A cytoplasmic serine protein kinase binds and may regulate the Fanconi anemia protein FANCA.

Science.gov (United States)

Yagasaki, H; Adachi, D; Oda, T; Garcia-Higuera, I; Tetteh, N; D'Andrea, A D; Futaki, M; Asano, S; Yamashita, T

2001-12-15

Fanconi anemia (FA) is an autosomal recessive disease with congenital anomalies, bone marrow failure, and susceptibility to leukemia. Patient cells show chromosome instability and hypersensitivity to DNA cross-linking agents. At least 8 complementation groups (A-G) have been identified and 6 FA genes (for subtypes A, C, D2, E, F, and G) have been cloned. Increasing evidence indicates that a protein complex assembly of multiple FA proteins, including FANCA and FANCG, plays a crucial role in the FA pathway. Previously, it was reported that FANCA was phosphorylated in lymphoblasts from normal controls, whereas the phosphorylation was defective in those derived from patients with FA of multiple complementation groups. The present study examined phosphorylation of FANCA ectopically expressed in FANCA(-) cells. Several patient-derived mutations abrogated in vivo phosphorylation of FANCA in this system, suggesting that FANCA phosphorylation is associated with its function. In vitro phosphorylation studies indicated that a physiologic protein kinase for FANCA (FANCA-PK) forms a complex with the substrate. Furthermore, at least a part of FANCA-PK as well as phosphorylated FANCA were included in the FANCA/FANCG complex. Thus, FANCA-PK appears to be another component of the FA protein complex and may regulate function of FANCA. FANCA-PK was characterized as a cytoplasmic serine kinase sensitive to wortmannin. Identification of the protein kinase is expected to elucidate regulatory mechanisms that control the FA pathway.

Region of Nipah virus C protein responsible for shuttling between the cytoplasm and nucleus

Energy Technology Data Exchange (ETDEWEB)

Horie, Ryo; Yoneda, Misako, E-mail: yone@ims.u-tokyo.ac.jp; Uchida, Shotaro; Sato, Hiroki; Kai, Chieko

2016-10-15

Nipah virus (NiV) causes severe encephalitis in humans, with high mortality. NiV nonstructural C protein (NiV-C) is essential for its pathogenicity, but its functions are unclear. In this study, we focused on NiV-C trafficking in cells and found that it localizes predominantly in the cytoplasm but partly in the nucleus. An analysis of NiV-C mutants showed that amino acids 2, 21–24 and 110–139 of NiV-C are important for its localization in the cytoplasm. Inhibitor treatment indicates that the nuclear export determinant is not a classical CRM1-dependent nuclear export signal. We also determined that amino acids 60–75 and 72–75 were important for nuclear localization of NiV-C. Furthermore, NiV-C mutants that had lost their capacity for nuclear localization inhibited the interferon (IFN) response more strongly than complete NiV-C. These results indicate that the IFN-antagonist activity of NiV-C occurs in the cytoplasm. -- Highlights: •Nipah virus (NiV) infection resulted in high mortality, but effective treatment has not been established. •Several reports revealed that NiV nonstructural C protein (NiV-C) was essential for NiV pathogenicity, however, whole of NiV-C function is still unknown. •Although nonstructural C proteins of other Paramyxoviruses are expressed in similar mechanism and exert similar activity, subcellular localization and cellular targets are different. In this study, we evaluated the subcellular localization of NiV-C. •To our knowledge, this is the first report showing that NiV-C shuttles between the nucleus and cytoplasm. We also clarified that NiV-C has nuclear export signal and nuclear localization signal using NiV-C deleted, alanine substitution mutants and enhanced green fluorescent protein (EGFP) fused proteins. •And we also showed that interferon (IFN) antagonist activity of NiV-C related to its subcellular localization. Our results indicate that NiV-C exert IFN antagonist activity in the cytoplasm.

The cytoplasmic tails of infectious bronchitis virus E and M proteins mediate their interaction

International Nuclear Information System (INIS)

Corse, Emily; Machamer, Carolyn E.

2003-01-01

Virus-like particle (VLP) formation by the coronavirus E and M proteins suggests that interactions between these proteins play a critical role in coronavirus assembly. We studied interactions between the infectious bronchitis virus (IBV) E and M proteins using in vivo crosslinking and VLP assembly assays. We show that IBV E and M can be crosslinked to each other in IBV-infected and transfected cells, indicating that they interact. The cytoplasmic tails of both proteins are important for this interaction. We also examined the ability of the mutant and chimeric E and M proteins to form VLPs. IBV M proteins that are missing portions of their cytoplasmic tails or transmembrane regions were not able to support VLP formation, regardless of their ability to be crosslinked to IBV E. Interactions between the E and M proteins and the membrane bilayer are likely to play an important role in VLP formation and virus budding

Nucleocapsid Protein from Fig Mosaic Virus Forms Cytoplasmic Agglomerates That Are Hauled by Endoplasmic Reticulum Streaming

Science.gov (United States)

Ishikawa, Kazuya; Miura, Chihiro; Maejima, Kensaku; Komatsu, Ken; Hashimoto, Masayoshi; Tomomitsu, Tatsuya; Fukuoka, Misato; Yusa, Akira; Yamaji, Yasuyuki

2014-01-01

throughout the cell, and we performed live imaging and ultrastructural analysis to identify the mechanism of motility. We provide evidence that cytoplasmic protein agglomerates were passively dragged by actomyosin-mediated streaming of the endoplasmic reticulum (ER) in plant cells. In virus-infected cells, NP agglomerates were surrounded by the ER membranes, indicating that NP agglomerates form the basis of enveloped virus particles in close proximity to the ER. Our work provides a sophisticated model of macromolecular trafficking in plant cells and improves our understanding of the formation of enveloped particles of negative-strand RNA viruses. PMID:25320328

Three major nucleolar proteins migrate from nucleolus to nucleoplasm and cytoplasm in root tip cells of Vicia faba L. exposed to aluminum.

Science.gov (United States)

Qin, Rong; Zhang, Huaning; Li, Shaoshan; Jiang, Wusheng; Liu, Donghua

2014-09-01

Results from our previous investigation indicated that Al could affect the nucleolus and induce extrusion of silver-staining nucleolar particles containing argyrophilic proteins from the nucleolus into the cytoplasm in root tip cells of Vicia faba L. So far, the nucleolar proteins involved have not been identified. It is well known that nucleophosmin (B23), nucleolin (C23), and fibrillarin are three major and multifunctional nucleolar proteins. Therefore, effects of Al on B23, C23, and fibrillarin in root tip cells of V. faba exposed to 100 μM Al for 48 h were observed and analyzed using indirect immunofluorescence microscopy and Western blotting. The results from this work demonstrated that after 100 μM of Al treatment for 48 h, B23 and C23 migrated from the nucleolus to the cytoplasm and fibrillarin from the nucleolus to the nucleoplasm. In some cells, fibrillarin was present only in the cytoplasm. Western blotting data revealed higher expression of the three major nucleolar proteins in Al-treated roots compared with the control and that the B23 content increased markedly. These findings confirmed our previous observations.

Cytoplasmic vitamin A binding proteins in chick embryo dermis and epidermis

International Nuclear Information System (INIS)

Gates, R.E.; King, L.E. Jr.

1985-01-01

Excess vitamin A has striking morphologic and developmental effects on chick embryo skin. While cytoplasmic retinoic acid-binding protein (CRABP) was known to be abundant in chick embryo skin, neither quantitative values nor the distribution between dermis and epidermis have been established. The authors determined CRABP levels in collagenase-separated dermis and epidermis from 8-day-old embryos using specific binding of all-trans-[11- 3 H]retinoic acid in cytosols prepared from gram quantities of these tissues. The level of CRABP in dermis was twice the level in epidermis whether calculated on the basis of wet weight, cytosol protein, or DNA. When averaged over many preparations, 3 times as much dermis as epidermis was recovered from a single piece of skin. Therefore, the dermis contained 85% of the extremely high CRABP levels found in collagenase-treated skin, while epidermis contributed only 15%. Cytoplasmic retinol binding protein (CRBP) was also detected in chick embryo skin, but the binding was low and the levels in epidermis and dermis were not significantly different. The amount of CRABP in chick embryo skin (1600 pmol/g wet weight or 100 pmol/mg cytosol protein) is the highest level reported in any tissue and suggests an important role for vitamin A in the normal development and maturation of skin

Endogenous Mouse Dicer Is an Exclusively Cytoplasmic Protein.

Directory of Open Access Journals (Sweden)

Christian Much

2016-06-01

Full Text Available Dicer is a large multi-domain protein responsible for the ultimate step of microRNA and short-interfering RNA biogenesis. In human and mouse cell lines, Dicer has been shown to be important in the nuclear clearance of dsRNA as well as the establishment of chromatin modifications. Here we set out to unambiguously define the cellular localization of Dicer in mice to understand if this is a conserved feature of mammalian Dicer in vivo. To this end, we utilized an endogenously epitope tagged Dicer knock-in mouse allele. From primary mouse cell lines and adult tissues, we determined with certainty by biochemical fractionation and confocal immunofluorescence microscopy that endogenous Dicer is exclusively cytoplasmic. We ruled out the possibility that a fraction of Dicer shuttles to and from the nucleus as well as that FGF or DNA damage signaling induce Dicer nuclear translocation. We also explored Dicer localization during the dynamic and developmental context of embryogenesis, where Dicer is ubiquitously expressed and strictly cytoplasmic in all three germ layers as well as extraembryonic tissues. Our data exclude a direct role for Dicer in the nuclear RNA processing in the mouse.

Endogenous Mouse Dicer Is an Exclusively Cytoplasmic Protein.

Science.gov (United States)

Much, Christian; Auchynnikava, Tania; Pavlinic, Dinko; Buness, Andreas; Rappsilber, Juri; Benes, Vladimir; Allshire, Robin; O'Carroll, Dónal

2016-06-01

Dicer is a large multi-domain protein responsible for the ultimate step of microRNA and short-interfering RNA biogenesis. In human and mouse cell lines, Dicer has been shown to be important in the nuclear clearance of dsRNA as well as the establishment of chromatin modifications. Here we set out to unambiguously define the cellular localization of Dicer in mice to understand if this is a conserved feature of mammalian Dicer in vivo. To this end, we utilized an endogenously epitope tagged Dicer knock-in mouse allele. From primary mouse cell lines and adult tissues, we determined with certainty by biochemical fractionation and confocal immunofluorescence microscopy that endogenous Dicer is exclusively cytoplasmic. We ruled out the possibility that a fraction of Dicer shuttles to and from the nucleus as well as that FGF or DNA damage signaling induce Dicer nuclear translocation. We also explored Dicer localization during the dynamic and developmental context of embryogenesis, where Dicer is ubiquitously expressed and strictly cytoplasmic in all three germ layers as well as extraembryonic tissues. Our data exclude a direct role for Dicer in the nuclear RNA processing in the mouse.

