilitate their clearance from the cell. Though PINK1-dependent phosphorylation of Ser65 is an important initial step, the molecular mechanisms underlying the activation of Parkin's enzymatic functions remain unclear. Using molecular modeling, we generated a complete structural model of human Parkin at all atom resolution. At steady state, the Ub ligase is maintained inactive in a closed, auto-inhibited conformation that results from intra-molecular interactions. Evidently, Parkin has to undergo major structural rearrangements in order to unleash its catalytic activity. As a spark, we have modeled PINK1-dependent Ser65 phosphorylation in silico and provide the first molecular dynamics simulation of Parkin conformations along a sequential unfolding pathway that could release its intertwined domains and enable its catalytic activity. We combined free (unbiased molecular dynamics simulation, Monte Carlo algorithms, and minimal-biasing methods with cell-based high content imaging and biochemical assays. Phosphorylation of Ser65 results in widening of a newly defined cleft and dissociation of the regulatory N-terminal UBL domain. This motion propagates through further opening conformations that allow binding of an Ub-loaded E2 co-enzyme. Subsequent spatial reorientation of the catalytic centers of both enzymes might facilitate the transfer of the Ub moiety to charge Parkin. Our structure-function study provides the basis to elucidate regulatory mechanisms and activity of the neuroprotective Parkin. This may open up new avenues for the development of small molecule Parkin

The Banana Fruit SINA Ubiquitin Ligase MaSINA1 Regulates the Stability of MaICE1 to be Negatively Involved in Cold Stress Response.

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Fan, Zhong-Qi; Chen, Jian-Ye; Kuang, Jian-Fei; Lu, Wang-Jin; Shan, Wei

2017-01-01

The regulation of ICE1 protein stability is important to ensure effective cold stress response, and is extensively studied in Arabidopsis . Currently, how ICE1 stability in fruits under cold stress is controlled remains largely unknown. Here, we reported the possible involvement of a SEVEN IN ABSENTIA (SINA) ubiquitin ligase MaSINA1 from banana fruit in affecting MaICE1 stability. MaSINA1 was identified based on a yeast two-hybrid screening using MaICE1 as bait. Further yeast two-hybrid, pull-down, bimolecular fluorescence complementation (BiFC) and co-immunoprecipitation (CoIP) assays confirmed that MaSINA1 interacted with MaICE1. The expression of MaSINA1 was repressed by cold stress. Subcellular localization analysis in tobacco leaves showed that MaSINA1 was localized predominantly in the nucleus. In vitro ubiquitination assay showed that MaSINA1 possessed E3 ubiquitin ligase activity. More importantly, in vitro and semi- in vivo experiments indicated that MaSINA1 can ubiquitinate MaICE1 for the 26S proteasome-dependent degradation, and therefore suppressed the transcriptional activation of MaICE1 to MaNAC1, an important regulator of cold stress response of banana fruit. Collectively, our data reveal a mechanism in banana fruit for control of the stability of ICE1 and for the negative regulation of cold stress response by a SINA E3 ligase via the ubiquitin proteasome system.

Protection against murine osteoarthritis by inhibition of the 26S proteasome and lysine-48 linked ubiquitination.

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Radwan, Marta; Wilkinson, David J; Hui, Wang; Destrument, Auriane P M; Charlton, Sarah H; Barter, Matt J; Gibson, Beth; Coulombe, Josée; Gray, Douglas A; Rowan, Andrew D; Young, David A

2015-08-01

To determine whether the process of ubiquitination and/or activity of the 26S proteasome are involved in the induction of osteoarthritis (OA). Bovine cartilage resorption assays, chondrocyte cell-line SW1353 and primary human articular chondrocytes were used with the general proteasome inhibitor MG132 or vehicle to identify a role of the ubiquitin-proteasome system (UPS) in cartilage destruction and matrix metalloproteinase-13 (MMP13) expression. In vivo, MG132 or vehicle, were delivered subcutaneously to mice following destabilisation of the medial meniscus (DMM)-induced OA. Subsequently, DMM was induced in Lys-to-Arg (K48R and K63R) mutant ubiquitin (Ub) transgenic mice. Cytokine signalling in SW1353s was monitored by immunoblotting and novel ubiquitinated substrates identified using Tandem Ubiquitin Binding Entities purification followed by mass spectrometry. The ubiquitination of TRAFD1 was assessed via immunoprecipitation and immunoblotting and its role in cytokine signal-transduction determined using RNA interference and real-time RT-PCR for MMP13 and interleukin-6 (IL6). Supplementation with the proteasome inhibitor MG132 protected cartilage from cytokine-mediated resorption and degradation in vivo in mice following DMM-induced OA. Using transgenic animals only K48R-mutated Ub partially protected against OA compared to wild-type or wild-type Ub transgenic mice, and this was only evident on the medial femoral condyle. After confirming ubiquitination was vital for NF-κB signalling and MMP13 expression, a screen for novel ubiquitinated substrates involved in cytokine-signalling identified TRAFD1; the depletion of which reduced inflammatory mediator-induced MMP13 and IL6 expression. Our data for the first time identifies a role for ubiquitination and the proteasome in the induction of OA via regulation of inflammatory mediator-induced MMP13 expression. These data open avenues of research to determine whether the proteasome, or K48-linked ubiquitination, are

USP7 Is a Suppressor of PCNA Ubiquitination and Oxidative-Stress-Induced Mutagenesis in Human Cells.

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Kashiwaba, Shu-ichiro; Kanao, Rie; Masuda, Yuji; Kusumoto-Matsuo, Rika; Hanaoka, Fumio; Masutani, Chikahide

2015-12-15

Mono-ubiquitinated PCNA activates error-prone DNA polymerases; therefore, strict regulation of PCNA mono-ubiquitination is crucial in avoiding undesired mutagenesis. In this study, we used an in vitro assay system to identify USP7 as a deubiquitinating enzyme of mono-ubiquitinated PCNA. Suppression of USP1, a previously identified PCNA deubiquitinase, or USP7 increased UV- and H2O2-induced PCNA mono-ubiquitination in a distinct and additive manner, suggesting that USP1 and USP7 make different contributions to PCNA deubiquitination in human cells. Cell-cycle-synchronization analyses revealed that USP7 suppression increased H2O2-induced PCNA ubiquitination throughout interphase, whereas USP1 suppression specifically increased ubiquitination in S-phase cells. UV-induced mutagenesis was elevated in USP1-suppressed cells, whereas H2O2-induced mutagenesis was elevated in USP7-suppressed cells. These results suggest that USP1 suppresses UV-induced mutations produced in a manner involving DNA replication, whereas USP7 suppresses H2O2-induced mutagenesis involving cell-cycle-independent processes such as DNA repair. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Pax3 stimulates p53 ubiquitination and degradation independent of transcription.

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Xiao Dan Wang

Full Text Available Pax3 is a developmental transcription factor that is required for neural tube and neural crest development. We previously showed that inactivating the p53 tumor suppressor protein prevents neural tube and cardiac neural crest defects in Pax3-mutant mouse embryos. This demonstrates that Pax3 regulates these processes by blocking p53 function. Here we investigated the mechanism by which Pax3 blocks p53 function.We employed murine embryonic stem cell (ESC-derived neuronal precursors as a cell culture model of embryonic neuroepithelium or neural crest. Pax3 reduced p53 protein stability, but had no effect on p53 mRNA levels or the rate of p53 synthesis. Full length Pax3 as well as fragments that contained either the DNA-binding paired box or the homeodomain, expressed as GST or FLAG fusion proteins, physically associated with p53 and Mdm2 both in vitro and in vivo. In contrast, Splotch Pax3, which causes neural tube and neural crest defects in homozygous embryos, bound weakly, or not at all, to p53 or Mdm2. The paired domain and homeodomain each stimulated Mdm2-mediated ubiquitination of p53 and p53 degradation in the absence of the Pax3 transcription regulatory domains, whereas Splotch Pax3 did not stimulate p53 ubiquitination or degradation.Pax3 inactivates p53 function by stimulating its ubiquitination and degradation. This process utilizes the Pax3 paired domain and homeodomain but is independent of DNA-binding and transcription regulation. Because inactivating p53 is the only required Pax3 function during neural tube closure and cardiac neural crest development, and inactivating p53 does not require Pax3-dependent transcription regulation, this indicates that Pax3 is not required to function as a transcription factor during neural tube closure and cardiac neural crest development. These findings further suggest novel explanations for PAX3 functions in human diseases, such as in neural crest-derived cancers and Waardenburg syndrome types 1 and 3.

The linear ubiquitin assembly complex (LUBAC) is essential for NLRP3 inflammasome activation

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Rodgers, Mary A.; Bowman, James W.; Fujita, Hiroaki; Orazio, Nicole; Shi, Mude; Liang, Qiming; Amatya, Rina; Kelly, Thomas J.; Iwai, Kazuhiro; Ting, Jenny

2014-01-01

Linear ubiquitination is a newly discovered posttranslational modification that is currently restricted to a small number of known protein substrates. The linear ubiquitination assembly complex (LUBAC), consisting of HOIL-1L, HOIP, and Sharpin, has been reported to activate NF-κB–mediated transcription in response to receptor signaling by ligating linear ubiquitin chains to Nemo and Rip1. Despite recent advances, the detailed roles of LUBAC in immune cells remain elusive. We demonstrate a novel HOIL-1L function as an essential regulator of the activation of the NLRP3/ASC inflammasome in primary bone marrow–derived macrophages (BMDMs) independently of NF-κB activation. Mechanistically, HOIL-1L is required for assembly of the NLRP3/ASC inflammasome and the linear ubiquitination of ASC, which we identify as a novel LUBAC substrate. Consequently, we find that HOIL-1L−/− mice have reduced IL-1β secretion in response to in vivo NLRP3 stimulation and survive lethal challenge with LPS. Together, these data demonstrate that linear ubiquitination is required for NLRP3 inflammasome activation, defining the molecular events of NLRP3 inflammasome activation and expanding the role of LUBAC as an innate immune regulator. Furthermore, our observation is clinically relevant because patients lacking HOIL-1L expression suffer from pyogenic bacterial immunodeficiency, providing a potential new therapeutic target for enhancing inflammation in immunodeficient patients. PMID:24958845

Ubiquitin regulates TORC1 in yeast Saccharomyces cerevisiae.

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Hu, Kejin; Guo, Shuguang; Yan, Gonghong; Yuan, Wenjie; Zheng, Yin; Jiang, Yu

2016-04-01

In the yeast Saccharomyces cerevisiae the TOR complex 1 (TORC1) controls many growth-related cellular processes and is essential for cell growth and proliferation. Macrolide antibiotic rapamycin, in complex with a cytosol protein named FKBP12, specifically inhibits TORC1, causing growth arrest. The FKBP12-rapamycin complex interferes with TORC1 function by binding to the FRB domain of the TOR proteins. In an attempt to understand the role of the FRB domain in TOR function, we identified a single point mutation (Tor2(W2041R) ) in the FRB domain of Tor2 that renders yeast cells rapamycin resistant and temperature sensitive. At the permissive temperature, the Tor2 mutant protein is partially defective for binding with Kog1 and TORC1 is impaired for membrane association. At the restrictive temperature, Kog1 but not the Tor2 mutant protein, is rapidly degraded. Overexpression of ubiquitin stabilizes Kog1 and suppresses the growth defect associated with the tor2 mutant at the nonpremissive temperature. We find that ubiquitin binds non-covalently to Kog1, prevents Kog1 from degradation and stabilizes TORC1. Our data reveal a unique role for ubiquitin in regulation of TORC1 and suggest that Kog1 requires association with the Tor proteins for stabilization. © 2016 John Wiley & Sons Ltd.

LUBAC-Recruited CYLD and A20 Regulate Gene Activation and Cell Death by Exerting Opposing Effects on Linear Ubiquitin in Signaling Complexes

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Peter Draber

2015-12-01

Full Text Available Ubiquitination and deubiquitination are crucial for assembly and disassembly of signaling complexes. LUBAC-generated linear (M1 ubiquitin is important for signaling via various immune receptors. We show here that the deubiquitinases CYLD and A20, but not OTULIN, are recruited to the TNFR1- and NOD2-associated signaling complexes (TNF-RSC and NOD2-SC, at which they cooperate to limit gene activation. Whereas CYLD recruitment depends on its interaction with LUBAC, but not on LUBAC’s M1-chain-forming capacity, A20 recruitment requires this activity. Intriguingly, CYLD and A20 exert opposing effects on M1 chain stability in the TNF-RSC and NOD2-SC. While CYLD cleaves M1 chains, and thereby sensitizes cells to TNF-induced death, A20 binding to them prevents their removal and, consequently, inhibits cell death. Thus, CYLD and A20 cooperatively restrict gene activation and regulate cell death via their respective activities on M1 chains. Hence, the interplay between LUBAC, M1-ubiquitin, CYLD, and A20 is central for physiological signaling through innate immune receptors.

Role of the ubiquitin-proteasome system in brain ischemia: friend or foe?

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Caldeira, Margarida V; Salazar, Ivan L; Curcio, Michele; Canzoniero, Lorella M T; Duarte, Carlos B

2014-01-01

The ubiquitin-proteasome system (UPS) is a catalytic machinery that targets numerous cellular proteins for degradation, thus being essential to control a wide range of basic cellular processes and cell survival. Degradation of intracellular proteins via the UPS is a tightly regulated process initiated by tagging a target protein with a specific ubiquitin chain. Neurons are particularly vulnerable to any change in protein composition, and therefore the UPS is a key regulator of neuronal physiology. Alterations in UPS activity may induce pathological responses, ultimately leading to neuronal cell death. Brain ischemia triggers a complex series of biochemical and molecular mechanisms, such as an inflammatory response, an exacerbated production of misfolded and oxidized proteins, due to oxidative stress, and the breakdown of cellular integrity mainly mediated by excitotoxic glutamatergic signaling. Brain ischemia also damages protein degradation pathways which, together with the overproduction of damaged proteins and consequent upregulation of ubiquitin-conjugated proteins, contribute to the accumulation of ubiquitin-containing proteinaceous deposits. Despite recent advances, the factors leading to deposition of such aggregates after cerebral ischemic injury remain poorly understood. This review discusses the current knowledge on the role of the UPS in brain function and the molecular mechanisms contributing to UPS dysfunction in brain ischemia with consequent accumulation of ubiquitin-containing proteins. Chemical inhibitors of the proteasome and small molecule inhibitors of deubiquitinating enzymes, which promote the degradation of proteins by the proteasome, were both shown to provide neuroprotection in brain ischemia, and this apparent contradiction is also discussed in this review. Copyright © 2013 Elsevier Ltd. All rights reserved.

Effects of major depression on remission and relapse of substance dependence.

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Hasin, Deborah; Liu, Xinhua; Nunes, Edward; McCloud, Steven; Samet, Sharon; Endicott, Jean

2002-04-01

The effects of major depressive disorder (MDD) on the course of substance dependence may differ depending on the temporal relationship of depression to dependence. We investigated the effects of MDD on the outcome of substance dependence under 3 circumstances: (1) lifetime onset of MDD prior to lifetime onset of dependence onset, (2) current MDD occurring during a period of abstinence, and (3) current MDD during substance use that exceeded the expected effects of intoxication or withdrawal. A sample of 250 inpatients with DSM-IV cocaine, heroin, and/or alcohol dependence were followed up at 6, 12, and 18 months. The Psychiatric Research Interview for Substance and Mental Disorders (PRISM) was used to make DSM-IV diagnoses. Using Cox proportional hazards models, stable remissions (those lasting at least 26 weeks) from DSM-IV cocaine, heroin, and/or alcohol dependence and from use were studied, as well as subsequent relapses of dependence and use. Patients with current substance-induced MDD were less likely to remit from dependence (adjusted hazards ratio, 0.11) than patients with no baseline MDD. A history of MDD prior to lifetime onset of substance dependence also reduced the likelihood of remission relative to the absence of such a history (adjusted hazard ratio, 0.49). Major depressive disorder during sustained abstinence predicted dependence relapse (adjusted hazards ratio, 3.07) and substance use after hospital discharge compared with those without abstinence MDD (adjusted hazards ratio, 1.45). The timing of depressive episodes relative to substance dependence served as an important factor in the remission and relapse of substance dependence and substance use.

Ubiquitination-Linked Phosphorylation of the FANCI S/TQ Cluster Contributes to Activation of the Fanconi Anemia I/D2 Complex

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Ronald S. Cheung

2017-06-01

Full Text Available Repair of interstrand crosslinks by the Fanconi anemia (FA pathway requires both monoubiquitination and de-ubiquitination of the FANCI/FANCD2 (FANCI/D2 complex. In the standing model, the phosphorylation of six sites in the FANCI S/TQ cluster domain occurs upstream of, and promotes, FANCI/D2 monoubiquitination. We generated phospho-specific antibodies against three different S/TQ cluster sites (serines 556, 559, and 565 on human FANCI and found that, in contrast to the standing model, distinct FANCI sites were phosphorylated either predominantly upstream (ubiquitination independent; serine 556 or downstream (ubiquitination-linked; serines 559 and 565 of FANCI/D2 monoubiquitination. Ubiquitination-linked FANCI phosphorylation inhibited FANCD2 de-ubiquitination and bypassed the need to de-ubiquitinate FANCD2 to achieve effective interstrand crosslink repair. USP1 depletion suppressed ubiquitination-linked FANCI phosphorylation despite increasing FANCI/D2 monoubiquitination, providing an explanation of why FANCD2 de-ubiquitination is important for function of the FA pathway. Our work results in a refined model of how FANCI phosphorylation activates the FANCI/D2 complex.

Proteins containing the UBA domain are able to bind to multi-ubiquitin chains

DEFF Research Database (Denmark)

Wilkinson, C R; Seeger, M; Hartmann-Petersen, R

2001-01-01

The UBA domain is a motif found in a variety of proteins, some of which are associated with the ubiquitin-proteasome system. We describe the isolation of a fission-yeast gene, mud1+, which encodes a UBA domain containing protein that is able to bind multi-ubiquitin chains. We show that the UBA do...

Smurf2 E3 ubiquitin ligase modulates proliferation and invasiveness of breast cancer cells in a CNKSR2 dependent manner

OpenAIRE

David, Diana; Jagadeeshan, Sankar; Hariharan, Ramkumar; Nair, Asha Sivakumari; Pillai, Radhakrishna Madhavan

2014-01-01

Background Smurf2 is a member of the HECT family of E3 ubiquitin ligases that play important roles in determining the competence of cells to respond to TGF- β/BMP signaling pathway. However, besides TGF-β/BMP pathway, Smurf2 regulates a repertoire of other signaling pathways ranging from planar cell polarity during embryonic development to cell proliferation, migration, differentiation and senescence. Expression of Smurf2 is found to be dysregulated in many cancers including breast cancer. Th...

Linear ubiquitination is involved in the pathogenesis of optineurin-associated amyotrophic lateral sclerosis

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Nakazawa, Seshiru; Oikawa, Daisuke; Ishii, Ryohei; Ayaki, Takashi; Takahashi, Hirotaka; Takeda, Hiroyuki; Ishitani, Ryuichiro; Kamei, Kiyoko; Takeyoshi, Izumi; Kawakami, Hideshi; Iwai, Kazuhiro; Hatada, Izuho; Sawasaki, Tatsuya; Ito, Hidefumi; Nureki, Osamu; Tokunaga, Fuminori

2016-01-01

Optineurin (OPTN) mutations cause neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and glaucoma. Although the ALS-associated E478G mutation in the UBAN domain of OPTN reportedly abolishes its NF-κB suppressive activity, the precise molecular basis in ALS pathogenesis still remains unclear. Here we report that the OPTN-UBAN domain is crucial for NF-κB suppression. Our crystal structure analysis reveals that OPTN-UBAN binds linear ubiquitin with homology to NEMO. TNF-α-mediated NF-κB activation is enhanced in OPTN-knockout cells, through increased ubiquitination and association of TNF receptor (TNFR) complex I components. Furthermore, OPTN binds caspase 8, and OPTN deficiency accelerates TNF-α-induced apoptosis by enhancing complex II formation. Immunohistochemical analyses of motor neurons from OPTN-associated ALS patients reveal that linear ubiquitin and activated NF-κB are partially co-localized with cytoplasmic inclusions, and that activation of caspases is elevated. Taken together, OPTN regulates both NF-κB activation and apoptosis via linear ubiquitin binding, and the loss of this ability may lead to ALS. PMID:27552911

Membrane-localized ubiquitin ligase ATL15 functions in sugar-responsive growth regulation in Arabidopsis.

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Aoyama, Shoki; Terada, Saki; Sanagi, Miho; Hasegawa, Yoko; Lu, Yu; Morita, Yoshie; Chiba, Yukako; Sato, Takeo; Yamaguchi, Junji

2017-09-09

Ubiquitin ligases play important roles in regulating various cellular processes by modulating the protein function of specific ubiquitination targets. The Arabidopsis Tóxicos en Levadura (ATL) family is a group of plant-specific RING-type ubiquitin ligases that localize to membranes via their N-terminal transmembrane-like domains. To date, 91 ATL isoforms have been identified in the Arabidopsis genome, with several ATLs reported to be involved in regulating plant responses to environmental stresses. However, the functions of most ATLs remain unknown. This study, involving transcriptome database analysis, identifies ATL15 as a sugar responsive ATL gene in Arabidopsis. ATL15 expression was rapidly down-regulated in the presence of sugar. The ATL15 protein showed ubiquitin ligase activity in vitro and localized to plasma membrane and endomembrane compartments. Further genetic analyses demonstrated that the atl15 knockout mutants are insensitive to high glucose concentrations, whereas ATL15 overexpression depresses plant growth. In addition, endogenous glucose and starch amounts were reciprocally affected in the atl15 knockout mutants and the ATL15 overexpressors. These results suggest that ATL15 protein plays a significant role as a membrane-localized ubiquitin ligase that regulates sugar-responsive plant growth in Arabidopsis. Copyright © 2017 Elsevier Inc. All rights reserved.

An extract of Artemisia dracunculus L. inhibits ubiquitin-proteasome activity and preserves skeletal muscle mass in a murine model of diabetes.

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Heather Kirk-Ballard

Full Text Available Impaired insulin signaling is a key feature of type 2 diabetes and is associated with increased ubiquitin-proteasome-dependent protein degradation in skeletal muscle. An extract of Artemisia dracunculus L. (termed PMI5011 improves insulin action by increasing insulin signaling in skeletal muscle. We sought to determine if the effect of PMI5011 on insulin signaling extends to regulation of the ubiquitin-proteasome system. C2C12 myotubes and the KK-A(y murine model of type 2 diabetes were used to evaluate the effect of PMI5011 on steady-state levels of ubiquitylation, proteasome activity and expression of Atrogin-1 and MuRF-1, muscle-specific ubiquitin ligases that are upregulated with impaired insulin signaling. Our results show that PMI5011 inhibits proteasome activity and steady-state ubiquitylation levels in vitro and in vivo. The effect of PMI5011 is mediated by PI3K/Akt signaling and correlates with decreased expression of Atrogin-1 and MuRF-1. Under in vitro conditions of hormonal or fatty acid-induced insulin resistance, PMI5011 improves insulin signaling and reduces Atrogin-1 and MuRF-1 protein levels. In the KK-A(y murine model of type 2 diabetes, skeletal muscle ubiquitylation and proteasome activity is inhibited and Atrogin-1 and MuRF-1 expression is decreased by PMI5011. PMI5011-mediated changes in the ubiquitin-proteasome system in vivo correlate with increased phosphorylation of Akt and FoxO3a and increased myofiber size. The changes in Atrogin-1 and MuRF-1 expression, ubiquitin-proteasome activity and myofiber size modulated by PMI5011 in the presence of insulin resistance indicate the botanical extract PMI5011 may have therapeutic potential in the preservation of muscle mass in type 2 diabetes.