Cytoplasmic assembly of snRNP particles from stored proteins and newly transcribed snRNA's in L929 mouse fibroblasts

International Nuclear Information System (INIS)

Sauterer, R.A.; Feeney, R.J.; Zieve, G.W.

1988-01-01

Newly synthesized snRNAs appear transiently in the cytoplasm where they assemble into ribonucleoprotein particles, the snRNP particles, before returning permanently to the interphase nucleus. In this report, bona fide cytoplasmic fractions, prepared by cell enucleation, are used for a quantitative analysis of snRNP assembly in growing mouse fibroblasts. The half-lives and abundances of the snRNP precursors in the cytoplasm and the rates of snRNP assembly are calculated in L929 cells. With the exception of U6, the major snRNAs are stable RNA species; U1 is almost totally stable while U2 has a half-life of about two cell cycles. In contrast, the majority of newly synthesized U6 decays with a half-life of about 15 h. The relative abundances of the newly synthesized snRNA species U1, U2, U3, U4 and U6 in the cytoplasm are determined by Northern hybridization using cloned probes and are approximately 2% of their nuclear abundance. The half-lives of the two major snRNA precursors in the cytoplasm (U1 and U2) are approximately 20 min as determined by labeling to steady state. The relative abundance of the snRNP B protein in the cytoplasm is determined by Western blotting with the Sm class of autoantibodies and is approximately 25% of the nuclear abundance. Kinetic studies, using the Sm antiserum to immunoprecipitate the methionine-labeled snRNP proteins, suggest that the B protein has a half-life of 90 to 120 min in the cytoplasm. These data are discussed and suggest that there is a large pool of more stable snRNP proteins in the cytoplasm available for assembly with the less abundant but more rapidly turning-over snRNAs

Correlation between the progressive cytoplasmic expression of a novel small heat shock protein (Hsp16.2) and malignancy in brain tumors

International Nuclear Information System (INIS)

Pozsgai, Eva; Gomori, Eva; Szigeti, Andras; Boronkai, Arpad; Gallyas, Ferenc Jr; Sumegi, Balazs; Bellyei, Szabolcs

2007-01-01

Small heat shock proteins are molecular chaperones that protect proteins against stress-induced aggregation. They have also been found to have anti-apoptotic activity and to play a part in the development of tumors. Recently, we identified a new small heat shock protein, Hsp16.2 which displayed increased expression in neuroectodermal tumors. Our aim was to investigate the expression of Hsp16.2 in different types of brain tumors and to correlate its expression with the histological grade of the tumor. Immunohistochemistry with a polyclonal antibody to Hsp16.2 was carried out on formalin-fixed, paraffin-wax-embedded sections using the streptavidin-biotin method. 91 samples were examined and their histological grade was defined. According to the intensity of Hsp16.2 immunoreactivity, low (+), moderate (++), high (+++) or none (-) scores were given. Immunoblotting was carried out on 30 samples of brain tumors using SDS-polyacrylamide gel electrophoresis and Western-blotting. Low grade (grades 1–2) brain tumors displayed low cytoplasmic Hsp16.2 immunoreactivity, grade 3 tumors showed moderate cytoplasmic staining, while high grade (grade 4) tumors exhibited intensive cytoplasmic Hsp16.2 staining. Immunoblotting supported the above mentioned results. Normal brain tissue acted as a negative control for the experiment, since the cytoplasm did not stain for Hsp16.2. There was a positive correlation between the level of Hsp16.2 expression and the level of anaplasia in different malignant tissue samples. Hsp16.2 expression was directly correlated with the histological grade of brain tumors, therefore Hsp16.2 may have relevance as becoming a possible tumor marker

The poly(rC)-binding protein αCP2 is a noncanonical factor in X. laevis cytoplasmic polyadenylation

Science.gov (United States)

Vishnu, Melanie R.; Sumaroka, Marina; Klein, Peter S.; Liebhaber, Stephen A.

2011-01-01

Post-transcriptional control of mRNA stability and translation is central to multiple developmental pathways. This control can be linked to cytoplasmic polyadenylation in certain settings. In maturing Xenopus oocytes, specific mRNAs are targeted for polyadenylation via recruitment of the Cytoplasmic Polyadenylation Element (CPE) binding protein (CPEB) to CPE(s) within the 3′ UTR. Cytoplasmic polyadenylation is also critical to early embryonic events, although corresponding determinants are less defined. Here, we demonstrate that the Xenopus ortholog of the poly(rC) binding protein αCP2 can recruit cytoplasmic poly(A) polymerase activity to mRNAs in Xenopus post-fertilization embryos, and that this recruitment relies on cis sequences recognized by αCP2. We find that the hα-globin 3′ UTR, a validated mammalian αCP2 target, constitutes an effective target for cytoplasmic polyadenylation in Xenopus embryos, but not during Xenopus oocyte maturation. We further demonstrate that the cytoplasmic polyadenylation activity is dependent on the action of the C-rich αCP-binding site in conjunction with the adjacent AAUAAA. Consistent with its ability to target mRNA for poly(A) addition, we find that XαCP2 associates with core components of the Xenopus cytoplasmic polyadenylation complex, including the cytoplasmic poly(A) polymerase XGLD2. Furthermore, we observe that the C-rich αCP-binding site can robustly enhance the activity of a weak canonical oocyte maturation CPE in early embryos, possibly via a direct interaction between XαCP2 and CPEB1. These studies establish XαCP2 as a novel cytoplasmic polyadenylation trans factor, indicate that C-rich sequences can function as noncanonical cytoplasmic polyadenylation elements, and expand our understanding of the complexities underlying cytoplasmic polyadenylation in specific developmental settings. PMID:21444632

Short Arginine Motifs Drive Protein Stickiness in the Escherichia coli Cytoplasm.

Science.gov (United States)

Kyne, Ciara; Crowley, Peter B

2017-09-19

Although essential to numerous biotech applications, knowledge of molecular recognition by arginine-rich motifs in live cells remains limited. 1 H, 15 N HSQC and 19 F NMR spectroscopies were used to investigate the effects of C-terminal -GR n (n = 1-5) motifs on GB1 interactions in Escherichia coli cells and cell extracts. While the "biologically inert" GB1 yields high-quality in-cell spectra, the -GR n fusions with n = 4 or 5 were undetectable. This result suggests that a tetra-arginine motif is sufficient to drive interactions between a test protein and macromolecules in the E. coli cytoplasm. The inclusion of a 12 residue flexible linker between GB1 and the -GR 5 motif did not improve detection of the "inert" domain. In contrast, all of the constructs were detectable in cell lysates and extracts, suggesting that the arginine-mediated complexes were weak. Together these data reveal the significance of weak interactions between short arginine-rich motifs and the E. coli cytoplasm and demonstrate the potential of such motifs to modify protein interactions in living cells. These interactions must be considered in the design of (in vivo) nanoscale assemblies that rely on arginine-rich sequences.

The 75-kilodalton cytoplasmic Chlamydia trachomatis L2 polypeptide is a DnaK-like protein

DEFF Research Database (Denmark)

Birkelund, Svend; Lundemose, AG; Christiansen, Gunna

1990-01-01

,980-base-pair open reading frame revealed 94% homology with a 75-kilodalton protein from C. trachomatis serovar D and 57% homology with the DnaK proteins of E. coli and of Bacillus megaterium, while amino acid homology with human heat shock protein 70 (hsp70) was 42%. The promoter region was identified......The gene coding for the 75-kilodalton cytoplasmic Chlamydia trachomatis L2 polypeptide has been cloned in Escherichia coli, and the nucleotide sequence has been determined. The cloned DNA fragment contained the coding region as well as the putative promoter. The deduced amino acid sequence of the 1...... by computer search and by primer extension of mRNA synthesized in recombinant E. coli. The promoter region which differed from the putative promoter region in serovar D was shown to be a mixed promoter type in which the -10 region showed a regular TATA box configuration while the -35 region showed high...

Functional role of the cytoplasmic tail domain of the major envelope fusion protein of group II baculoviruses

NARCIS (Netherlands)

Long, G.; Pan, M.; Westenberg, M.; Vlak, J.M.

2006-01-01

F proteins from baculovirus nucleopolyhedrovirus (NPV) group II members are the major budded virus (BV) viral envelope fusion proteins. They undergo furin-like proteolysis processing in order to be functional. F proteins from different baculovirus species have a long cytoplasmic tail domain (CTD),

Correlation between the progressive cytoplasmic expression of a novel small heat shock protein (Hsp16.2 and malignancy in brain tumors

Directory of Open Access Journals (Sweden)

Gallyas Ferenc

2007-12-01

Full Text Available Abstract Background Small heat shock proteins are molecular chaperones that protect proteins against stress-induced aggregation. They have also been found to have anti-apoptotic activity and to play a part in the development of tumors. Recently, we identified a new small heat shock protein, Hsp16.2 which displayed increased expression in neuroectodermal tumors. Our aim was to investigate the expression of Hsp16.2 in different types of brain tumors and to correlate its expression with the histological grade of the tumor. Methods Immunohistochemistry with a polyclonal antibody to Hsp16.2 was carried out on formalin-fixed, paraffin-wax-embedded sections using the streptavidin-biotin method. 91 samples were examined and their histological grade was defined. According to the intensity of Hsp16.2 immunoreactivity, low (+, moderate (++, high (+++ or none (- scores were given. Immunoblotting was carried out on 30 samples of brain tumors using SDS-polyacrylamide gel electrophoresis and Western-blotting. Results Low grade (grades 1–2 brain tumors displayed low cytoplasmic Hsp16.2 immunoreactivity, grade 3 tumors showed moderate cytoplasmic staining, while high grade (grade 4 tumors exhibited intensive cytoplasmic Hsp16.2 staining. Immunoblotting supported the above mentioned results. Normal brain tissue acted as a negative control for the experiment, since the cytoplasm did not stain for Hsp16.2. There was a positive correlation between the level of Hsp16.2 expression and the level of anaplasia in different malignant tissue samples. Conclusion Hsp16.2 expression was directly correlated with the histological grade of brain tumors, therefore Hsp16.2 may have relevance as becoming a possible tumor marker.

Protein synthesis and the recovery of both survival and cytoplasmic "petite" mutation in ultraviolet-treated yeast cells. II. Mitochondrial protein synthesis.