The ubiquitin C-terminal hydrolase UCH-L1 promotes bacterial invasion by altering the dynamics of the actin cytoskeleton

DEFF Research Database (Denmark)

Basseres, Eugene; Coppotelli, Giuseppe; Pfirrmann, Thorsten

2010-01-01

Invasion of eukaryotic target cells by pathogenic bacteria requires extensive remodelling of the membrane and actin cytoskeleton. Here we show that the remodelling process is regulated by the ubiquitin C-terminal hydrolase UCH-L1 that promotes the invasion of epithelial cells by Listeria monocyto......Invasion of eukaryotic target cells by pathogenic bacteria requires extensive remodelling of the membrane and actin cytoskeleton. Here we show that the remodelling process is regulated by the ubiquitin C-terminal hydrolase UCH-L1 that promotes the invasion of epithelial cells by Listeria...... of downstream ERK1/2- and AKT-dependent signalling in response to the natural ligand Hepatocyte Growth Factor (HGF). The regulation of cytoskeleton dynamics was further confirmed by the induction of actin stress fibres in HeLa expressing the active enzyme but not the catalytic mutant UCH-L1(C90S...

Poly(ADP-ribosyl)ation links the chromatin remodeler SMARCA5/SNF2H to RNF168-dependent DNA damage signaling

DEFF Research Database (Denmark)

Smeenk, G.; Wiegant, W.W.; Luijsterburg, M.S.

2013-01-01

Ionizing radiation (IR)-induced DNA double-strand breaks (DSBs) arising in native chromatin elicit an RNF8/RNF168-dependent ubiquitylation response, which triggers the recruitment of various repair factors. Precisely how this response is regulated in the context of chromatin remains largely...... unexplored. Here, we show that SMARCA5/SNF2H, the catalytic subunit of ISWI chromatin remodeling complexes, is recruited to DSBs in a poly(ADP-ribose) polymerase 1 (PARP1)-dependent manner. Remarkably, PARP activity, although dispensable for the efficient spreading of νH2AX into damaged chromatin......, selectively promotes spreading of SMARCA5, the E3 ubiquitin ligase RNF168, ubiquitin conjugates and the ubiquitin-binding factors RAD18 and the RAP80-BRCA1 complex throughout DSB-flanking chromatin. This suggests that PARP regulates the spatial organization of the RNF168-driven ubiquitin response to DNA...

The Ubiquitin E3 Ligase TRAF6 Exacerbates Ischemic Stroke by Ubiquitinating and Activating Rac1.

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Li, Tao; Qin, Juan-Juan; Yang, Xia; Ji, Yan-Xiao; Guo, Fangliang; Cheng, Wen-Lin; Wu, Xiaolin; Gong, Fu-Han; Hong, Ying; Zhu, Xue-Yong; Gong, Jun; Wang, Zhihua; Huang, Zan; She, Zhi-Gang; Li, Hongliang

2017-12-13

Stroke is one of the leading causes of morbidity and mortality worldwide. Inflammation, oxidative stress, apoptosis, and excitotoxicity contribute to neuronal death during ischemic stroke; however, the mechanisms underlying these complicated pathophysiological processes remain to be fully elucidated. Here, we found that the expression of tumor necrosis factor receptor-associated factor 6 (TRAF6) was markedly increased after cerebral ischemia/reperfusion (I/R) in mice. TRAF6 ablation in male mice decreased the infarct volume and neurological deficit scores and decreased proinflammatory signaling, oxidative stress, and neuronal death after cerebral I/R, whereas transgenic overexpression of TRAF6 in male mice exhibited the opposite effects. Mechanistically, we demonstrated that TRAF6 induced Rac1 activation and consequently promoted I/R injury by directly binding and ubiquitinating Rac1. Either functionally mutating the TRAF6 ubiquitination site on Rac1 or inactivating Rac1 with a specific inhibitor reversed the deleterious effects of TRAF6 overexpression during I/R injury. In conclusion, our study demonstrated that TRAF6 is a key promoter of ischemic signaling cascades and neuronal death after cerebral I/R injury. Therefore, the TRAF6/Rac1 pathway might be a promising target to attenuate cerebral I/R injury. SIGNIFICANCE STATEMENT Stroke is one of the most severe and devastating neurological diseases globally. The complicated pathophysiological processes restrict the translation of potential therapeutic targets into medicine. Further elucidating the molecular mechanisms underlying cerebral ischemia/reperfusion injury may open a new window for pharmacological interventions to promote recovery from stroke. Our study revealed that ischemia-induced tumor necrosis factor receptor-associated factor 6 (TRAF6) upregulation binds and ubiquitinates Rac1 directly, which promotes neuron death through neuroinflammation and neuro-oxidative signals. Therefore, precisely targeting

BRCA1 Is a Histone-H2A-Specific Ubiquitin Ligase

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Reinhard Kalb

2014-08-01

Full Text Available The RING domain proteins BRCA1 and BARD1 comprise a heterodimeric ubiquitin (E3 ligase that is required for the accumulation of ubiquitin conjugates at sites of DNA damage and for silencing at DNA satellite repeat regions. Despite its links to chromatin, the substrate and underlying function of the BRCA1/BARD1 ubiquitin ligase remain unclear. Here, we show that BRCA1/BARD1 specifically ubiquitylates histone H2A in its C-terminal tail on lysines 127 and 129 in vitro and in vivo. The specificity for K127-129 is acquired only when H2A is within a nucleosomal context. Moreover, site-specific targeting of the BRCA1/BARD1 RING domains to chromatin is sufficient for H2Aub foci formation in vivo. Our data establish BRCA1/BARD1 as a histone-H2A-specific E3 ligase, helping to explain its localization and activities on chromatin in cells.

Strategic role of the ubiquitin-dependent segregase p97 (VCP or Cdc48) in DNA replication.

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Ramadan, Kristijan; Halder, Swagata; Wiseman, Katherine; Vaz, Bruno

2017-02-01

Genome amplification (DNA synthesis) is one of the most demanding cellular processes in all proliferative cells. The DNA replication machinery (also known as the replisome) orchestrates genome amplification during S-phase of the cell cycle. Genetic material is particularly vulnerable to various events that can challenge the replisome during its assembly, activation (firing), progression (elongation) and disassembly from chromatin (termination). Any disturbance of the replisome leads to stalling of the DNA replication fork and firing of dormant replication origins, a process known as DNA replication stress. DNA replication stress is considered to be one of the main causes of sporadic cancers and other pathologies related to tissue degeneration and ageing. The mechanisms of replisome assembly and elongation during DNA synthesis are well understood. However, once DNA synthesis is complete, the process of replisome disassembly, and its removal from chromatin, remains unclear. In recent years, a growing body of evidence has alluded to a central role in replisome regulation for the ubiquitin-dependent protein segregase p97, also known as valosin-containing protein (VCP) in metazoans and Cdc48 in lower eukaryotes. By orchestrating the spatiotemporal turnover of the replisome, p97 plays an essential role in DNA replication. In this review, we will summarise our current knowledge about how p97 controls the replisome from replication initiation, to elongation and finally termination. We will also further examine the more recent findings concerning the role of p97 and how mutations in p97 cofactors, also known as adaptors, cause DNA replication stress induced genomic instability that leads to cancer and accelerated ageing. To our knowledge, this is the first comprehensive review concerning the mechanisms involved in the regulation of DNA replication by p97.

Dissecting the function of Cullin-RING ubiquitin ligase complex genes in planarian regeneration.

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Strand, Nicholas S; Allen, John M; Ghulam, Mahjoobah; Taylor, Matthew R; Munday, Roma K; Carrillo, Melissa; Movsesyan, Artem; Zayas, Ricardo M

2018-01-15

The ubiquitin system plays a role in nearly every aspect of eukaryotic cell biology. The enzymes responsible for transferring ubiquitin onto specific substrates are the E3 ubiquitin ligases, a large and diverse family of proteins, for which biological roles and target substrates remain largely undefined. Studies using model organisms indicate that ubiquitin signaling mediates key steps in developmental processes and tissue regeneration. Here, we used the freshwater planarian, Schmidtea mediterranea, to investigate the role of Cullin-RING ubiquitin ligase (CRL) complexes in stem cell regulation during regeneration. We identified six S. mediterranea cullin genes, and used RNAi to uncover roles for homologs of Cullin-1, -3 and -4 in planarian regeneration. The cullin-1 RNAi phenotype included defects in blastema formation, organ regeneration, lesions, and lysis. To further investigate the function of cullin-1-mediated cellular processes in planarians, we examined genes encoding the adaptor protein Skp1 and F-box substrate-recognition proteins that are predicted to partner with Cullin-1. RNAi against skp1 resulted in phenotypes similar to cullin-1 RNAi, and an RNAi screen of the F-box genes identified 19 genes that recapitulated aspects of cullin-1 RNAi, including ones that in mammals are involved in stem cell regulation and cancer biology. Our data provides evidence that CRLs play discrete roles in regenerative processes and provide a platform to investigate how CRLs regulate stem cells in vivo. Copyright © 2017 Elsevier Inc. All rights reserved.

Origin and diversification of TRIM ubiquitin ligases.

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Ignacio Marín

Full Text Available Most proteins of the TRIM family (also known as RBCC family are ubiquitin ligases that share a peculiar protein structure, characterized by including an N-terminal RING finger domain closely followed by one or two B-boxes. Additional protein domains found at their C termini have been used to classify TRIM proteins into classes. TRIMs are involved in multiple cellular processes and many of them are essential components of the innate immunity system of animal species. In humans, it has been shown that mutations in several TRIM-encoding genes lead to diverse genetic diseases and contribute to several types of cancer. They had been hitherto detected only in animals. In this work, by comprehensively analyzing the available diversity of TRIM and TRIM-like protein sequences and evaluating their evolutionary patterns, an improved classification of the TRIM family is obtained. Members of one of the TRIM subfamilies defined, called Subfamily A, turn to be present not only in animals, but also in many other eukaryotes, such as fungi, apusozoans, alveolates, excavates and plants. The rest of subfamilies are animal-specific and several of them originated only recently. Subfamily A proteins are characterized by containing a MATH domain, suggesting a potential evolutionary connection between TRIM proteins and a different type of ubiquitin ligases, known as TRAFs, which contain quite similar MATH domains. These results indicate that the TRIM family emerged much earlier than so far thought and contribute to our understanding of its origin and diversification. The structural and evolutionary links with the TRAF family of ubiquitin ligases can be experimentally explored to determine whether functional connections also exist.

ERAD-dependent control of the Wnt secretory factor Evi.

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Glaeser, Kathrin; Urban, Manuela; Fenech, Emma; Voloshanenko, Oksana; Kranz, Dominique; Lari, Federica; Christianson, John C; Boutros, Michael

2018-02-15

Active regulation of protein abundance is an essential strategy to modulate cellular signaling pathways. Within the Wnt signaling cascade, regulated degradation of β-catenin by the ubiquitin-proteasome system (UPS) affects the outcome of canonical Wnt signaling. Here, we found that abundance of the Wnt cargo receptor Evi (Wls/GPR177), which is required for Wnt protein secretion, is also regulated by the UPS through endoplasmic reticulum (ER)-associated degradation (ERAD). In the absence of Wnt ligands, Evi is ubiquitinated and targeted for ERAD in a VCP-dependent manner. Ubiquitination of Evi involves the E2-conjugating enzyme UBE2J2 and the E3-ligase CGRRF1. Furthermore, we show that a triaging complex of Porcn and VCP determines whether Evi enters the secretory or the ERAD pathway. In this way, ERAD-dependent control of Evi availability impacts the scale of Wnt protein secretion by adjusting the amount of Evi to meet the requirement of Wnt protein export. As Wnt and Evi protein levels are often dysregulated in cancer, targeting regulatory ERAD components might be a useful approach for therapeutic interventions. © 2018 The Authors. Published under the terms of the CC BY 4.0 license.