Science.gov (United States)

Heude, M; Chanet, R

1975-04-01

The contribution of mitochondrial proteins in the repair of UV-induced lethal and cytoplasmic genetic damages was studied in dark liquid held exponential and stationary phase yeast cells. This was performed by using the specific inhibitors, erythromycin (ER) anc chloramphenicol (CAP). It was shown that mitochondrial proteins are involved in the recovery of stationary phase cells. Mitochondrial proteins are partly implicated in the mechanisms leading to the restoration of the (see article) genotype in UV-irradiated dark liquid held exponential phase cells. Here again, in stationary phase cells, mitochondrial enzymes do not seem to participate in the negative liquid holding (NLH) process for the (see article) induction, as shown by inhibiting mitochondrial protein synthesis or both mitochondrial and nuclear protein synthesis. When cells are grown in glycerol, the response after dark liquid holding of UV-treated cells in the different growth stages are similar to that found for glucose-grown cells. In other words, the fate of cytoplasmic genetic damage, in particular, is not correlated with the repressed or derepressed state of the mitochondria.

Mutant p53 protein localized in the cytoplasm inhibits autophagy.

Science.gov (United States)

Morselli, Eugenia; Tasdemir, Ezgi; Maiuri, Maria Chiara; Galluzzi, Lorenzo; Kepp, Oliver; Criollo, Alfredo; Vicencio, José Miguel; Soussi, Thierry; Kroemer, Guido

2008-10-01

The knockout, knockdown or chemical inhibition of p53 stimulates autophagy. Moreover, autophagy-inducing stimuli such as nutrient depletion, rapamycin or lithium cause the depletion of cytoplasmic p53, which in turn is required for the induction of autophagy. Here, we show that retransfection of p53(-/-) HCT 116 colon carcinoma cells with wild type p53 decreases autophagy down to baseline levels. Surprisingly, one third among a panel of 22 cancer-associated p53 single amino acid mutants also inhibited autophagy when transfected into p53(-/-) cells. Those variants of p53 that preferentially localize to the cytoplasm effectively repressed autophagy, whereas p53 mutants that display a prominently nuclear distribution failed to inhibit autophagy. The investigation of a series of deletion mutants revealed that removal of the DNA-binding domain from p53 fails to interfere with its role in the regulation of autophagy. Altogether, these results identify the cytoplasmic localization of p53 as the most important feature for p53-mediated autophagy inhibition. Moreover, the structural requirements for the two biological activities of extranuclear p53, namely induction of apoptosis and inhibition of autophagy, are manifestly different.

Characterization of phosphorylation sites in the cytoplasmic domain of the 300 kDa mannose-6-phosphate receptor

DEFF Research Database (Denmark)

Rosorius, O; Mieskes, G; Issinger, O G

1993-01-01

The human 300 kDa mannose-6-phosphate receptor (MPR 300) is phosphorylated in vivo at serine residues of its cytoplasmic domain. Two-dimensional separation can resolve tryptic phosphopeptides into four major species. To identify the kinases involved in MPR 300 phosphorylation and the phosphorylat......The human 300 kDa mannose-6-phosphate receptor (MPR 300) is phosphorylated in vivo at serine residues of its cytoplasmic domain. Two-dimensional separation can resolve tryptic phosphopeptides into four major species. To identify the kinases involved in MPR 300 phosphorylation...... and the phosphorylation sites the entire coding sequence of the cytoplasmic tail was expressed in Escherichia coli. The isolated cytoplasmic domain was used as a substrate for four purified serine/threonine kinases [casein kinase II (CK II), protein kinase A (PKA), protein kinase C and Ca2+/calmodulin kinase]. All...... kinases phosphorylate the cytoplasmic tail exclusively on serine residues. Inhibition studies using synthetic peptides, partial sequencing of isolated tryptic phosphopeptides and co-migration with tryptic phosphopeptides from MPR 300 labelled in vivo showed that (i) PKA phosphorylates the cytoplasmic MPR...

Quantitative proteomic view on secreted, cell surface-associated, and cytoplasmic proteins of the methicillin-resistant human pathogen Staphylococcus aureus under iron-limited conditions.

Science.gov (United States)

Hempel, Kristina; Herbst, Florian-Alexander; Moche, Martin; Hecker, Michael; Becher, Dörte

2011-04-01

Staphylococcus aureus is capable of colonizing and infecting humans by its arsenal of surface-exposed and secreted proteins. Iron-limited conditions in mammalian body fluids serve as a major environmental signal to bacteria to express virulence determinants. Here we present a comprehensive, gel-free, and GeLC-MS/MS-based quantitative proteome profiling of S. aureus under this infection-relevant situation. (14)N(15)N metabolic labeling and three complementing approaches were combined for relative quantitative analyses of surface-associated proteins. The surface-exposed and secreted proteome profiling approaches comprise trypsin shaving, biotinylation, and precipitation of the supernatant. By analysis of the outer subproteomic and cytoplasmic protein fraction, 1210 proteins could be identified including 221 surface-associated proteins. Thus, access was enabled to 70% of the predicted cell wall-associated proteins, 80% of the predicted sortase substrates, two/thirds of lipoproteins and more than 50% of secreted and cytoplasmic proteins. For iron-deficiency, 158 surface-associated proteins were quantified. Twenty-nine proteins were found in altered amounts showing particularly surface-exposed proteins strongly induced, such as the iron-regulated surface determinant proteins IsdA, IsdB, IsdC and IsdD as well as lipid-anchored iron compound-binding proteins. The work presents a crucial subject for understanding S. aureus pathophysiology by the use of methods that allow quantitative surface proteome profiling.

An ABC transporter B family protein, ABCB19, is required for cytoplasmic streaming and gravitropism of the inflorescence stems.

Science.gov (United States)

Okamoto, Keishi; Ueda, Haruko; Shimada, Tomoo; Tamura, Kentaro; Koumoto, Yasuko; Tasaka, Masao; Morita, Miyo Terao; Hara-Nishimura, Ikuko

2016-01-01

A significant feature of plant cells is the extensive motility of organelles and the cytosol, which was originally defined as cytoplasmic streaming. We suggested previously that a three-way interaction between plant-specific motor proteins myosin XIs, actin filaments, and the endoplasmic reticulum (ER) was responsible for cytoplasmic streaming. (1) Currently, however, there are no reports of molecular components for cytoplasmic streaming other than the actin-myosin-cytoskeleton and ER-related proteins. In the present study, we found that elongated cells of inflorescence stems of Arabidopsis thaliana exhibit vigorous cytoplasmic streaming. Statistical analysis showed that the maximal velocity of plastid movements is 7.26 µm/s, which is much faster than the previously reported velocities of organelles. Surprisingly, the maximal velocity of streaming in the inflorescence stem cells was significantly reduced to 1.11 µm/s in an Arabidopsis mutant, abcb19-101, which lacks ATP BINDING CASSETTE SUBFAMILY B19 (ABCB19) that mediates the polar transport of the phytohormone auxin together with PIN-FORMED (PIN) proteins. Polar auxin transport establishes the auxin concentration gradient essential for plant development and tropisms. Deficiency of ABCB19 activity eventually caused enhanced gravitropic responses of the inflorescence stems and abnormally flexed inflorescence stems. These results suggest that ABCB19-mediated auxin transport plays a role not only in tropism regulation, but also in cytoplasmic streaming.

Integrin cytoplasmic domain-associated protein-1 (ICAP-1) interacts with the ROCK-I kinase at the plasma membrane

NARCIS (Netherlands)

Stroeken, Peter J. M.; Alvarez, Belén; van Rheenen, Jacco; Wijnands, Yvonne M.; Geerts, Dirk; Jalink, Kees; Roos, Ed

2006-01-01

The integrin cytoplasmic domain-associated protein-1 (ICAP-1) binds via its C-terminal PTB (phosphotyrosine-binding) domain to the cytoplasmic tails of beta1 but not other integrins. Using the yeast two-hybrid assay, we found that ICAP-1 binds the ROCK-I kinase, an effector of the RhoA GTPase. By

Ubiquitin-regulated nuclear-cytoplasmic trafficking of the Nipah virus matrix protein is important for viral budding.

Directory of Open Access Journals (Sweden)

Yao E Wang

2010-11-01

Full Text Available Paramyxoviruses are known to replicate in the cytoplasm and bud from the plasma membrane. Matrix is the major structural protein in paramyxoviruses that mediates viral assembly and budding. Curiously, the matrix proteins of a few paramyxoviruses have been found in the nucleus, although the biological function associated with this nuclear localization remains obscure. We report here that the nuclear-cytoplasmic trafficking of the Nipah virus matrix (NiV-M protein and associated post-translational modification play a critical role in matrix-mediated virus budding. Nipah virus (NiV is a highly pathogenic emerging paramyxovirus that causes fatal encephalitis in humans, and is classified as a Biosafety Level 4 (BSL4 pathogen. During live NiV infection, NiV-M was first detected in the nucleus at early stages of infection before subsequent localization to the cytoplasm and the plasma membrane. Mutations in the putative bipartite nuclear localization signal (NLS and the leucine-rich nuclear export signal (NES found in NiV-M impaired its nuclear-cytoplasmic trafficking and also abolished NiV-M budding. A highly conserved lysine residue in the NLS served dual functions: its positive charge was important for mediating nuclear import, and it was also a potential site for monoubiquitination which regulates nuclear export of the protein. Concordantly, overexpression of ubiquitin enhanced NiV-M budding whereas depletion of free ubiquitin in the cell (via proteasome inhibitors resulted in nuclear retention of NiV-M and blocked viral budding. Live Nipah virus budding was exquisitely sensitive to proteasome inhibitors: bortezomib, an FDA-approved proteasome inhibitor for treating multiple myeloma, reduced viral titers with an IC(50 of 2.7 nM, which is 100-fold less than the peak plasma concentration that can be achieved in humans. This opens up the possibility of using an "off-the-shelf" therapeutic against acute NiV infection.

Cytoplasmic Localization of HTLV-1 HBZ Protein: A Biomarker of HTLV-1-Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP).

Science.gov (United States)

Baratella, Marco; Forlani, Greta; Raval, Goutham U; Tedeschi, Alessandra; Gout, Olivier; Gessain, Antoine; Tosi, Giovanna; Accolla, Roberto S

2017-01-01

HTLV-1 is the causative agent of a severe form of adult T cell leukemia/Lymphoma (ATL), and of a chronic progressive neuromyelopathy designated HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). Two important HTLV-1-encoded proteins, Tax-1 and HBZ, play crucial roles in the generation and maintenance of the oncogenic process. Less information is instead available on the molecular and cellular mechanisms leading to HAM/TSP. More importantly, no single specific biomarker has been described that unambiguously define the status of HAM/TSP. Here we report for the first time the finding that HBZ, described until now as an exclusive nuclear protein both in chronically infected and in ATL cells, is instead exclusively localized in the cytoplasm of peripheral blood mononuclear cells (PBMC) from patients suffering of HAM/TSP. Interestingly, at the single cell level, HBZ and Tax-1 proteins are never found co-expressed in the same cell, suggesting the existence of mechanisms of expression uncoupling of these two important HTLV-1 viral products in HAM/TSP patients. Cells expressing cytoplasmic HBZ were almost exclusively found in the CD4+ T cell compartment that was not, at least in a representative HAM/TSP patient, expressing the CD25 marker. Less than 1 percent CD8+ T cells were fond positive for HBZ, while B cells and NK cells were found negative for HBZ in HAM/TSP patients. Our results identify the cytoplasmic localization of HBZ in HAM/TSP patient as a possible biomarker of this rather neglected tropical disease, and raise important hypotheses on the role of HBZ in the pathogenesis of the neuromyelopathy associated to HTLV-1 infection.

Cytoplasmic Localization of HTLV-1 HBZ Protein: A Biomarker of HTLV-1-Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP.

Directory of Open Access Journals (Sweden)

Marco Baratella

2017-01-01

Full Text Available HTLV-1 is the causative agent of a severe form of adult T cell leukemia/Lymphoma (ATL, and of a chronic progressive neuromyelopathy designated HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP. Two important HTLV-1-encoded proteins, Tax-1 and HBZ, play crucial roles in the generation and maintenance of the oncogenic process. Less information is instead available on the molecular and cellular mechanisms leading to HAM/TSP. More importantly, no single specific biomarker has been described that unambiguously define the status of HAM/TSP. Here we report for the first time the finding that HBZ, described until now as an exclusive nuclear protein both in chronically infected and in ATL cells, is instead exclusively localized in the cytoplasm of peripheral blood mononuclear cells (PBMC from patients suffering of HAM/TSP. Interestingly, at the single cell level, HBZ and Tax-1 proteins are never found co-expressed in the same cell, suggesting the existence of mechanisms of expression uncoupling of these two important HTLV-1 viral products in HAM/TSP patients. Cells expressing cytoplasmic HBZ were almost exclusively found in the CD4+ T cell compartment that was not, at least in a representative HAM/TSP patient, expressing the CD25 marker. Less than 1 percent CD8+ T cells were fond positive for HBZ, while B cells and NK cells were found negative for HBZ in HAM/TSP patients. Our results identify the cytoplasmic localization of HBZ in HAM/TSP patient as a possible biomarker of this rather neglected tropical disease, and raise important hypotheses on the role of HBZ in the pathogenesis of the neuromyelopathy associated to HTLV-1 infection.

Stabilization and Degradation Mechanisms of Cytoplasmic Ataxin-1

Directory of Open Access Journals (Sweden)

Mayumi F. Kohiyama

2015-01-01

Full Text Available Aggregation-prone proteins in neurodegenerative disease disrupt cellular protein stabilization and degradation pathways. The neurodegenerative disease spinocerebellar ataxia type 1 (SCA1 is caused by a coding polyglutamine expansion in the Ataxin-1 gene ( ATXN1 , which gives rise to the aggregation-prone mutant form of ATXN1 protein. Cerebellar Purkinje neurons, preferentially vulnerable in SCA1, produce ATXN1 protein in both cytoplasmic and nuclear compartments. Cytoplasmic stabilization of ATXN1 by phosphorylation and 14-3-3-mediated mechanisms ultimately drive translocation of the protein to the nucleus where aggregation may occur. However, experimental inhibition of phosphorylation and 14-3-3 binding results in rapid degradation of ATXN1, thus preventing nuclear translocation and cellular toxicity. The exact mechanism of cytoplasmic ATXN1 degradation is currently unknown; further investigation of degradation may provide future therapeutic targets. This review examines the present understanding of cytoplasmic ATXN1 stabilization and potential degradation mechanisms during normal and pathogenic states.

Efficient soluble expression of disulfide bonded proteins in the cytoplasm of Escherichia coli in fed-batch fermentations on chemically defined minimal media.

Science.gov (United States)

Gąciarz, Anna; Khatri, Narendar Kumar; Velez-Suberbie, M Lourdes; Saaranen, Mirva J; Uchida, Yuko; Keshavarz-Moore, Eli; Ruddock, Lloyd W

2017-06-15

The production of recombinant proteins containing disulfide bonds in Escherichia coli is challenging. In most cases the protein of interest needs to be either targeted to the oxidizing periplasm or expressed in the cytoplasm in the form of inclusion bodies, then solubilized and re-folded in vitro. Both of these approaches have limitations. Previously we showed that soluble expression of disulfide bonded proteins in the cytoplasm of E. coli is possible at shake flask scale with a system, known as CyDisCo, which is based on co-expression of a protein of interest along with a sulfhydryl oxidase and a disulfide bond isomerase. With CyDisCo it is possible to produce disulfide bonded proteins in the presence of intact reducing pathways in the cytoplasm. Here we scaled up production of four disulfide bonded proteins to stirred tank bioreactors and achieved high cell densities and protein yields in glucose fed-batch fermentations, using an E. coli strain (BW25113) with the cytoplasmic reducing pathways intact. Even without process optimization production of purified human single chain IgA 1 antibody fragment reached 139 mg/L and hen avidin 71 mg/L, while purified yields of human growth hormone 1 and interleukin 6 were around 1 g/L. Preliminary results show that human growth hormone 1 was also efficiently produced in fermentations of W3110 strain and when glucose was replaced with glycerol as the carbon source. Our results show for the first time that efficient production of high yields of soluble disulfide bonded proteins in the cytoplasm of E. coli with the reducing pathways intact is feasible to scale-up to bioreactor cultivations on chemically defined minimal media.

Protein synthesis and the recovery of both survival and cytoplasmic ''petite'' mutation in ultraviolet-treated yeast cells

International Nuclear Information System (INIS)

Heude, M.; Chanet, R.

1975-01-01

The contribution of mitochondrial proteins in the repair of UV-induced lethal and cytoplasmic genetic damages was studied in dark liquid-held exponential and stationary phase yeast cells. This was performed by using the specific inhibitors, erythromycin and chloramphenicol. It was shown that mitochondrial proteins are involved in the recovery and survival of UV-treated exponential phase cells, but not in the recovery of stationary phase cells. Mitochondrial proteins are partly implicated in the mechanisms leading to the restoration of the e + genotype in UV-irradiated dark liquid-held exponential phase cells. Here again, in stationary phase cells, mitochondrial enzymes do not seem to participate in the negative liquid-holding process for the e - induction, as shown by inhibiting mitochondrial protein synthesis of both mitochondrial and nuclear protein synthesis. When cells are grown in glycerol, the response after dark liquid-holding of the UV-treated cells in the different growth stages are similar to that found for glucose-grown cells. In other words, the fate of cytoplasmic genetic damage in particular is not correlated with the repressed or derepressed state of the mitochondria

TRIM5α association with cytoplasmic bodies is not required for antiretroviral activity

International Nuclear Information System (INIS)

Song, Byeongwoon; Diaz-Griffero, Felipe; Park, Do Hyun; Rogers, Thomas; Stremlau, Matthew; Sodroski, Joseph

2005-01-01

The tripartite motif (TRIM) protein, TRIM5α, restricts infection by particular retroviruses. Many TRIM proteins form cytoplasmic bodies of unknown function. We investigated the relationship between cytoplasmic body formation and the structure and antiretroviral activity of TRIM5α. In addition to diffuse cytoplasmic staining, the TRIM5α proteins from several primate species were located in cytoplasmic bodies of different sizes; by contrast, TRIM5α from spider monkeys did not form cytoplasmic bodies. Despite these differences, all of the TRIM5α proteins exhibited the ability to restrict infection by particular retroviruses. Treatment of cells with geldanamycin, an Hsp90 inhibitor, resulted in disappearance or reduction of the TRIM5α-associated cytoplasmic bodies, yet exerted little effect on the restriction of retroviral infection. Studies of green fluorescent protein-TRIM5α fusion proteins indicated that no TRIM5α domain is specifically required for association with cytoplasmic bodies. Apparently, the formation of cytoplasmic bodies is not required for the antiretroviral activity of TRIM5α

In-Depth, Label-Free Analysis of the Erythrocyte Cytoplasmic Proteome in Diamond Blackfan Anemia Identifies a Unique Inflammatory Signature.

Directory of Open Access Journals (Sweden)

Esther N Pesciotta

Full Text Available Diamond Blackfan Anemia (DBA is a rare, congenital erythrocyte aplasia that is usually caused by haploinsufficiency of ribosomal proteins due to diverse mutations in one of several ribosomal genes. A striking feature of this disease is that a range of different mutations in ribosomal proteins results in similar disease phenotypes primarily characterized by erythrocyte abnormalities and macrocytic anemia, while most other cell types in the body are minimally affected. Previously, we analyzed the erythrocyte membrane proteomes of several DBA patients and identified several proteins that are not typically associated with this cell type and that suggested inflammatory mechanisms contribute to the pathogenesis of DBA. In this study, we evaluated the erythrocyte cytosolic proteome of DBA patients through in-depth analysis of hemoglobin-depleted erythrocyte cytosols. Simple, reproducible, hemoglobin depletion using nickel columns enabled in-depth analysis of over 1000 cytosolic erythrocyte proteins with only moderate total analysis time per proteome. Label-free quantitation and statistical analysis identified 29 proteins with significantly altered abundance levels in DBA patients compared to matched healthy control donors. Proteins that were significantly increased in DBA erythrocyte cytoplasms included three proteasome subunit beta proteins that make up the immunoproteasome and proteins induced by interferon-γ such as n-myc interactor and interferon-induced 35 kDa protein [NMI and IFI35 respectively]. Pathway analysis confirmed the presence of an inflammatory signature in erythrocytes of DBA patients and predicted key upstream regulators including mitogen activated kinase 1, interferon-γ, tumor suppressor p53, and tumor necrosis factor. These results show that erythrocytes in DBA patients are intrinsically different from those in healthy controls which may be due to an inflammatory response resulting from the inherent molecular defect of ribosomal

Ultrastructural and biochemical evidence for the presence of mature steroidogenic acute regulatory protein (StAR) in the cytoplasm of human luteal cells.