Determination of the Ubiquitin Fitness Landscape under Seventeen Chemical Conditions in a Classroom Setting

Science.gov (United States)

Mavor, David Carl

2017-01-01

Ubiquitin is essential for eukaryotic life and varies in only 3 amino acid positions between yeast and humans. However, recent deep sequencing studies indicate that ubiquitin is highly tolerant to single mutations. We hypothesized that this tolerance would be reduced by chemically induced physiologic perturbations. To test this hypothesis, a class…

The F-box Protein FBXO44 Mediates BRCA1 Ubiquitination and Degradation*

Science.gov (United States)

Lu, Yunzhe; Li, Jiezhi; Cheng, Dongmei; Parameswaran, Balaji; Zhang, Shaohua; Jiang, Zefei; Yew, P. Renee; Peng, Junmin; Ye, Qinong; Hu, Yanfen

2012-01-01

BRCA1 mutations account for a significant proportion of familial breast and ovarian cancers. In addition, reduced BRCA1 protein is associated with sporadic cancer cases in these tissues. At the cellular level, BRCA1 plays a critical role in multiple cellular functions such as DNA repair and cell cycle checkpoint control. Its protein level is regulated in a cell cycle-dependent manner. However, regulation of BRCA1 protein stability is not fully understood. Our earlier study showed that the amino terminus of BRCA1 harbors a degron sequence that is sufficient and necessary for conferring BRCA1 degradation. In the current study, we used mass spectrometry to identify Skp1 that regulates BRCA1 protein stability. Small interfering RNA screening that targets all human F-box proteins uncovered FBXO44 as an important protein that influences BRCA1 protein level. The Skp1-Cul1-F-box-protein44 (SCFFBXO44) complex ubiquitinates full-length BRCA1 in vitro. Furthermore, the N terminus of BRCA1 mediates the interaction between BRCA1 and FBXO44. Overexpression of SCFFBXO44 reduces BRCA1 protein level. Taken together, our work strongly suggests that SCFFBXO44 is an E3 ubiquitin ligase responsible for BRCA1 degradation. In addition, FBXO44 expression pattern in breast carcinomas suggests that SCFFBXO44-mediated BRCA1 degradation might contribute to sporadic breast tumor development. PMID:23086937

Ubiquitome Analysis Reveals PCNA-Associated Factor 15 (PAF15) as a Specific Ubiquitination Target of UHRF1 in Embryonic Stem Cells.

Science.gov (United States)

Karg, Elisabeth; Smets, Martha; Ryan, Joel; Forné, Ignasi; Qin, Weihua; Mulholland, Christopher B; Kalideris, Georgia; Imhof, Axel; Bultmann, Sebastian; Leonhardt, Heinrich

2017-12-08

Ubiquitination is a multifunctional posttranslational modification controlling the activity, subcellular localization and stability of proteins. The E3 ubiquitin ligase ubiquitin-like PHD and RING finger domain-containing protein 1 (UHRF1) is an essential epigenetic factor that recognizes repressive histone marks as well as hemi-methylated DNA and recruits DNA methyltransferase 1. To explore enzymatic functions of UHRF1 beyond epigenetic regulation, we conducted a comprehensive screen in mouse embryonic stem cells to identify novel ubiquitination targets of UHRF1 and its paralogue UHRF2. We found differentially ubiquitinated peptides associated with a variety of biological processes such as transcriptional regulation and DNA damage response. Most prominently, we identified PCNA-associated factor 15 (PAF15; also known as Pclaf, Ns5atp9, KIAA0101 and OEATC-1) as a specific ubiquitination target of UHRF1. Although the function of PAF15 ubiquitination in translesion DNA synthesis is well characterized, the respective E3 ligase had been unknown. We could show that UHRF1 ubiquitinates PAF15 at Lys 15 and Lys 24 and promotes its binding to PCNA during late S-phase. In summary, we identified novel ubiquitination targets that link UHRF1 to transcriptional regulation and DNA damage response. Copyright © 2017. Published by Elsevier Ltd.

Global-genome Nucleotide Excision Repair Controlled by Ubiquitin/Sumo Modifiers

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Peter eRuethemann

2016-04-01

Full Text Available Global-genome nucleotide excision repair (GG-NER prevents genome instability by excising a wide range of structurally unrelated DNA base adducts and crosslinks induced by chemical carcinogens, ultraviolet (UV radiation or intracellular metabolic by-products. As a versatile damage sensor, xeroderma pigmentosum group C (XPC protein initiates this generic defense reaction by locating the damage and recruiting the subunits of a large lesion demarcation complex that, in turn, triggers the excision of aberrant DNA by endonucleases. In the very special case of a DNA repair response to UV radiation, the function of this XPC initiator is tightly controlled by the dual action of cullin-type CRL4DDB2 and sumo-targeted RNF111 ubiquitin ligases. This twofold protein ubiquitination system promotes GG-NER reactions by spatially and temporally regulating the interaction of XPC protein with damaged DNA across the nucleosome landscape of chromatin. In the absence of either CRL4DDB2 or RNF111, the DNA excision repair of UV lesions is inefficient, indicating that these two ubiquitin ligases play a critical role in mitigating the adverse biological effects of UV light in the exposed skin.

Comparison of altered expression of histocompatibility antigens with altered immune function in murine spleen cells treated with ultraviolet radiation and/or TPA

International Nuclear Information System (INIS)

Pretell, J.O.; Cone, R.E.

1985-01-01

Previous studies in our laboratory demonstrated that several treatments that inhibited the ability of cells to stimulate the mixed lymphocyte reaction (MLR) also blocked the shedding of histocompatibility antigens and Ia antigens from murine spleen cells. In the present studies, one of these treatments, ultraviolet radiation (UV), was shown to cause an initial loss in the density of H-2K, IA, and IE antigens prior to the block in shedding observed after culture of these cells. Further analysis revealed that the UV-induced loss of antigens could be prevented by the presence of colchicine during irradiation. Biosynthetic analyses revealed the IA antigen synthesis was also inhibited in the UV-irradiated cells. Examination of the effects of a second agent, 12-0-tetradecanoylphorbol-13-acetate (TPA) on the turnover of histocompatibility antigens revealed that the biosynthesis and shedding of these antigens were accelerated by this agent. However, addition of TPA to UV-irradiated cells did not result in a reversal of the UV-induced block in biosynthesis of IA antigens. Results of immune function assays correlated with the biochemical studies: UV-irradiation inhibited the generation of the MLR, but TPA enhanced this reaction, and addition of TPA to mixed lymphocyte cultures with UV-irradiated stimulators did not reverse the UV-induced inhibition. These results suggest that, although the turnover of histocompatibility antigens may be affected by TPA and UV in an antagonistic fashion, additional factors other than the expression of histocompatibility antigens are operating in the inhibition of stimulation of an MLR by UV radiation or its enhancement by TPA

Bioinformatics analysis identifies several intrinsically disordered human E3 ubiquitin-protein ligases

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Wouter Boomsma

2016-02-01

Full Text Available The ubiquitin-proteasome system targets misfolded proteins for degradation. Since the accumulation of such proteins is potentially harmful for the cell, their prompt removal is important. E3 ubiquitin-protein ligases mediate substrate ubiquitination by bringing together the substrate with an E2 ubiquitin-conjugating enzyme, which transfers ubiquitin to the substrate. For misfolded proteins, substrate recognition is generally delegated to molecular chaperones that subsequently interact with specific E3 ligases. An important exception is San1, a yeast E3 ligase. San1 harbors extensive regions of intrinsic disorder, which provide both conformational flexibility and sites for direct recognition of misfolded targets of vastly different conformations. So far, no mammalian ortholog of San1 is known, nor is it clear whether other E3 ligases utilize disordered regions for substrate recognition. Here, we conduct a bioinformatics analysis to examine >600 human and S. cerevisiae E3 ligases to identify enzymes that are similar to San1 in terms of function and/or mechanism of substrate recognition. An initial sequence-based database search was found to detect candidates primarily based on the homology of their ordered regions, and did not capture the unique disorder patterns that encode the functional mechanism of San1. However, by searching specifically for key features of the San1 sequence, such as long regions of intrinsic disorder embedded with short stretches predicted to be suitable for substrate interaction, we identified several E3 ligases with these characteristics. Our initial analysis revealed that another remarkable trait of San1 is shared with several candidate E3 ligases: long stretches of complete lysine suppression, which in San1 limits auto-ubiquitination. We encode these characteristic features into a San1 similarity-score, and present a set of proteins that are plausible candidates as San1 counterparts in humans. In conclusion, our work

ISG15 inhibits Nedd4 ubiquitin E3 activity and enhances the innate antiviral response.

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Malakhova, Oxana A; Zhang, Dong-Er

2008-04-04

Interferons regulate diverse immune functions through the transcriptional activation of hundreds of genes involved in anti-viral responses. The interferon-inducible ubiquitin-like protein ISG15 is expressed in cells in response to a variety of stress conditions like viral or bacterial infection and is present in its free form or is conjugated to cellular proteins. In addition, protein ubiquitination plays a regulatory role in the immune system. Many viruses modulate the ubiquitin (Ub) pathway to alter cellular signaling and the antiviral response. Ubiquitination of retroviral group-specific antigen precursors and matrix proteins of the Ebola, vesicular stomatitis, and rabies viruses by Nedd4 family HECT domain E3 ligases is an important step in facilitating viral release. We found that Nedd4 is negatively regulated by ISG15. Free ISG15 specifically bound to Nedd4 and blocked its interaction with Ub-E2 molecules, thus preventing further Ub transfer from E2 to E3. Furthermore, overexpression of ISG15 diminished the ability of Nedd4 to ubiquitinate viral matrix proteins and led to a decrease in the release of Ebola VP40 virus-like particles from the cells. These results point to a mechanistically novel function of ISG15 in the enhancement of the innate anti-viral response through specific inhibition of Nedd4 Ub-E3 activity. To our knowledge, this is the first example of a Ub-like protein with the ability to interfere with Ub-E2 and E3 interaction to inhibit protein ubiquitination.

Establishment of a Wheat Cell-Free Synthesized Protein Array Containing 250 Human and Mouse E3 Ubiquitin Ligases to Identify Novel Interaction between E3 Ligases and Substrate Proteins.

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Hirotaka Takahashi

Full Text Available Ubiquitination is a key post-translational modification in the regulation of numerous biological processes in eukaryotes. The primary roles of ubiquitination are thought to be the triggering of protein degradation and the regulation of signal transduction. During protein ubiquitination, substrate specificity is mainly determined by E3 ubiquitin ligase (E3. Although more than 600 genes in the human genome encode E3, the E3s of many target proteins remain unidentified owing to E3 diversity and the instability of ubiquitinated proteins in cell. We demonstrate herein a novel biochemical analysis for the identification of E3s targeting specific proteins. Using wheat cell-free protein synthesis system, a protein array containing 227 human and 23 mouse recombinant E3s was synthesized. To establish the high-throughput binding assay using AlphaScreen technology, we selected MDM2 and p53 as the model combination of E3 and its target protein. The AlphaScreen assay specifically detected the binding of p53 and MDM2 in a crude translation mixture. Then, a comprehensive binding assay using the E3 protein array was performed. Eleven of the E3s showed high binding activity, including four previously reported E3s (e.g., MDM2, MDM4, and WWP1 targeting p53. This result demonstrated the reliability of the assay. Another interactors, RNF6 and DZIP3-which there have been no report to bind p53-were found to ubiquitinate p53 in vitro. Further analysis showed that RNF6 decreased the amount of p53 in H1299 cells in E3 activity-dependent manner. These results suggest the possibility that the RNF6 ubiquitinates and degrades p53 in cells. The novel in vitro screening system established herein is a powerful tool for finding novel E3s of a target protein.

Pseudosubstrate regulation of the SCF(beta-TrCP) ubiquitin ligase by hnRNP-U

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Davis, Matti; Hatzubai, Ada; Andersen, Jens S

2002-01-01

in the nucleus. Here we report the isolation of the major E3RS-associated protein, hnRNP-U, an abundant nuclear phosphoprotein. This protein occupies E3RS in a specific and stoichiometric manner, stabilizes the E3 component, and is likely responsible for its nuclear localization. hnRNP-U binding was abolished....... Consequently, hnRNP-U engages a highly neddylated active SCF(beta-TrCP), which dissociates in the presence of a high-affinity substrate, resulting in ubiquitination of the latter. Our study points to a novel regulatory mechanism, which secures the localization, stability, substrate binding threshold...