Science.gov (United States)

Sierralta, Walter D; Kohen, Paulina; Castro, Olga; Muñoz, Alex; Strauss, Jerome F; Devoto, Luigi

2005-10-20

The distribution of the steroidogenic acute regulatory protein (StAR) inside thecal and granulosa-lutein cells of human corpus luteum (CL) was assessed by immunoelectron microscopy. We found greater levels of StAR immunolabeling in steroidogenic cells from early- and mid-than in late luteal phase CL and lower levels in cells from women treated with a GnRH antagonist in the mid-luteal phase. Immunoelectron microscopy revealed significant levels of StAR antigen in the mitochondria and in the cytoplasm of luteal cells. The 30 kDa mature StAR protein was present in both mitochondria and cytosol (post-mitochondrial) fractions from homogenates of CL at different ages, whereas cytochrome c and mitochondrial HSP70 were detected only in the mitochondrial fraction. Therefore, we hypothesized that either appreciable processing of StAR 37 kDa pre-protein occurs outside the mitochondria, or mature StAR protein is selectively released into the cytoplasm after mitochondrial processing. The presence of mature StAR in the cytoplasm is consonant with the notion that StAR acts on the outer mitochondrial membrane to effect sterol import, and that StAR may interact with other cytoplasmic proteins involved in cholesterol metabolism, including hormone sensitive lipase.

Changes in protein metabolism after irradiation. Pt. 1. Protease activity, protease pattern, protein and free amino acids in cytoplasm and cell organelles of the rat spleen after 600 R whole body x irradiation

Energy Technology Data Exchange (ETDEWEB)

Valet, G [Max-Planck-Institut fuer Biochemie, Muenchen (F.R. Germany). Abt. fuer Experimentelle Medizin

1975-12-01

The protease activity of cytoplasm and cell organelles of the rat spleen against spleen protein and hemoglobin as a substrate increases during a initial reaction phase of the organism on the first day after 600 R whole body X-irradiation. The alkaline protease in the cytoplasm and the acid protease in the cell organelles increase, whereas the protease activity against externally added hemoglobin as substrate decreases below the initial values. The protein, the protease activity and the free amino acids of the cytoplasm and the cell organelles decrease during the disease phase on day 3 and 4 after irradiation. The protein loss of the spleen is therefore not explained by an increased protease activity. Acid proteases appear in the cytoplasm which derive probably from the cell organelles. The protease activity and the free amino acids are increased in the cytoplasm and the cell organelles during the regeneration phase of the organism between day 15 and 18 after irradiation.

Prediction of Chlamydia pneumoniae protein localization in host mitochondria and cytoplasm and possible involvements in lung cancer etiology: a computational approach

Directory of Open Access Journals (Sweden)

Aws Alshamsan

2017-12-01

Full Text Available Collecting evidence suggests that the intercellular infection of Chlamydia pneumoniae in lungs contributes to the etiology of lung cancer. Many proteins of Chlamydia pneumoniae outmanoeuvre the various system of the host. The infection may regulate various factors, which can influence the growth of lung cancer in affected persons. In this in-silico study, we predict potential targeting of Chlamydia pneumoniae proteins in mitochondrial and cytoplasmic comportments of host cell and their possible involvement in growth and development of lung cancer. Various cellular activities are controlled in mitochondria and cytoplasm, where the localization of Chlamydia pneumoniae proteins may alter the normal functioning of host cells. The rationale of this study is to find out and explain the connection between Chlamydia pneumoniae infection and lung cancer. A sum of 183 and 513 proteins were predicted to target in mitochondria and cytoplasm of host cell out of total 1112 proteins of Chlamydia pneumoniae. In particular, many targeted proteins may interfere with normal growth behaviour of host cells, thereby altering the decision of program cell death. Present article provides a potential connection of Chlamydia pneumoniae protein targeting and proposed that various targeted proteins may play crucial role in lung cancer etiology through diverse mechanisms.

A model for the dynamic nuclear/nucleolar/cytoplasmic trafficking of the porcine reproductive and respiratory syndrome virus (PRRSV) nucleocapsid protein based on live cell imaging

International Nuclear Information System (INIS)

You, Jae-Hwan; Howell, Gareth; Pattnaik, Asit K.; Osorio, Fernando A.; Hiscox, Julian A.

2008-01-01

Porcine reproductive and respiratory syndrome virus (PRRSV), an arterivirus, in common with many other positive strand RNA viruses, encodes a nucleocapsid (N) protein which can localise not only to the cytoplasm but also to the nucleolus in virus-infected cells and cells over-expressing N protein. The dynamic trafficking of positive strand RNA virus nucleocapsid proteins and PRRSV N protein in particular between the cytoplasm and nucleolus is unknown. In this study live imaging of permissive and non-permissive cell lines, in conjunction with photo-bleaching (FRAP and FLIP), was used to investigate the trafficking of fluorescent labeled (EGFP) PRRSV-N protein. The data indicated that EGFP-PRRSV-N protein was not permanently sequestered to the nucleolus and had equivalent mobility to cellular nucleolar proteins. Further the nuclear import of N protein appeared to occur faster than nuclear export, which may account for the observed relative distribution of N protein between the cytoplasm and the nucleolus

Hydrodynamic property of the cytoplasm is sufficient to mediate cytoplasmic streaming in the Caenorhabiditis elegans embryo

Science.gov (United States)

Niwayama, Ritsuya; Shinohara, Kyosuke; Kimura, Akatsuki

2011-01-01

Cytoplasmic streaming is a type of intracellular transport widely seen in nature. Cytoplasmic streaming in Caenorhabditis elegans at the one-cell stage is bidirectional; the flow near the cortex (“cortical flow”) is oriented toward the anterior, whereas the flow in the central region (“cytoplasmic flow”) is oriented toward the posterior. Both cortical flow and cytoplasmic flow depend on non-muscle-myosin II (NMY-2), which primarily localizes in the cortex. The manner in which NMY-2 proteins drive cytoplasmic flow in the opposite direction from remote locations has not been fully understood. In this study, we demonstrated that the hydrodynamic properties of the cytoplasm are sufficient to mediate the forces generated by the cortical myosin to drive bidirectional streaming throughout the cytoplasm. We quantified the flow velocities of cytoplasmic streaming using particle image velocimetry (PIV) and conducted a three-dimensional hydrodynamic simulation using the moving particle semiimplicit method. Our simulation quantitatively reconstructed the quantified flow velocity distribution resolved through PIV analysis. Furthermore, our PIV analyses detected microtubule-dependent flows during the pronuclear migration stage. These flows were reproduced via hydrodynamic interactions between moving pronuclei and the cytoplasm. The agreement of flow dynamics in vivo and in simulation indicates that the hydrodynamic properties of the cytoplasm are sufficient to mediate cytoplasmic streaming in C. elegans embryos. PMID:21730185

Detergent/nanodisc screening for high-resolution NMR studies of an integral membrane protein containing a cytoplasmic domain.

Directory of Open Access Journals (Sweden)

Christos Tzitzilonis

Full Text Available Because membrane proteins need to be extracted from their natural environment and reconstituted in artificial milieus for the 3D structure determination by X-ray crystallography or NMR, the search for membrane mimetic that conserve the native structure and functional activities remains challenging. We demonstrate here a detergent/nanodisc screening study by NMR of the bacterial α-helical membrane protein YgaP containing a cytoplasmic rhodanese domain. The analysis of 2D [(15N,(1H]-TROSY spectra shows that only a careful usage of low amounts of mixed detergents did not perturb the cytoplasmic domain while solubilizing in parallel the transmembrane segments with good spectral quality. In contrast, the incorporation of YgaP into nanodiscs appeared to be straightforward and yielded a surprisingly high quality [(15N,(1H]-TROSY spectrum opening an avenue for the structural studies of a helical membrane protein in a bilayer system by solution state NMR.

The application of protein markers in conversion of maize inbred lines to the cytoplasmic male sterility basis

Directory of Open Access Journals (Sweden)

Stevanovic Milan

2016-01-01

Full Text Available A total of seven maize inbred lines of different origin and maturity group were used in the trial set up according to the split-plot randomized complete block design in five environments. Each inbred was observed in five variants: original inbred (N; cytoplasmic male sterile C-type (CMS-C; restorer for CMS-C (RfC; cytoplasmic male sterile S-type (CMS-S and restorer for CMS-S (RfS. The objective was to compare grain yield of original inbreds and their CMS and Rf variants and to apply Isoelectric focusing (IEF to determine whether the conversion of original inbreds to their CMS and Rf counterparts have been done completely. Protein markers have shown that conversion of almost all inbreds was done good and completely. Only original inbreds ZPL2 and ZPL5 did not concur on banding patterns with their RfC variants. The type of cytoplasm had a very significant impact on grain yield. Namely, CMS-C counterparts significantly out yielded their CMS-S versions, while the inbreds with C and S cytoplasm over yielded inbreds with N cytoplasm, as well as their RfC and RfS versions.

Majority of cellular fatty acid acylated proteins are localized to the cytoplasmic surface of the plasma membrane

International Nuclear Information System (INIS)

Wilcox, C.A.; Olson, E.N.

1987-01-01

The BC 2 Hl muscle cell line was previously reported to contain a broad array of fatty acid acylated proteins. Palmitate was shown to be attached to membrane proteins posttranslationally through thiol ester linkages, whereas myristate was attached cotranslationally, or within seconds thereafter, to soluble and membrane-bound proteins through amide linkages. The temporal and subcellular differences between palmitate and myristate acylation suggested that these two classes of acyl proteins might follow different intracellular pathways to distinct subcellular membrane systems or organelles. In this study, the authors examined the subcellular localization of the major fatty acylated proteins in BC 4 Hl cells. Palmitate-containing proteins were localized to the plasma membrane, but only a subset of myristate-containing proteins was localized to this membrane fraction. The majority of acyl proteins were nonglycosylated and resistant to digestion with extracellular proteases, suggesting that they were not exposed to the external surface of the plasma membrane. Many proteins were, however, digested during incubation of isolated membranes with proteases, which indicates that these proteins were, however, digested during incubation of isolated membranes with proteases, which indicates that these proteins face the cytoplasm. Two-dimensional gel electrophoresis of proteins labeled with [ 3 H]palmitate and [ 3 H]myristate revealed that individual proteins were modified by only one of the two fatty acids and did not undergo both N-linked myristylation and ester-linked palmitylation. Together, these results suggest that the majority of cellular acyl proteins are routed to the cytoplasmic surface of the plasma membrane, and they raise the possibility that fatty acid acylation may play a role in intracellular sorting of nontransmembranous, nonglycosylated membrane proteins

Inhibition of host protein synthesis by Sindbis virus: correlation with viral RNA replication and release of nuclear proteins to the cytoplasm.