Rictor forms a complex with Cullin-1 to promote SGK1 ubiquitination and destruction

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Gao, Daming; Wan, Lixin; Inuzuka, Hiroyuki; Berg, Anders H.; Tseng, Alan; Zhai, Bo; Shaik, Shavali; Bennett, Eric; Tron, Adriana E.; Gasser, Jessica A.; Lau, Alan; Gygi, Steven; Harper, J. Wade; DeCaprio, James A.; Toker, Alex; Wei, Wenyi

2010-01-01

Summary The Rictor/mTOR complex (also known as mTORC2) plays a critical role in cellular homeostasis by phosphorylating AGC kinases such as Akt and SGK at their hydrophobic motifs to activate downstream signaling. However, the regulation of mTORC2 and whether it has additional function(s), remains largely unknown. Here we report that Rictor associates with Cullin-1 to form a functional E3 ubiquitin ligase. Rictor, but not Raptor or mTOR alone promotes SGK1 ubiquitination. Loss of Rictor/Cullin-1-mediated ubiquitination leads to increased SGK1 protein levels as detected in Rictor null cells. Moreover, as part of a feedback mechanism, phosphorylation of Rictor at T1135 by multiple AGC kinases disrupts the interaction between Rictor and Cullin-1 to impair SGK1 ubiquitination. These findings indicate that the Rictor/Cullin-1 E3 ligase activity is regulated by a specific signal relay cascade and that misregulation of this mechanism may contribute to the frequent overexpression of SGK1 in various human cancers. PMID:20832730

Cul3-mediated Nrf2 ubiquitination and antioxidant response element (ARE) activation are dependent on the partial molar volume at position 151 of Keap1.

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Eggler, Aimee L; Small, Evan; Hannink, Mark; Mesecar, Andrew D

2009-07-29

Nrf2 (nuclear factor erythroid 2-related factor 2) is a transcription factor that activates transcription of a battery of cytoprotective genes by binding to the ARE (antioxidant response element). Nrf2 is repressed by the cysteine-rich Keap1 (kelch-like ECH-associated protein 1) protein, which targets Nrf2 for ubiquitination and subsequent degradation by a Cul3 (cullin 3)-mediated ubiquitination complex. We find that modification of Cys(151) of human Keap1, by mutation to a tryptophan, relieves the repression by Keap1 and allows activation of the ARE by Nrf2. The Keap1 C151W substitution has a decreased affinity for Cul3, and can no longer serve to target Nrf2 for ubiquitination, though it retains its affinity for Nrf2. A series of 12 mutant Keap1 proteins, each containing a different residue at position 151, was constructed to explore the chemistry required for this effect. The series reveals that the extent to which Keap1 loses the ability to target Nrf2 for degradation, and hence the ability to repress ARE activation, correlates well with the partial molar volume of the residue. Other physico-chemical properties do not appear to contribute significantly to the effect. Based on this finding, a structural model is proposed whereby large residues at position 151 cause steric clashes that lead to alteration of the Keap1-Cul3 interaction. This model has significant implications for how electrophiles which modify Cys(151), disrupt the repressive function of Keap1.

Calmodulin promotes matrix metalloproteinase 9 production and cell migration by inhibiting the ubiquitination and degradation of TBC1D3 oncoprotein in human breast cancer cells.

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Zhao, Huzi; Zhang, Lina; Zhang, Yongchen; Zhao, Lei; Wan, Qing; Wang, Bei; Bu, Xiaodong; Wan, Meiling; Shen, Chuanlu

2017-05-30

The hominoid oncoprotein TBC1D3 enhances growth factor (GF) signaling and GF signaling, conversely, induces the ubiquitination and subsequent degradation of TBC1D3. However, little is known regarding the regulation of this degradation, and the role of TBC1D3 in the progression of tumors has also not been defined. In the present study, we demonstrated that calmodulin (CaM), a ubiquitous cellular calcium sensor, specifically interacted with TBC1D3 in a Ca2+-dependent manner and inhibited GF signaling-induced ubiquitination and degradation of the oncoprotein in both cytoplasm and nucleus of human breast cancer cells. The CaM-interacting site of TBC1D3 was mapped to amino acids 157~171, which comprises two 1-14 hydrophobic motifs and one lysine residue (K166). Deletion of these motifs was shown to abolish interaction between TBC1D3 and CaM. Surprisingly, this deletion mutation caused inability of GF signaling to induce the ubiquitination and subsequent degradation of TBC1D3. In agreement with this, we identified lysine residue 166 within the CaM-interacting motifs of TBC1D3 as the actual site for the GF signaling-induced ubiquitination using mutational analysis. Point mutation of this lysine residue exhibited the same effect on TBC1D3 as the deletion mutant, suggesting that CaM inhibits GF signaling-induced degradation of TBC1D3 by occluding its ubiquitination at K166. Notably, we found that TBC1D3 promoted the expression and activation of MMP-9 and the migration of MCF-7 cells. Furthermore, interaction with CaM considerably enhanced such effect of TBC1D3. Taken together, our work reveals a novel model by which CaM promotes cell migration through inhibiting the ubiquitination and degradation of TBC1D3.

An ontology for major histocompatibility restriction.

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Vita, Randi; Overton, James A; Seymour, Emily; Sidney, John; Kaufman, Jim; Tallmadge, Rebecca L; Ellis, Shirley; Hammond, John; Butcher, Geoff W; Sette, Alessandro; Peters, Bjoern

2016-01-01

MHC molecules are a highly diverse family of proteins that play a key role in cellular immune recognition. Over time, different techniques and terminologies have been developed to identify the specific type(s) of MHC molecule involved in a specific immune recognition context. No consistent nomenclature exists across different vertebrate species. To correctly represent MHC related data in The Immune Epitope Database (IEDB), we built upon a previously established MHC ontology and created an ontology to represent MHC molecules as they relate to immunological experiments. This ontology models MHC protein chains from 16 species, deals with different approaches used to identify MHC, such as direct sequencing verses serotyping, relates engineered MHC molecules to naturally occurring ones, connects genetic loci, alleles, protein chains and multi-chain proteins, and establishes evidence codes for MHC restriction. Where available, this work is based on existing ontologies from the OBO foundry. Overall, representing MHC molecules provides a challenging and practically important test case for ontology building, and could serve as an example of how to integrate other ontology building efforts into web resources.

The ubiquitin ligase ASB4 promotes trophoblast differentiation through the degradation of ID2.

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W H Davin Townley-Tilson

Full Text Available Vascularization of the placenta is a critical developmental process that ensures fetal viability. Although the vascular health of the placenta affects both maternal and fetal well being, relatively little is known about the early stages of placental vascular development. The ubiquitin ligase Ankyrin repeat, SOCS box-containing 4 (ASB4 promotes embryonic stem cell differentiation to vascular lineages and is highly expressed early in placental development. The transcriptional regulator Inhibitor of DNA binding 2 (ID2 negatively regulates vascular differentiation during development and is a target of many ubiquitin ligases. Due to their overlapping spatiotemporal expression pattern in the placenta and contrasting effects on vascular differentiation, we investigated whether ASB4 regulates ID2 through its ligase activity in the placenta and whether this activity mediates vascular differentiation. In mouse placentas, ASB4 expression is restricted to a subset of cells that express both stem cell and endothelial markers. Placentas that lack Asb4 display immature vascular patterning and retain expression of placental progenitor markers, including ID2 expression. Using JAR placental cells, we determined that ASB4 ubiquitinates and represses ID2 expression in a proteasome-dependent fashion. Expression of ASB4 in JAR cells and primary isolated trophoblast stem cells promotes the expression of differentiation markers. In functional endothelial co-culture assays, JAR cells ectopically expressing ASB4 increased endothelial cell turnover and stabilized endothelial tube formation, both of which are hallmarks of vascular differentiation within the placenta. Co-transfection of a degradation-resistant Id2 mutant with Asb4 inhibits both differentiation and functional responses. Lastly, deletion of Asb4 in mice induces a pathology that phenocopies human pre-eclampsia, including hypertension and proteinuria in late-stage pregnant females. These results indicate that

Degradation of human Lipin-1 by BTRC E3 ubiquitin ligase.

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Ishimoto, Kenji; Hayase, Ayaka; Kumagai, Fumiko; Kawai, Megumi; Okuno, Hiroko; Hino, Nobumasa; Okada, Yoshiaki; Kawamura, Takeshi; Tanaka, Toshiya; Hamakubo, Takao; Sakai, Juro; Kodama, Tatsuhiko; Tachibana, Keisuke; Doi, Takefumi

2017-06-17

Lipin-1 has dual functions in the regulation of lipid and energy metabolism according to its subcellular localization, which is tightly controlled. However, it is unclear how Lipin-1 degradation is regulated. Here, we demonstrate that Lipin-1 is degraded through its DSGXXS motif. We show that Lipin-1 interacts with either of two E3 ubiquitin ligases, BTRC or FBXW11, and that this interaction is DSGXXS-dependent and mediates the attachment of polyubiquitin chains. Further, we demonstrate that degradation of Lipin-1 is regulated by BTRC in the cytoplasm and on membranes. These novel insights into the regulation of human Lipin-1 stability will be useful in planning further studies to elucidate its metabolic processes. Copyright © 2017 Elsevier Inc. All rights reserved.

Hijacking of the host SCF ubiquitin ligase machinery by plant pathogens

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Shimpei eMagori

2011-11-01

Full Text Available The SCF (SKP1-CUL1-F-box protein ubiquitin ligase complex mediates polyubiquitination of proteins targeted for degradation, thereby controlling a plethora of biological processes in eukaryotic cells. Although this ubiquitination machinery is found and functional only in eukaryotes, many non-eukaryotic pathogens also encode F-box proteins, the critical subunits of the SCF complex. Increasing evidence indicates that such non-eukaryotic F-box proteins play an essential role in subverting or exploiting the host ubiquitin/proteasome system for efficient pathogen infection. A recent bioinformatic analysis has identified more than 70 F-box proteins in 22 different bacterial species, suggesting that use of pathogen-encoded F-box effectors in the host cell may be a widespread infection strategy. In this review, we focus on plant pathogen-encoded F-box effectors, such as VirF of Agrobacterium tumefaciens, GALAs of Ralstonia solanacearum, and P0 of Poleroviruses, and discuss the molecular mechanism by which plant pathogens use these factors to manipulate the host cell for their own benefit.

KF-1 ubiquitin ligase: an anxiety suppressor

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Tamotsu Hashimoto-Gotoh

2009-05-01

Full Text Available Anxiety is an instinct that may have developed to promote adaptive survival by evading unnecessary danger. However, excessive anxiety is disruptive and can be a basic disorder of other psychiatric diseases such as depression. The KF-1, a ubiquitin ligase located to the endoplasmic reticulum (ER, may prevent excessive anxiety; kf-1−/− mice exhibit selectively elevated anxiety-like behavior against light or heights. Thus, KF-1 may degrade some target proteins, responsible for promoting anxiety, through the ER-associated degradation pathway, similar to Parkin in Parkinson's disease (PD. Parkin, another ER-ubiquitin ligase, prevents the degeneration of dopaminergic neurons by degrading the target proteins responsible for PD. Molecular phylogenetic studies have revealed that the prototype of kf-1 appeared in the very early phase of animal evolution but was lost, unlike parkin, in the lineage leading up to Drosophila. Therefore, kf-1−/− mice, be a powerful tool for elucidating the molecular mechanisms involved in emotional regulation, and for screening novel anxiolytic/antidepressant compounds.

Deciphering the ubiquitin-mediated pathway in apicomplexan parasites: a potential strategy to interfere with parasite virulence.

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Ponts, Nadia; Yang, Jianfeng; Chung, Duk-Won Doug; Prudhomme, Jacques; Girke, Thomas; Horrocks, Paul; Le Roch, Karine G

2008-06-11

Reversible modification of proteins through the attachment of ubiquitin or ubiquitin-like modifiers is an essential post-translational regulatory mechanism in eukaryotes. The conjugation of ubiquitin or ubiquitin-like proteins has been demonstrated to play roles in growth, adaptation and homeostasis in all eukaryotes, with perturbation of ubiquitin-mediated systems associated with the pathogenesis of many human diseases, including cancer and neurodegenerative disorders. Here we describe the use of an HMM search of functional Pfam domains found in the key components of the ubiquitin-mediated pathway necessary to activate and reversibly modify target proteins in eight apicomplexan parasitic protozoa for which complete or late-stage genome projects exist. In parallel, the same search was conducted on five model organisms, single-celled and metazoans, to generate data to validate both the search parameters employed and aid paralog classification in Apicomplexa. For each of the 13 species investigated, a set of proteins predicted to be involved in the ubiquitylation pathway has been identified and demonstrates increasing component members of the ubiquitylation pathway correlating with organism and genome complexity. Sequence homology and domain architecture analyses facilitated prediction of apicomplexan-specific protein function, particularly those involved in regulating cell division during these parasite's complex life cycles. This study provides a comprehensive analysis of proteins predicted to be involved in the apicomplexan ubiquitin-mediated pathway. Given the importance of such pathway in a wide variety of cellular processes, our data is a key step in elucidating the biological networks that, in part, direct the pathogenicity of these parasites resulting in a massive impact on global health. Moreover, apicomplexan-specific adaptations of the ubiquitylation pathway may represent new therapeutic targets for much needed drugs against apicomplexan parasites.

Discovery of Ubiquitin Deamidases in the Pathogenic Arsenal of Legionella pneumophila

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Dylan Valleau

2018-04-01

Full Text Available Summary: Legionella pneumophila translocates the largest known arsenal of over 330 pathogenic factors, called “effectors,” into host cells during infection, enabling L. pneumophila to establish a replicative niche inside diverse amebas and human macrophages. Here, we reveal that the L. pneumophila effectors MavC (Lpg2147 and MvcA (Lpg2148 are structural homologs of cycle inhibiting factor (Cif effectors and that the adjacent gene, lpg2149, produces a protein that directly inhibits their activity. In contrast to canonical Cifs, both MavC and MvcA contain an insertion domain and deamidate the residue Gln40 of ubiquitin but not Gln40 of NEDD8. MavC and MvcA are functionally diverse, with only MavC interacting with the human E2-conjugating enzyme UBE2N (Ubc13. MavC deamidates the UBE2N∼Ub conjugate, disrupting Lys63 ubiquitination and dampening NF-κB signaling. Combined, our data reveal a molecular mechanism of host manipulation by pathogenic bacteria and highlight the complex regulatory mechanisms integral to L. pneumophila’s pathogenic strategy. : Legionella pneumophila, possessing the largest known arsenal of effectors, continues to reveal unique approaches to host cell control. Valleau et al. decrypt the functions of a trio of effectors, discovering a pair of ubiquitin-specific deamidases, their regulation by a neighboring dual-specificity protein inhibitor, and a mechanism of NF-κB suppression. Keywords: pathogen-host interaction, ubiquitination, Legionella, UBE2N/Ubc13, NF-κB signaling, Type IV secretion system, effectors, metaeffector, cycle inhibiting factor

Roles of Ubiquitination and SUMOylation on Prostate Cancer: Mechanisms and Clinical Implications

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Zhenbang Chen

2015-02-01

Full Text Available The initiation and progression of human prostate cancer are highly associated with aberrant dysregulations of tumor suppressors and proto-oncogenes. Despite that deletions and mutations of tumor suppressors and aberrant elevations of oncogenes at the genetic level are reported to cause cancers, emerging evidence has revealed that cancer progression is largely regulated by posttranslational modifications (PTMs and epigenetic alterations. PTMs play critical roles in gene regulation, cellular functions, tissue development, diseases, malignant progression and drug resistance. Recent discoveries demonstrate that ubiquitination and SUMOylation are complicated but highly-regulated PTMs, and make essential contributions to diseases and cancers by regulation of key factors and signaling pathways. Ubiquitination and SUMOylation pathways can be differentially modulated under various stimuli or stresses in order to produce the sustained oncogenic potentials. In this review, we discuss some new insights about molecular mechanisms on ubiquitination and SUMOylation, their associations with diseases, oncogenic impact on prostate cancer (PCa and clinical implications for PCa treatment.

The FA Core Complex Contains a Homo-dimeric Catalytic Module for the Symmetric Mono-ubiquitination of FANCI-FANCD2

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Paolo Swuec

2017-01-01

Full Text Available Activation of the main DNA interstrand crosslink repair pathway in higher eukaryotes requires mono-ubiquitination of FANCI and FANCD2 by FANCL, the E3 ligase subunit of the Fanconi anemia core complex. FANCI and FANCD2 form a stable complex; however, the molecular basis of their ubiquitination is ill defined. FANCD2 mono-ubiquitination by FANCL is stimulated by the presence of the FANCB and FAAP100 core complex components, through an unknown mechanism. How FANCI mono-ubiquitination is achieved remains unclear. Here, we use structural electron microscopy, combined with crosslink-coupled mass spectrometry, to find that FANCB, FANCL, and FAAP100 form a dimer of trimers, containing two FANCL molecules that are ideally poised to target both FANCI and FANCD2 for mono-ubiquitination. The FANCC-FANCE-FANCF subunits bridge between FANCB-FANCL-FAAP100 and the FANCI-FANCD2 substrate. A transient interaction with FANCC-FANCE-FANCF alters the FANCI-FANCD2 configuration, stabilizing the dimerization interface. Our data provide a model to explain how equivalent mono-ubiquitination of FANCI and FANCD2 occurs.

Genes Outside the Major Histocompatibility Complex Locus Are Linked to the Development of Thyroid Autoantibodies and Thyroiditis in NOD.H2h4 Mice.

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McLachlan, Sandra M; Lesage, Sylvie; Collin, Roxanne; Banuelos, Bianca; Aliesky, Holly A; Rapoport, Basil

2017-04-01

Thyroiditis and autoantibodies to thyroglobulin (TgAb) and thyroid peroxidase (TPOAb) develop spontaneously in NOD.H2h4 mice, a phenotype enhanced by dietary iodine. NOD.H2h4 mice were derived by introducing the major histocompatibility class (MHC) molecule I-Ak from B10.A(4R) mice to nonobese diabetic (NOD) mice. Apart from I-Ak, the genes responsible for the NOD.H2h4 phenotype are unknown. Extending serendipitous observations from crossing BALB/c to NOD.H2h4 mice, thyroid autoimmunity was investigated in both genders of the F1, F2, and the second-generation backcross of F1 to NOD.H2h4 (N2). Medium-density linkage analysis was performed on thyroid autoimmunity traits in F2 and N2 progeny. TgAb develop before TPOAb and were measured after 8 and 16 weeks of iodide exposure; TPOAb and thyroiditis were studied at 16 weeks. TgAb, TPOAb, and thyroiditis, absent in BALB/c and F1 mice, developed in most NOD.H2h4 and in more N2 than F2 progeny. No linkages were observed in F2 progeny, probably because of the small number of autoantibody-positive mice. In N2 progeny (equal numbers of males and females), a chromosome 17 locus is linked to thyroiditis and TgAb and is suggestively linked to TPOAb. This locus includes MHC region genes from B10.A(4R) mice (such as I-Ak and Tnf, the latter involved in thyrocyte apoptosis) and genes from NOD mice such as Satb1, which most likely plays a role in immune tolerance. In conclusion, MHC and non-MHC genes, encoded within the chromosome 17 locus from both B10.A(4R) and NOD strains, are most likely responsible for the Hashimoto disease-like phenotype of NOD.H2h4 mice. Copyright © 2017 Endocrine Society.

Human cytomegalovirus IE1 downregulates Hes1 in neural progenitor cells as a potential E3 ubiquitin ligase.

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Xi-Juan Liu

2017-07-01

Full Text Available Congenital human cytomegalovirus (HCMV infection is the leading cause of neurological disabilities in children worldwide, but the mechanisms underlying these disorders are far from well-defined. HCMV infection has been shown to dysregulate the Notch signaling pathway in human neural progenitor cells (NPCs. As an important downstream effector of Notch signaling, the transcriptional regulator Hairy and Enhancer of Split 1 (Hes1 is essential for governing NPC fate and fetal brain development. In the present study, we report that HCMV infection downregulates Hes1 protein levels in infected NPCs. The HCMV 72-kDa immediate-early 1 protein (IE1 is involved in Hes1 degradation by assembling a ubiquitination complex and promoting Hes1 ubiquitination as a potential E3 ubiquitin ligase, followed by proteasomal degradation of Hes1. Sp100A, an important component of PML nuclear bodies, is identified to be another target of IE1-mediated ubiquitination. A C-terminal acidic region in IE1, spanning amino acids 451 to 475, is required for IE1/Hes1 physical interaction and IE1-mediated Hes1 ubiquitination, but is dispensable for IE1/Sp100A interaction and ubiquitination. Our study suggests a novel mechanism linking downregulation of Hes1 protein to neurodevelopmental disorders caused by HCMV infection. Our findings also complement the current knowledge of herpesviruses by identifying IE1 as the first potential HCMV-encoded E3 ubiquitin ligase.

Proteolysis targeting peptide (PROTAP) strategy for protein ubiquitination and degradation.

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Zheng, Jing; Tan, Chunyan; Xue, Pengcheng; Cao, Jiakun; Liu, Feng; Tan, Ying; Jiang, Yuyang

2016-02-19

Ubiquitination proteasome pathway (UPP) is the most important and selective way to degrade proteins in vivo. Here, a novel proteolysis targeting peptide (PROTAP) strategy, composed of a target protein binding peptide, a linker and a ubiquitin E3 ligase recognition peptide, was designed to recruit both target protein and E3 ligase and then induce polyubiquitination and degradation of the target protein through UPP. In our study, the PROTAP strategy was proved to be a general method with high specificity using Bcl-xL protein as model target in vitro and in cells, which indicates that the strategy has great potential for in vivo application. Copyright © 2016 Elsevier Inc. All rights reserved.

Beyond ubiquitination: the atypical functions of Fbxo7 and other F-box proteins.

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Nelson, David E; Randle, Suzanne J; Laman, Heike

2013-10-09

F-box proteins (FBPs) are substrate-recruiting subunits of Skp1-cullin1-FBP (SCF)-type E3 ubiquitin ligases. To date, 69 FBPs have been identified in humans, but ubiquitinated substrates have only been identified for a few, with the majority of FBPs remaining 'orphans'. In recent years, a growing body of work has identified non-canonical, SCF-independent roles for about 12% of the human FBPs. These atypical FBPs affect processes as diverse as transcription, cell cycle regulation, mitochondrial dynamics and intracellular trafficking. Here, we provide a general review of FBPs, with a particular emphasis on these expanded functions. We review Fbxo7 as an exemplar of this special group as it has well-defined roles in both SCF and non-SCF complexes. We review its function as a cell cycle regulator, via its ability to stabilize p27 protein and Cdk6 complexes, and as a proteasome regulator, owing to its high affinity binding to PI31. We also highlight recent advances in our understanding of Fbxo7 function in Parkinson's disease, where it functions in the regulation of mitophagy with PINK1 and Parkin. We postulate that a few extraordinary FBPs act as platforms that seamlessly segue their canonical and non-canonical functions to integrate different cellular pathways and link their regulation.

Genetic variants are major determinants of CSF antibody levels in multiple sclerosis.