Science.gov (United States)

Sanz, Miguel A; García-Moreno, Manuel; Carrasco, Luis

2015-04-01

Infection of mammalian cells by Sindbis virus (SINV) profoundly blocks cellular mRNA translation. Experimental evidence points to viral non-structural proteins (nsPs), in particular nsP2, as the mediator of this inhibition. However, individual expression of nsP1, nsP2, nsP3 or nsP1-4 does not block cellular protein synthesis in BHK cells. Trans-complementation of a defective SINV replicon lacking most of the coding region for nsPs by the co-expression of nsP1-4 propitiates viral RNA replication at low levels, and inhibition of cellular translation is not observed. Exit of nuclear proteins including T-cell intracellular antigen and polypyrimidine tract-binding protein is clearly detected in SINV-infected cells, but not upon the expression of nsPs, even when the defective replicon was complemented. Analysis of a SINV variant with a point mutation in nsP2, exhibiting defects in the shut-off of host protein synthesis, indicates that both viral RNA replication and the release of nuclear proteins to the cytoplasm are greatly inhibited. Furthermore, nucleoside analogues that inhibit cellular and viral RNA synthesis impede the blockade of host mRNA translation, in addition to the release of nuclear proteins. Prevention of the shut-off of host mRNA translation by nucleoside analogues is not due to the inhibition of eIF2α phosphorylation, as this prevention is also observed in PKR(-/-) mouse embryonic fibroblasts that do not phosphorylate eIF2α after SINV infection. Collectively, our observations are consistent with the concept that for the inhibition of cellular protein synthesis to occur, viral RNA replication must take place at control levels, leading to the release of nuclear proteins to the cytoplasm. © 2014 John Wiley & Sons Ltd.

Changes in protein metabolism after irradiation. Pt. 2. Protease activity, protease pattern, protein and free amino acids in cytoplasm and cell organelles of the rat liver after 600 R whole body X-irradiation

Energy Technology Data Exchange (ETDEWEB)

Valet, G [Max-Planck-Institut fuer Biochemie, Muenchen (F.R. Germany). Abt. fuer Experimentelle Medizin

1976-01-01

The protease activity of cytoplasm and cell organelles of the rat liver against liver protein and hemoglobin as a substrate increases during an initial reaction phase on the first day after 600 R whole body x irradiation. This is probably a consequence of the degradation of cellular debris. The protein, the protease activity and the free amino acids of the cytoplasm and the cell organelles decrease during the disease phase on day 3 and 4 after irradiation. The protein loss of the liver is therefore not explained by an increased protease activity. The protease activity and the free amino acids are increased in the cytoplasm and the cell organelles during the regeneration phase of the organism between day 15 and 18 after irradiation.

Complete genome sequence and integrated protein localization and interaction map for alfalfa dwarf virus, which combines properties of both cytoplasmic and nuclear plant rhabdoviruses

Energy Technology Data Exchange (ETDEWEB)

Bejerman, Nicolás, E-mail: n.bejerman@uq.edu.au [Instituto de Patología Vegetal (IPAVE), Centro de Investigaciones Agropecuarias (CIAP), Instituto Nacional de Tecnología Agropecuaria INTA, Camino a 60 Cuadras k 5,5, Córdoba X5020ICA (Argentina); Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, St Lucia, QLD 4072 (Australia); Giolitti, Fabián; Breuil, Soledad de; Trucco, Verónica; Nome, Claudia; Lenardon, Sergio [Instituto de Patología Vegetal (IPAVE), Centro de Investigaciones Agropecuarias (CIAP), Instituto Nacional de Tecnología Agropecuaria INTA, Camino a 60 Cuadras k 5,5, Córdoba X5020ICA (Argentina); Dietzgen, Ralf G. [Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, St Lucia, QLD 4072 (Australia)

2015-09-15

Summary: We have determined the full-length 14,491-nucleotide genome sequence of a new plant rhabdovirus, alfalfa dwarf virus (ADV). Seven open reading frames (ORFs) were identified in the antigenomic orientation of the negative-sense, single-stranded viral RNA, in the order 3′-N-P-P3-M-G-P6-L-5′. The ORFs are separated by conserved intergenic regions and the genome coding region is flanked by complementary 3′ leader and 5′ trailer sequences. Phylogenetic analysis of the nucleoprotein amino acid sequence indicated that this alfalfa-infecting rhabdovirus is related to viruses in the genus Cytorhabdovirus. When transiently expressed as GFP fusions in Nicotiana benthamiana leaves, most ADV proteins accumulated in the cell periphery, but unexpectedly P protein was localized exclusively in the nucleus. ADV P protein was shown to have a homotypic, and heterotypic nuclear interactions with N, P3 and M proteins by bimolecular fluorescence complementation. ADV appears unique in that it combines properties of both cytoplasmic and nuclear plant rhabdoviruses. - Highlights: • The complete genome of alfalfa dwarf virus is obtained. • An integrated localization and interaction map for ADV is determined. • ADV has a genome sequence similarity and evolutionary links with cytorhabdoviruses. • ADV protein localization and interaction data show an association with the nucleus. • ADV combines properties of both cytoplasmic and nuclear plant rhabdoviruses.

Complete genome sequence and integrated protein localization and interaction map for alfalfa dwarf virus, which combines properties of both cytoplasmic and nuclear plant rhabdoviruses

International Nuclear Information System (INIS)

Bejerman, Nicolás; Giolitti, Fabián; Breuil, Soledad de; Trucco, Verónica; Nome, Claudia; Lenardon, Sergio; Dietzgen, Ralf G.

2015-01-01

Summary: We have determined the full-length 14,491-nucleotide genome sequence of a new plant rhabdovirus, alfalfa dwarf virus (ADV). Seven open reading frames (ORFs) were identified in the antigenomic orientation of the negative-sense, single-stranded viral RNA, in the order 3′-N-P-P3-M-G-P6-L-5′. The ORFs are separated by conserved intergenic regions and the genome coding region is flanked by complementary 3′ leader and 5′ trailer sequences. Phylogenetic analysis of the nucleoprotein amino acid sequence indicated that this alfalfa-infecting rhabdovirus is related to viruses in the genus Cytorhabdovirus. When transiently expressed as GFP fusions in Nicotiana benthamiana leaves, most ADV proteins accumulated in the cell periphery, but unexpectedly P protein was localized exclusively in the nucleus. ADV P protein was shown to have a homotypic, and heterotypic nuclear interactions with N, P3 and M proteins by bimolecular fluorescence complementation. ADV appears unique in that it combines properties of both cytoplasmic and nuclear plant rhabdoviruses. - Highlights: • The complete genome of alfalfa dwarf virus is obtained. • An integrated localization and interaction map for ADV is determined. • ADV has a genome sequence similarity and evolutionary links with cytorhabdoviruses. • ADV protein localization and interaction data show an association with the nucleus. • ADV combines properties of both cytoplasmic and nuclear plant rhabdoviruses

Cytoplasmic male sterility-associated chimeric open reading frames identified by mitochondrial genome sequencing of four Cajanus genotypes.

Science.gov (United States)

Tuteja, Reetu; Saxena, Rachit K; Davila, Jaime; Shah, Trushar; Chen, Wenbin; Xiao, Yong-Li; Fan, Guangyi; Saxena, K B; Alverson, Andrew J; Spillane, Charles; Town, Christopher; Varshney, Rajeev K

2013-10-01

The hybrid pigeonpea (Cajanus cajan) breeding technology based on cytoplasmic male sterility (CMS) is currently unique among legumes and displays major potential for yield increase. CMS is defined as a condition in which a plant is unable to produce functional pollen grains. The novel chimeric open reading frames (ORFs) produced as a results of mitochondrial genome rearrangements are considered to be the main cause of CMS. To identify these CMS-related ORFs in pigeonpea, we sequenced the mitochondrial genomes of three C. cajan lines (the male-sterile line ICPA 2039, the maintainer line ICPB 2039, and the hybrid line ICPH 2433) and of the wild relative (Cajanus cajanifolius ICPW 29). A single, circular-mapping molecule of length 545.7 kb was assembled and annotated for the ICPA 2039 line. Sequence annotation predicted 51 genes, including 34 protein-coding and 17 RNA genes. Comparison of the mitochondrial genomes from different Cajanus genotypes identified 31 ORFs, which differ between lines within which CMS is present or absent. Among these chimeric ORFs, 13 were identified by comparison of the related male-sterile and maintainer lines. These ORFs display features that are known to trigger CMS in other plant species and to represent the most promising candidates for CMS-related mitochondrial rearrangements in pigeonpea.

Cellular Subcompartments through Cytoplasmic Streaming.

Science.gov (United States)

Pieuchot, Laurent; Lai, Julian; Loh, Rachel Ann; Leong, Fong Yew; Chiam, Keng-Hwee; Stajich, Jason; Jedd, Gregory

2015-08-24

Cytoplasmic streaming occurs in diverse cell types, where it generally serves a transport function. Here, we examine streaming in multicellular fungal hyphae and identify an additional function wherein regimented streaming forms distinct cytoplasmic subcompartments. In the hypha, cytoplasm flows directionally from cell to cell through septal pores. Using live-cell imaging and computer simulations, we identify a flow pattern that produces vortices (eddies) on the upstream side of the septum. Nuclei can be immobilized in these microfluidic eddies, where they form multinucleate aggregates and accumulate foci of the HDA-2 histone deacetylase-associated factor, SPA-19. Pores experiencing flow degenerate in the absence of SPA-19, suggesting that eddy-trapped nuclei function to reinforce the septum. Together, our data show that eddies comprise a subcellular niche favoring nuclear differentiation and that subcompartments can be self-organized as a consequence of regimented cytoplasmic streaming. Copyright © 2015 Elsevier Inc. All rights reserved.

Leukemia-Associated Nup214 Fusion Proteins Disturb the XPO1-Mediated Nuclear-Cytoplasmic Transport Pathway and Thereby the NF-κB Signaling Pathway.