Science.gov (United States)

Goris, An; Pauwels, Ine; Gustavsen, Marte W; van Son, Brechtje; Hilven, Kelly; Bos, Steffan D; Celius, Elisabeth Gulowsen; Berg-Hansen, Pål; Aarseth, Jan; Myhr, Kjell-Morten; D'Alfonso, Sandra; Barizzone, Nadia; Leone, Maurizio A; Martinelli Boneschi, Filippo; Sorosina, Melissa; Liberatore, Giuseppe; Kockum, Ingrid; Olsson, Tomas; Hillert, Jan; Alfredsson, Lars; Bedri, Sahl Khalid; Hemmer, Bernhard; Buck, Dorothea; Berthele, Achim; Knier, Benjamin; Biberacher, Viola; van Pesch, Vincent; Sindic, Christian; Bang Oturai, Annette; Søndergaard, Helle Bach; Sellebjerg, Finn; Jensen, Poul Erik H; Comabella, Manuel; Montalban, Xavier; Pérez-Boza, Jennifer; Malhotra, Sunny; Lechner-Scott, Jeannette; Broadley, Simon; Slee, Mark; Taylor, Bruce; Kermode, Allan G; Gourraud, Pierre-Antoine; Sawcer, Stephen J; Andreassen, Bettina Kullle; Dubois, Bénédicte; Harbo, Hanne F

2015-03-01

Immunological hallmarks of multiple sclerosis include the production of antibodies in the central nervous system, expressed as presence of oligoclonal bands and/or an increased immunoglobulin G index-the level of immunoglobulin G in the cerebrospinal fluid compared to serum. However, the underlying differences between oligoclonal band-positive and -negative patients with multiple sclerosis and reasons for variability in immunoglobulin G index are not known. To identify genetic factors influencing the variation in the antibody levels in the cerebrospinal fluid in multiple sclerosis, we have performed a genome-wide association screen in patients collected from nine countries for two traits, presence or absence of oligoclonal bands (n = 3026) and immunoglobulin G index levels (n = 938), followed by a replication in 3891 additional patients. We replicate previously suggested association signals for oligoclonal band status in the major histocompatibility complex region for the rs9271640*A-rs6457617*G haplotype, correlated with HLA-DRB1*1501, and rs34083746*G, correlated with HLA-DQA1*0301 (P comparing two haplotypes = 8.88 × 10(-16)). Furthermore, we identify a novel association signal of rs9807334, near the ELAC1/SMAD4 genes, for oligoclonal band status (P = 8.45 × 10(-7)). The previously reported association of the immunoglobulin heavy chain locus with immunoglobulin G index reaches strong evidence for association in this data set (P = 3.79 × 10(-37)). We identify two novel associations in the major histocompatibility complex region with immunoglobulin G index: the rs9271640*A-rs6457617*G haplotype (P = 1.59 × 10(-22)), shared with oligoclonal band status, and an additional independent effect of rs6457617*G (P = 3.68 × 10(-6)). Variants identified in this study account for up to 2-fold differences in the odds of being oligoclonal band positive and 7.75% of the variation in immunoglobulin G index. Both traits are associated with clinical features of disease such

Genetic variants are major determinants of CSF antibody levels in multiple sclerosis

Science.gov (United States)

Pauwels, Ine; Gustavsen, Marte W.; van Son, Brechtje; Hilven, Kelly; Bos, Steffan D.; Celius, Elisabeth Gulowsen; Berg-Hansen, Pål; Aarseth, Jan; Myhr, Kjell-Morten; D’Alfonso, Sandra; Barizzone, Nadia; Leone, Maurizio A.; Martinelli Boneschi, Filippo; Sorosina, Melissa; Liberatore, Giuseppe; Kockum, Ingrid; Olsson, Tomas; Hillert, Jan; Alfredsson, Lars; Bedri, Sahl Khalid; Hemmer, Bernhard; Buck, Dorothea; Berthele, Achim; Knier, Benjamin; Biberacher, Viola; van Pesch, Vincent; Sindic, Christian; Bang Oturai, Annette; Søndergaard, Helle Bach; Sellebjerg, Finn; Jensen, Poul Erik H.; Comabella, Manuel; Montalban, Xavier; Pérez-Boza, Jennifer; Malhotra, Sunny; Lechner-Scott, Jeannette; Broadley, Simon; Slee, Mark; Taylor, Bruce; Kermode, Allan G.; Gourraud, Pierre-Antoine; Sawcer, Stephen J.; Andreassen, Bettina Kullle; Dubois, Bénédicte; Harbo, Hanne F.

2015-01-01

Immunological hallmarks of multiple sclerosis include the production of antibodies in the central nervous system, expressed as presence of oligoclonal bands and/or an increased immunoglobulin G index—the level of immunoglobulin G in the cerebrospinal fluid compared to serum. However, the underlying differences between oligoclonal band-positive and -negative patients with multiple sclerosis and reasons for variability in immunoglobulin G index are not known. To identify genetic factors influencing the variation in the antibody levels in the cerebrospinal fluid in multiple sclerosis, we have performed a genome-wide association screen in patients collected from nine countries for two traits, presence or absence of oligoclonal bands (n = 3026) and immunoglobulin G index levels (n = 938), followed by a replication in 3891 additional patients. We replicate previously suggested association signals for oligoclonal band status in the major histocompatibility complex region for the rs9271640*A-rs6457617*G haplotype, correlated with HLA-DRB1*1501, and rs34083746*G, correlated with HLA-DQA1*0301 (P comparing two haplotypes = 8.88 × 10−16). Furthermore, we identify a novel association signal of rs9807334, near the ELAC1/SMAD4 genes, for oligoclonal band status (P = 8.45 × 10−7). The previously reported association of the immunoglobulin heavy chain locus with immunoglobulin G index reaches strong evidence for association in this data set (P = 3.79 × 10−37). We identify two novel associations in the major histocompatibility complex region with immunoglobulin G index: the rs9271640*A-rs6457617*G haplotype (P = 1.59 × 10−22), shared with oligoclonal band status, and an additional independent effect of rs6457617*G (P = 3.68 × 10−6). Variants identified in this study account for up to 2-fold differences in the odds of being oligoclonal band positive and 7.75% of the variation in immunoglobulin G index. Both traits are associated with clinical features of disease such

A new non-catalytic role for ubiquitin ligase RNF8 in unfolding higher-order chromatin structure

DEFF Research Database (Denmark)

Luijsterburg, Martijn S; Acs, Klara; Ackermann, Leena

2012-01-01

The ubiquitin ligases RNF8 and RNF168 orchestrate DNA damage signalling through the ubiquitylation of histone H2A and the recruitment of downstream repair factors. Here, we demonstrate that RNF8, but not RNF168 or the canonical H2A ubiquitin ligase RNF2, mediates extensive chromatin decondensation....... Our data show that CHD4, the catalytic subunit of the NuRD complex, interacts with RNF8 and is essential for RNF8-mediated chromatin unfolding. The chromatin remodelling activity of CHD4 promotes efficient ubiquitin conjugation and assembly of RNF168 and BRCA1 at DNA double-strand breaks....... Interestingly, RNF8-mediated recruitment of CHD4 and subsequent chromatin remodelling were independent of the ubiquitin-ligase activity of RNF8, but involved a non-canonical interaction with the forkhead-associated (FHA) domain. Our study reveals a new mechanism of chromatin remodelling-assisted ubiquitylation...

The Major Genetic Determinants of HIV-1 Control Affect HLA Class I Peptide Presentation

OpenAIRE

Pereyra, Florencia; Jia, Xiaoming; McLaren, Paul J.; Telenti, Amalio; de Bakker, Paul I.W.; Walker, Bruce D.; Jia, Xiaoming; McLaren, Paul J.; Ripke, Stephan; Brumme, Chanson J.; Pulit, Sara L.; Telenti, Amalio; Carrington, Mary; Kadie, Carl M.; Carlson, Jonathan M.

2010-01-01

Infectious and inflammatory diseases have repeatedly shown strong genetic associations within the major histocompatibility complex (MHC); however, the basis for these associations remains elusive. To define host genetic effects on the outcome of a chronic viral infection, we performed genome-wide association analysis in a multiethnic cohort of HIV-1 controllers and progressors, and we analyzed the effects of individual amino acids within the classical human leukocyte antigen (HLA) proteins. W...

RavN is a member of a previously unrecognized group of Legionella pneumophila E3 ubiquitin ligases

Science.gov (United States)

Lin, Yi-Han; Evans, Timothy R.; Doms, Alexandra G.; Beauchene, Nicole A.; Hierro, Aitor

2018-01-01

The eukaryotic ubiquitylation machinery catalyzes the covalent attachment of the small protein modifier ubiquitin to cellular target proteins in order to alter their fate. Microbial pathogens exploit this post-translational modification process by encoding molecular mimics of E3 ubiquitin ligases, eukaryotic enzymes that catalyze the final step in the ubiquitylation cascade. Here, we show that the Legionella pneumophila effector protein RavN belongs to a growing class of bacterial proteins that mimic host cell E3 ligases to exploit the ubiquitylation pathway. The E3 ligase activity of RavN was located within its N-terminal region and was dependent upon interaction with a defined subset of E2 ubiquitin-conjugating enzymes. The crystal structure of the N-terminal region of RavN revealed a U-box-like motif that was only remotely similar to other U-box domains, indicating that RavN is an E3 ligase relic that has undergone significant evolutionary alteration. Substitution of residues within the predicted E2 binding interface rendered RavN inactive, indicating that, despite significant structural changes, the mode of E2 recognition has remained conserved. Using hidden Markov model-based secondary structure analyses, we identified and experimentally validated four additional L. pneumophila effectors that were not previously recognized to possess E3 ligase activity, including Lpg2452/SdcB, a new paralog of SidC. Our study provides strong evidence that L. pneumophila is dedicating a considerable fraction of its effector arsenal to the manipulation of the host ubiquitylation pathway. PMID:29415051

Entropy Transfer between Residue Pairs and Allostery in Proteins: Quantifying Allosteric Communication in Ubiquitin.

Directory of Open Access Journals (Sweden)

Aysima Hacisuleyman

2017-01-01

Full Text Available It has recently been proposed by Gunasakaran et al. that allostery may be an intrinsic property of all proteins. Here, we develop a computational method that can determine and quantify allosteric activity in any given protein. Based on Schreiber's transfer entropy formulation, our approach leads to an information transfer landscape for the protein that shows the presence of entropy sinks and sources and explains how pairs of residues communicate with each other using entropy transfer. The model can identify the residues that drive the fluctuations of others. We apply the model to Ubiquitin, whose allosteric activity has not been emphasized until recently, and show that there are indeed systematic pathways of entropy and information transfer between residues that correlate well with the activities of the protein. We use 600 nanosecond molecular dynamics trajectories for Ubiquitin and its complex with human polymerase iota and evaluate entropy transfer between all pairs of residues of Ubiquitin and quantify the binding susceptibility changes upon complex formation. We explain the complex formation propensities of Ubiquitin in terms of entropy transfer. Important residues taking part in allosteric communication in Ubiquitin predicted by our approach are in agreement with results of NMR relaxation dispersion experiments. Finally, we show that time delayed correlation of fluctuations of two interacting residues possesses an intrinsic causality that tells which residue controls the interaction and which one is controlled. Our work shows that time delayed correlations, entropy transfer and causality are the required new concepts for explaining allosteric communication in proteins.

RYBP Is a K63-Ubiquitin-Chain-Binding Protein that Inhibits Homologous Recombination Repair

Directory of Open Access Journals (Sweden)

Mohammad A.M. Ali

2018-01-01

Full Text Available Summary: Ring1-YY1-binding protein (RYBP is a member of the non-canonical polycomb repressive complex 1 (PRC1, and like other PRC1 members, it is best described as a transcriptional regulator. However, several PRC1 members were recently shown to function in DNA repair. Here, we report that RYBP preferentially binds K63-ubiquitin chains via its Npl4 zinc finger (NZF domain. Since K63-linked ubiquitin chains are assembled at DNA double-strand breaks (DSBs, we examined the contribution of RYBP to DSB repair. Surprisingly, we find that RYBP is K48 polyubiquitylated by RNF8 and rapidly removed from chromatin upon DNA damage by the VCP/p97 segregase. High expression of RYBP competitively inhibits recruitment of BRCA1 repair complex to DSBs, reducing DNA end resection and homologous recombination (HR repair. Moreover, breast cancer cell lines expressing high endogenous RYBP levels show increased sensitivity to DNA-damaging agents and poly ADP-ribose polymerase (PARP inhibition. These data suggest that RYBP negatively regulates HR repair by competing for K63-ubiquitin chain binding. : Ali et al. find that RYBP binds K63-linked ubiquitin chains and is removed from DNA damage sites. This K63-ubiquitin binding allows RYBP to hinder the recruitment of BRCA1 and Rad51 to DNA double-strand breaks, thus inhibiting homologous recombination repair. Accordingly, cancer cells expressing high RYBP are more sensitive to DNA-damaging therapies. Keywords: DNA damage response, homologous recombination, ubiquitylation, RYBP, polycomb proteins, double-strand break repair, chromatin, histone modification

Entropy Transfer between Residue Pairs and Allostery in Proteins: Quantifying Allosteric Communication in Ubiquitin.

Science.gov (United States)

Hacisuleyman, Aysima; Erman, Burak

2017-01-01

It has recently been proposed by Gunasakaran et al. that allostery may be an intrinsic property of all proteins. Here, we develop a computational method that can determine and quantify allosteric activity in any given protein. Based on Schreiber's transfer entropy formulation, our approach leads to an information transfer landscape for the protein that shows the presence of entropy sinks and sources and explains how pairs of residues communicate with each other using entropy transfer. The model can identify the residues that drive the fluctuations of others. We apply the model to Ubiquitin, whose allosteric activity has not been emphasized until recently, and show that there are indeed systematic pathways of entropy and information transfer between residues that correlate well with the activities of the protein. We use 600 nanosecond molecular dynamics trajectories for Ubiquitin and its complex with human polymerase iota and evaluate entropy transfer between all pairs of residues of Ubiquitin and quantify the binding susceptibility changes upon complex formation. We explain the complex formation propensities of Ubiquitin in terms of entropy transfer. Important residues taking part in allosteric communication in Ubiquitin predicted by our approach are in agreement with results of NMR relaxation dispersion experiments. Finally, we show that time delayed correlation of fluctuations of two interacting residues possesses an intrinsic causality that tells which residue controls the interaction and which one is controlled. Our work shows that time delayed correlations, entropy transfer and causality are the required new concepts for explaining allosteric communication in proteins.

The F-box protein FBXO44 mediates BRCA1 ubiquitination and degradation.

Science.gov (United States)

Lu, Yunzhe; Li, Jiezhi; Cheng, Dongmei; Parameswaran, Balaji; Zhang, Shaohua; Jiang, Zefei; Yew, P Renee; Peng, Junmin; Ye, Qinong; Hu, Yanfen

2012-11-30

BRCA1 mutations account for a significant proportion of familial breast and ovarian cancers. In addition, reduced BRCA1 protein is associated with sporadic cancer cases in these tissues. At the cellular level, BRCA1 plays a critical role in multiple cellular functions such as DNA repair and cell cycle checkpoint control. Its protein level is regulated in a cell cycle-dependent manner. However, regulation of BRCA1 protein stability is not fully understood. Our earlier study showed that the amino terminus of BRCA1 harbors a degron sequence that is sufficient and necessary for conferring BRCA1 degradation. In the current study, we used mass spectrometry to identify Skp1 that regulates BRCA1 protein stability. Small interfering RNA screening that targets all human F-box proteins uncovered FBXO44 as an important protein that influences BRCA1 protein level. The Skp1-Cul1-F-box-protein44 (SCF(FBXO44)) complex ubiquitinates full-length BRCA1 in vitro. Furthermore, the N terminus of BRCA1 mediates the interaction between BRCA1 and FBXO44. Overexpression of SCF(FBXO44) reduces BRCA1 protein level. Taken together, our work strongly suggests that SCF(FBXO44) is an E3 ubiquitin ligase responsible for BRCA1 degradation. In addition, FBXO44 expression pattern in breast carcinomas suggests that SCF(FBXO44)-mediated BRCA1 degradation might contribute to sporadic breast tumor development.

Examining the evidence for major histocompatibility complex-dependent mate selection in humans and nonhuman primates

Czech Academy of Sciences Publication Activity Database

Winternitz, Jamie Caroline; Abbate, J. L.

2015-01-01

Roč. 6, 13 May (2015), s. 73-88 ISSN 1179-7274 R&D Projects: GA MŠk(CZ) EE2.3.30.0048 Institutional support: RVO:67985939 Keywords : secual selection * olfaction * facial attraction * inbreeding avoidance * parasite resistance Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Genetics and heredity (medical genetics to be 3)

Interplay between Ubiquitin, SUMO, and Poly(ADP-Ribose) in the Cellular Response to Genotoxic Stress

Science.gov (United States)

Pellegrino, Stefania; Altmeyer, Matthias

2016-01-01

Cells employ a complex network of molecular pathways to cope with endogenous and exogenous genotoxic stress. This multilayered response ensures that genomic lesions are efficiently detected and faithfully repaired in order to safeguard genome integrity. The molecular choreography at sites of DNA damage relies heavily on post-translational modifications (PTMs). Protein modifications with ubiquitin and the small ubiquitin-like modifier SUMO have recently emerged as important regulatory means to coordinate DNA damage signaling and repair. Both ubiquitylation and SUMOylation can lead to extensive chain-like protein modifications, a feature that is shared with yet another DNA damage-induced PTM, the modification of proteins with poly(ADP-ribose) (PAR). Chains of ubiquitin, SUMO, and PAR all contribute to the multi-protein assemblies found at sites of DNA damage and regulate their spatio-temporal dynamics. Here, we review recent advancements in our understanding of how ubiquitin, SUMO, and PAR coordinate the DNA damage response and highlight emerging examples of an intricate interplay between these chain-like modifications during the cellular response to genotoxic stress. PMID:27148359

Ubiquitin-Mediated Regulation of Endocytosis by Proteins of the Arrestin Family

Directory of Open Access Journals (Sweden)

Michel Becuwe

2012-01-01

Full Text Available In metazoans, proteins of the arrestin family are key players of G-protein-coupled receptors (GPCRS signaling and trafficking. Following stimulation, activated receptors are phosphorylated, thus allowing the binding of arrestins and hence an “arrest” of receptor signaling. Arrestins act by uncoupling receptors from G proteins and contribute to the recruitment of endocytic proteins, such as clathrin, to direct receptor trafficking into the endocytic pathway. Arrestins also serve as adaptor proteins by promoting the recruitment of ubiquitin ligases and participate in the agonist-induced ubiquitylation of receptors, known to have impact on their subcellular localization and stability. Recently, the arrestin family has expanded following the discovery of arrestin-related proteins in other eukaryotes such as yeasts or fungi. Surprisingly, most of these proteins are also involved in the ubiquitylation and endocytosis of plasma membrane proteins, thus suggesting that the role of arrestins as ubiquitin ligase adaptors is at the core of these proteins' functions. Importantly, arrestins are themselves ubiquitylated, and this modification is crucial for their function. In this paper, we discuss recent data on the intricate connections between arrestins and the ubiquitin pathway in the control of endocytosis.

Inhibition of Siah2 ubiquitin ligase by vitamin K3 (menadione) attenuates hypoxia and MAPK signaling and blocks melanoma tumorigenesis.

Science.gov (United States)

Shah, Meera; Stebbins, John L; Dewing, Antimone; Qi, Jianfei; Pellecchia, Maurizio; Ronai, Ze'ev A

2009-12-01

The E3 ubiquitin ligase Siah2 has been implicated in the regulation of the hypoxia response, as well as in the control of Ras, JNK/p38/NF-kappaB signaling pathways. Both Ras/mitogen-activated protein kinase (MAPK) and hypoxia pathways are important for melanoma development and progression, pointing to the possible use of Siah2 as target for treatment of this tumor type. In the present study, we have established a high-throughput electro-chemiluninescent-based assay in order to screen and identify inhibitors of Siah2 ubiquitin ligase activity. Of 1840 compounds screened, we identified and characterized menadione (MEN) as a specific inhibitor of Siah2 ligase activity. MEN attenuated Siah2 self-ubiquitination, and increased expression of its substrates PHD3 and Sprouty2, with concomitant decrease in levels of HIF-1alpha and pERK, the respective downstream effectors. MEN treatment no longer affected PHD3 or Sprouty2 in Siah-KO cells, pointing to its Siah-dependent effects. Further, MEN inhibition of Siah2 was not attenuated by free radical scavenger, suggesting it is ROS-independent. Significantly, growth of xenograft melanoma tumors was inhibited following the administration of MEN or its derivative. These findings reveal an efficient platform for the identification of Siah inhibitors while identifying and characterizing MEN as Siah inhibitor that attenuates hypoxia and MAPK signaling, and inhibits melanoma tumorigenesis.

Cellular Ubc2/Rad6 E2 ubiquitin-conjugating enzyme facilitates tombusvirus replication in yeast and plants

International Nuclear Information System (INIS)

Imura, Yoshiyuki; Molho, Melissa; Chuang, Chingkai; Nagy, Peter D.

2015-01-01

Mono- and multi-ubiquitination alters the functions and subcellular localization of many cellular and viral proteins. Viruses can co-opt or actively manipulate the ubiquitin network to support viral processes or suppress innate immunity. Using yeast (Saccharomyces cerevisiae) model host, we show that the yeast Rad6p (radiation sensitive 6) E2 ubiquitin-conjugating enzyme and its plant ortholog, AtUbc2, interact with two tombusviral replication proteins and these E2 ubiquitin-conjugating enzymes could be co-purified with the tombusvirus replicase. We demonstrate that TBSV RNA replication and the mono- and bi-ubiquitination level of p33 is decreased in rad6Δ yeast. However, plasmid-based expression of AtUbc2p could complement both defects in rad6Δ yeast. Knockdown of UBC2 expression in plants also decreases tombusvirus accumulation and reduces symptom severity, suggesting that Ubc2p is critical for virus replication in plants. We provide evidence that Rad6p is involved in promoting the subversion of Vps23p and Vps4p ESCRT proteins for viral replicase complex assembly. - Highlights: • Tombusvirus p33 replication protein interacts with cellular RAD6/Ubc2 E2 enzymes. • Deletion of RAD6 reduces tombusvirus replication in yeast. • Silencing of UBC2 in plants inhibits tombusvirus replication. • Mono- and bi-ubiquitination of p33 replication protein in yeast and in vitro. • Rad6p promotes the recruitment of cellular ESCRT proteins into the tombusvirus replicase

Cellular Ubc2/Rad6 E2 ubiquitin-conjugating enzyme facilitates tombusvirus replication in yeast and plants

Energy Technology Data Exchange (ETDEWEB)

Imura, Yoshiyuki, E-mail: imura@brs.nihon-u.ac.jp; Molho, Melissa; Chuang, Chingkai; Nagy, Peter D., E-mail: pdnagy2@uky.edu

2015-10-15

Mono- and multi-ubiquitination alters the functions and subcellular localization of many cellular and viral proteins. Viruses can co-opt or actively manipulate the ubiquitin network to support viral processes or suppress innate immunity. Using yeast (Saccharomyces cerevisiae) model host, we show that the yeast Rad6p (radiation sensitive 6) E2 ubiquitin-conjugating enzyme and its plant ortholog, AtUbc2, interact with two tombusviral replication proteins and these E2 ubiquitin-conjugating enzymes could be co-purified with the tombusvirus replicase. We demonstrate that TBSV RNA replication and the mono- and bi-ubiquitination level of p33 is decreased in rad6Δ yeast. However, plasmid-based expression of AtUbc2p could complement both defects in rad6Δ yeast. Knockdown of UBC2 expression in plants also decreases tombusvirus accumulation and reduces symptom severity, suggesting that Ubc2p is critical for virus replication in plants. We provide evidence that Rad6p is involved in promoting the subversion of Vps23p and Vps4p ESCRT proteins for viral replicase complex assembly. - Highlights: • Tombusvirus p33 replication protein interacts with cellular RAD6/Ubc2 E2 enzymes. • Deletion of RAD6 reduces tombusvirus replication in yeast. • Silencing of UBC2 in plants inhibits tombusvirus replication. • Mono- and bi-ubiquitination of p33 replication protein in yeast and in vitro. • Rad6p promotes the recruitment of cellular ESCRT proteins into the tombusvirus replicase.

Dynamic ubiquitin signaling in cell cycle regulation.

Science.gov (United States)

Gilberto, Samuel; Peter, Matthias

2017-08-07

The cell division cycle is driven by a collection of enzymes that coordinate DNA duplication and separation, ensuring that genomic information is faithfully and perpetually maintained. The activity of the effector proteins that perform and coordinate these biological processes oscillates by regulated expression and/or posttranslational modifications. Ubiquitylation is a cardinal cellular modification and is long known for driving cell cycle transitions. In this review, we emphasize emerging concepts of how ubiquitylation brings the necessary dynamicity and plasticity that underlie the processes of DNA replication and mitosis. New studies, often focusing on the regulation of chromosomal proteins like DNA polymerases or kinetochore kinases, are demonstrating that ubiquitylation is a versatile modification that can be used to fine-tune these cell cycle events, frequently through processes that do not involve proteasomal degradation. Understanding how the increasing variety of identified ubiquitin signals are transduced will allow us to develop a deeper mechanistic perception of how the multiple factors come together to faithfully propagate genomic information. Here, we discuss these and additional conceptual challenges that are currently under study toward understanding how ubiquitin governs cell cycle regulation. © 2017 Gilberto and Peter.

Eating disorder risk, exercise dependence, and body weight dissatisfaction among female nutrition and exercise science university majors.