Science.gov (United States)

Saito, Shoko; Cigdem, Sadik; Okuwaki, Mitsuru; Nagata, Kyosuke

2016-07-01

Nuclear-cytoplasmic transport through nuclear pore complexes is mediated by nuclear transport receptors. Previous reports have suggested that aberrant nuclear-cytoplasmic transport due to mutations or overexpression of nuclear pore complexes and nuclear transport receptors is closely linked to diseases. Nup214, a component of nuclear pore complexes, has been found as chimeric fusion proteins in leukemia. Among various Nup214 fusion proteins, SET-Nup214 and DEK-Nup214 have been shown to be engaged in tumorigenesis, but their oncogenic mechanisms remain unclear. In this study, we examined the functions of the Nup214 fusion proteins by focusing on their effects on nuclear-cytoplasmic transport. We found that SET-Nup214 and DEK-Nup214 interact with exportin-1 (XPO1)/CRM1 and nuclear RNA export factor 1 (NXF1)/TAP, which mediate leucine-rich nuclear export signal (NES)-dependent protein export and mRNA export, respectively. SET-Nup214 and DEK-Nup214 decreased the XPO1-mediated nuclear export of NES proteins such as cyclin B and proteins involved in the NF-κB signaling pathway by tethering XPO1 onto nuclear dots where Nup214 fusion proteins are localized. We also demonstrated that SET-Nup214 and DEK-Nup214 expression inhibited NF-κB-mediated transcription by abnormal tethering of the complex containing p65 and its inhibitor, IκB, in the nucleus. These results suggest that SET-Nup214 and DEK-Nup214 perturb the regulation of gene expression through alteration of the nuclear-cytoplasmic transport system. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Lessons from Animal Models of Cytoplasmic Intermediate Filament Proteins.

Science.gov (United States)

Bouameur, Jamal-Eddine; Magin, Thomas M

Cytoplasmic intermediate filaments (IFs) represent a major cytoskeletal network contributing to cell shape, adhesion and migration as well as to tissue resilience and renewal in numerous bilaterians, including mammals. The observation that IFs are dispensable in cultured mammalian cells, but cause tissue-specific, life-threatening disorders, has pushed the need to investigate their function in vivo. In keeping with human disease, the deletion or mutation of murine IF genes resulted in highly specific pathologies. Epidermal keratins, together with desmin, are essential to protect corresponding tissues against mechanical force but also participate in stabilizing cell adhesion and in inflammatory signalling. Surprisingly, other IF proteins contribute to tissue integrity to a much lesser extent than anticipated, pointing towards their role in stress situations. In support, the overexpression of small chaperones or the interference with inflammatory signalling in several settings has been shown to rescue severe tissue pathologies that resulted from the expression of mutant IF proteins. It stills remains an open issue whether the wide range of IF disorders share similar pathomechanisms. Moreover, we lack an understanding how IF proteins participate in signalling processes. Now, with a large number of mouse models in hand, the next challenge will be to develop organotypic cell culture models to dissect pathomechanisms at the molecular level, to employ Crispr/Cas-mediated genome engineering to optimize models and, finally, to combine available animal models with medicinal chemistry for the development of molecular therapies.

SH2 and SH3 domains: elements that control interactions of cytoplasmic signaling proteins.

Science.gov (United States)

Koch, C A; Anderson, D; Moran, M F; Ellis, C; Pawson, T

1991-05-03

Src homology (SH) regions 2 and 3 are noncatalytic domains that are conserved among a series of cytoplasmic signaling proteins regulated by receptor protein-tyrosine kinases, including phospholipase C-gamma, Ras GTPase (guanosine triphosphatase)-activating protein, and Src-like tyrosine kinases. The SH2 domains of these signaling proteins bind tyrosine phosphorylated polypeptides, implicated in normal signaling and cellular transformation. Tyrosine phosphorylation acts as a switch to induce the binding of SH2 domains, thereby mediating the formation of heteromeric protein complexes at or near the plasma membrane. The formation of these complexes is likely to control the activation of signal transduction pathways by tyrosine kinases. The SH3 domain is a distinct motif that, together with SH2, may modulate interactions with the cytoskeleton and membrane. Some signaling and transforming proteins contain SH2 and SH3 domains unattached to any known catalytic element. These noncatalytic proteins may serve as adaptors to link tyrosine kinases to specific target proteins. These observations suggest that SH2 and SH3 domains participate in the control of intracellular responses to growth factor stimulation.

Coulomb interactions between cytoplasmic electric fields and phosphorylated messenger proteins optimize information flow in cells.

Directory of Open Access Journals (Sweden)

Robert A Gatenby

2010-08-01

Full Text Available Normal cell function requires timely and accurate transmission of information from receptors on the cell membrane (CM to the nucleus. Movement of messenger proteins in the cytoplasm is thought to be dependent on random walk. However, Brownian motion will disperse messenger proteins throughout the cytosol resulting in slow and highly variable transit times. We propose that a critical component of information transfer is an intracellular electric field generated by distribution of charge on the nuclear membrane (NM. While the latter has been demonstrated experimentally for decades, the role of the consequent electric field has been assumed to be minimal due to a Debye length of about 1 nanometer that results from screening by intracellular Cl- and K+. We propose inclusion of these inorganic ions in the Debye-Huckel equation is incorrect because nuclear pores allow transit through the membrane at a rate far faster than the time to thermodynamic equilibrium. In our model, only the charged, mobile messenger proteins contribute to the Debye length.Using this revised model and published data, we estimate the NM possesses a Debye-Huckel length of a few microns and find this is consistent with recent measurement using intracellular nano-voltmeters. We demonstrate the field will accelerate isolated messenger proteins toward the nucleus through Coulomb interactions with negative charges added by phosphorylation. We calculate transit times as short as 0.01 sec. When large numbers of phosphorylated messenger proteins are generated by increasing concentrations of extracellular ligands, we demonstrate they generate a self-screening environment that regionally attenuates the cytoplasmic field, slowing movement but permitting greater cross talk among pathways. Preliminary experimental results with phosphorylated RAF are consistent with model predictions.This work demonstrates that previously unrecognized Coulomb interactions between phosphorylated messenger

Cytoplasmic Domains and Voltage-Dependent Potassium Channel Gating

Science.gov (United States)

Barros, Francisco; Domínguez, Pedro; de la Peña, Pilar

2012-01-01

The basic architecture of the voltage-dependent K+ channels (Kv channels) corresponds to a transmembrane protein core in which the permeation pore, the voltage-sensing components and the gating machinery (cytoplasmic facing gate and sensor–gate coupler) reside. Usually, large protein tails are attached to this core, hanging toward the inside of the cell. These cytoplasmic regions are essential for normal channel function and, due to their accessibility to the cytoplasmic environment, constitute obvious targets for cell-physiological control of channel behavior. Here we review the present knowledge about the molecular organization of these intracellular channel regions and their role in both setting and controlling Kv voltage-dependent gating properties. This includes the influence that they exert on Kv rapid/N-type inactivation and on activation/deactivation gating of Shaker-like and eag-type Kv channels. Some illustrative examples about the relevance of these cytoplasmic domains determining the possibilities for modulation of Kv channel gating by cellular components are also considered. PMID:22470342

Changes in Cellular mRNA Stability, Splicing, and Polyadenylation through HuR Protein Sequestration by a Cytoplasmic RNA Virus

Directory of Open Access Journals (Sweden)

Michael D. Barnhart

2013-11-01

Full Text Available The impact of RNA viruses on the posttranscriptional regulation of cellular gene expression is unclear. Sindbis virus causes a dramatic relocalization of the cellular HuR protein from the nucleus to the cytoplasm in infected cells. This is to the result of the expression of large amounts of viral RNAs that contain high-affinity HuR binding sites in their 3′ UTRs effectively serving as a sponge for the HuR protein. Sequestration of HuR by Sindbis virus is associated with destabilization of cellular mRNAs that normally bind HuR and rely on it to regulate their expression. Furthermore, significant changes can be observed in nuclear alternative polyadenylation and splicing events on cellular pre-mRNAs as a result of sequestration of HuR protein by the 3′ UTR of transcripts of this cytoplasmic RNA virus. These studies suggest a molecular mechanism of virus-host interaction that probably has a significant impact on virus replication, cytopathology, and pathogenesis.

Cytoplasmic Dynein Is Required for the Spatial Organization of Protein Aggregates in Filamentous Fungi

Directory of Open Access Journals (Sweden)

Martin J. Egan

2015-04-01

Full Text Available Eukaryotes have evolved multiple strategies for maintaining cellular protein homeostasis. One such mechanism involves neutralization of deleterious protein aggregates via their defined spatial segregation. Here, using the molecular disaggregase Hsp104 as a marker for protein aggregation, we describe the spatial and temporal dynamics of protein aggregates in the filamentous fungus Aspergillus nidulans. Filamentous fungi, such as A. nidulans, are a diverse group of species of major health and economic importance and also serve as model systems for studying highly polarized eukaryotic cells. We find that microtubules promote the formation of Hsp104-positive aggregates, which coalesce into discrete subcellular structures in a process dependent on the microtubule-based motor cytoplasmic dynein. Finally, we find that impaired clearance of these inclusions negatively impacts retrograde trafficking of endosomes, a conventional dynein cargo, indicating that microtubule-based transport can be overwhelmed by chronic cellular stress.

Mining the Human Complexome Database Identifies RBM14 as an XPO1-Associated Protein Involved in HIV-1 Rev Function

OpenAIRE

Budhiraja, Sona; Liu, Hongbing; Couturier, Jacob; Malovannaya, Anna; Qin, Jun; Lewis, Dorothy E.; Rice, Andrew P.

2015-01-01

By recruiting the host protein XPO1 (CRM1), the HIV-1 Rev protein mediates the nuclear export of incompletely spliced viral transcripts. We mined data from the recently described human nuclear complexome to identify a host protein, RBM14, which associates with XPO1 and Rev and is involved in Rev function. Using a Rev-dependent p24 reporter plasmid, we found that RBM14 depletion decreased Rev activity and Rev-mediated enhancement of the cytoplasmic levels of unspliced viral transcripts. RBM14 ...

The intermediate filament protein vimentin binds specifically to a recombinant integrin α2/β1 cytoplasmic tail complex and co-localizes with native α2/β1 in endothelial cell focal adhesions

International Nuclear Information System (INIS)

Kreis, Stephanie; Schoenfeld, Hans-Joachim; Melchior, Chantal; Steiner, Beat; Kieffer, Nelly

2005-01-01

Integrin receptors are crucial players in cell adhesion and migration. Identification and characterization of cellular proteins that interact with their short α and β cytoplasmic tails will help to elucidate the molecular mechanisms by which integrins mediate bi-directional signaling across the plasma membrane. Integrin α2β1 is a major collagen receptor but to date, only few proteins have been shown to interact with the α2 cytoplasmic tail or with the α2β1 complex. In order to identify novel binding partners of a α2β1cytoplasmic domain complex, we have generated recombinant GST-fusion proteins, incorporating the leucine zipper heterodimerization cassettes of Jun and Fos. To ascertain proper functionality of the recombinant proteins, interaction with natural binding partners was tested. GST-α2 and GST-Jun α2 bound His-tagged calreticulin while GST-β1 and GST-Fos β1 proteins bound talin. In screening assays for novel binding partners, the immobilized GST-Jun α2/GST-Fos β1 heterodimeric complex, but not the single subunits, interacted specifically with endothelial cell-derived vimentin. Vimentin, an abundant intermediate filament protein, has previously been shown to co-localize with αvβ3-positive focal contacts. Here, we provide evidence that this interaction also occurs with α2β1-enriched focal adhesions and we further show that this association is lost after prolonged adhesion of endothelial cells to collagen

Late-assembly of human ribosomal protein S20 in the cytoplasm is essential for the functioning of the small subunit ribosome

International Nuclear Information System (INIS)

Tai, Lin-Ru; Chou, Chang-Wei; Wu, Jing-Ying; Kirby, Ralph; Lin, Alan

2013-01-01

Using immuno-fluorescent probing and Western blotting analysis, we reveal the exclusive cytoplasm nature of the small subunit ribosomal protein S20. To illustrate the importance of the cellular compartmentation of S20 to the function of small subunit 40S, we created a nuclear resident S20 NLS mutant gene and examined polysome profile of cells that had been transfected with the S20 NLS gene. As a result, we observed the formation of recombinant 40S carried S20 NLS but this recombinant 40S was never found in the polysome, suggesting such a recombinant 40S was translation incompetent. Moreover, by the tactic of the energy depletion and restoration, we were able to restrain the nuclear-resided S20 NLS in the cytoplasm. Yet, along a progressive energy restoration, we observed the presence of recombinant 40S subunits carrying the S20 NLS in the polysome. This proves that S20 needs to be cytoplasmic in order to make a functional 40S subunit. Furthermore, it also implies that the assembly order of ribosomal protein in eukaryote is orderly regulated. - Highlights: • The step of S20 assembled on 40S is happened in the cytoplasm. • A small subunit assembled with a nuclear S20 NLS is translational incompetence. • Using energy depletion and recovery to manipulate the cellular compartment of S20 NLS . • Cytoplasm-retained S20 NLS is crucial for creating a functional small subunit

The Nonreceptor Protein Tyrosine Phosphatase PTP1B Binds to the Cytoplasmic Domain of N-Cadherin and Regulates the Cadherin–Actin Linkage

Science.gov (United States)

Balsamo, Janne; Arregui, Carlos; Leung, TinChung; Lilien, Jack

1998-01-01

Cadherin-mediated adhesion depends on the association of its cytoplasmic domain with the actin-containing cytoskeleton. This interaction is mediated by a group of cytoplasmic proteins: α-and β- or γ- catenin. Phosphorylation of β-catenin on tyrosine residues plays a role in controlling this association and, therefore, cadherin function. Previous work from our laboratory suggested that a nonreceptor protein tyrosine phosphatase, bound to the cytoplasmic domain of N-cadherin, is responsible for removing tyrosine-bound phosphate residues from β-catenin, thus maintaining the cadherin–actin connection (Balsamo et al., 1996). Here we report the molecular cloning of the cadherin-associated tyrosine phosphatase and identify it as PTP1B. To definitively establish a causal relationship between the function of cadherin-bound PTP1B and cadherin-mediated adhesion, we tested the effect of expressing a catalytically inactive form of PTP1B in L cells constitutively expressing N-cadherin. We find that expression of the catalytically inactive PTP1B results in reduced cadherin-mediated adhesion. Furthermore, cadherin is uncoupled from its association with actin, and β-catenin shows increased phosphorylation on tyrosine residues when compared with parental cells or cells transfected with the wild-type PTP1B. Both the transfected wild-type and the mutant PTP1B are found associated with N-cadherin, and recombinant mutant PTP1B binds to N-cadherin in vitro, indicating that the catalytically inactive form acts as a dominant negative, displacing endogenous PTP1B, and rendering cadherin nonfunctional. Our results demonstrate a role for PTP1B in regulating cadherin-mediated cell adhesion. PMID:9786960

Late-assembly of human ribosomal protein S20 in the cytoplasm is essential for the functioning of the small subunit ribosome

Energy Technology Data Exchange (ETDEWEB)

Tai, Lin-Ru [Institute of Genome Sciences, School of Life Sciences, National Yang-Ming University, Taipei, Taiwan, ROC (China); Chou, Chang-Wei [Department of Dentistry, School of Dentistry, National Yang-Ming University, Taipei, Taiwan, ROC (China); Wu, Jing-Ying; Kirby, Ralph [Institute of Genome Sciences, School of Life Sciences, National Yang-Ming University, Taipei, Taiwan, ROC (China); Lin, Alan, E-mail: alin@ym.edu.tw [Institute of Genome Sciences, School of Life Sciences, National Yang-Ming University, Taipei, Taiwan, ROC (China); Department of Dentistry, School of Dentistry, National Yang-Ming University, Taipei, Taiwan, ROC (China)

2013-11-15

Using immuno-fluorescent probing and Western blotting analysis, we reveal the exclusive cytoplasm nature of the small subunit ribosomal protein S20. To illustrate the importance of the cellular compartmentation of S20 to the function of small subunit 40S, we created a nuclear resident S20{sub NLS} mutant gene and examined polysome profile of cells that had been transfected with the S20{sub NLS} gene. As a result, we observed the formation of recombinant 40S carried S20{sub NLS} but this recombinant 40S was never found in the polysome, suggesting such a recombinant 40S was translation incompetent. Moreover, by the tactic of the energy depletion and restoration, we were able to restrain the nuclear-resided S20{sub NLS} in the cytoplasm. Yet, along a progressive energy restoration, we observed the presence of recombinant 40S subunits carrying the S20{sub NLS} in the polysome. This proves that S20 needs to be cytoplasmic in order to make a functional 40S subunit. Furthermore, it also implies that the assembly order of ribosomal protein in eukaryote is orderly regulated. - Highlights: • The step of S20 assembled on 40S is happened in the cytoplasm. • A small subunit assembled with a nuclear S20{sub NLS} is translational incompetence. • Using energy depletion and recovery to manipulate the cellular compartment of S20{sub NLS}. • Cytoplasm-retained S20{sub NLS} is crucial for creating a functional small subunit.

Pyridoxal phosphate as a probe of the cytoplasmic domains of transmembrane proteins: Application to the nicotinic acetylcholine receptor

International Nuclear Information System (INIS)

Perez-Ramirez, B.; Martinez-Carrion, M.

1989-01-01

A novel procedure has been developed to specifically label the cytoplasmic domains of transmembrane proteins with the aldehyde pyridoxal 5-phosphate (PLP). Torpedo californica acetylcholine receptor (AcChR) vesicles were loaded with [ 3 H]pyridoxine 5-phosphate ([ 3 H]PNP) and pyridoxine-5-phosphate oxidase, followed by intravesicular enzymatic oxidation of [ 3 H]PNP at 37 degree C in the presence of externally added cytochrome c as a scavenger of possible leaking PLP product. The four receptor subunits were labeled whether the reaction was carried out on the internal surface or separately designed to mark the external one. On the other hand, the relative pyridoxylation of the subunits differed in both cases, reflecting differences in accessible lysyl residues in each side of the membrane. Even though there are no large differences in the total lysine content among the subunits and there are two copies of the α-subunit, internal surface labeling by PLP was greatest for the highest molecular weight (δ) subunit, reinforcing the concept that the four receptor subunits are transmembranous and may protrude into the cytoplasmic face in a fashion that is proportional to their subunit molecular weight. Yet, the labeling data do not fit well to any of the models proposed for AcChR subunit folding. The method described can be used for selective labeling of the cytoplasmic domains of transmembrane proteins in sealed membrane vesicles

Activation gating kinetics of GIRK channels are mediated by cytoplasmic residues adjacent to transmembrane domains.

Science.gov (United States)

Sadja, Rona; Reuveny, Eitan

2009-01-01

G-protein-coupled inwardly rectifying potassium channels (GIRK/Kir3.x) are involved in neurotransmission-mediated reduction of excitability. The gating mechanism following G protein activation of these channels likely proceeds from movement of inner transmembrane helices to allow K(+) ions movement through the pore of the channel. There is limited understanding of how the binding of G-protein betagamma subunits to cytoplasmic regions of the channel transduces the signal to the transmembrane regions. In this study, we examined the molecular basis that governs the activation kinetics of these channels, using a chimeric approach. We identified two regions as being important in determining the kinetics of activation. One region is the bottom of the outer transmembrane helix (TM1) and the cytoplasmic domain immediately adjacent (the slide helix); and the second region is the bottom of the inner transmembrane helix (TM2) and the cytoplasmic domain immediately adjacent. Interestingly, both of these regions are sufficient in mediating the kinetics of fast activation gating. This result suggests that there is a cooperative movement of either one of these domains to allow fast and efficient activation gating of GIRK channels.

The SPF27 homologue Num1 connects splicing and kinesin 1-dependent cytoplasmic trafficking in Ustilago maydis.

Directory of Open Access Journals (Sweden)

Nikola Kellner

2014-01-01

Full Text Available The conserved NineTeen protein complex (NTC is an integral subunit of the spliceosome and required for intron removal during pre-mRNA splicing. The complex associates with the spliceosome and participates in the regulation of conformational changes of core spliceosomal components, stabilizing RNA-RNA- as well as RNA-protein interactions. In addition, the NTC is involved in cell cycle checkpoint control, response to DNA damage, as well as formation and export of mRNP-particles. We have identified the Num1 protein as the homologue of SPF27, one of NTC core components, in the basidiomycetous fungus Ustilago maydis. Num1 is required for polarized growth of the fungal hyphae, and, in line with the described NTC functions, the num1 mutation affects the cell cycle and cell division. The num1 deletion influences splicing in U. maydis on a global scale, as RNA-Seq analysis revealed increased intron retention rates. Surprisingly, we identified in a screen for Num1 interacting proteins not only NTC core components as Prp19 and Cef1, but several proteins with putative functions during vesicle-mediated transport processes. Among others, Num1 interacts with the motor protein Kin1 in the cytoplasm. Similar phenotypes with respect to filamentous and polar growth, vacuolar morphology, as well as the motility of early endosomes corroborate the genetic interaction between Num1 and Kin1. Our data implicate a previously unidentified connection between a component of the splicing machinery and cytoplasmic transport processes. As the num1 deletion also affects cytoplasmic mRNA transport, the protein may constitute a novel functional interconnection between the two disparate processes of splicing and trafficking.

Phosphorylation of the cytoplasmic tail of the 300-kDa mannose 6-phosphate receptor is required for the interaction with a cytosolic protein

DEFF Research Database (Denmark)

Rosorius, O; Issinger, O G; Braulke, T

1993-01-01

The cytoplasmic tail of the human 300-kDa mannose 6-phosphate receptor (MPR 300-CT) is an excellent substrate for casein kinase II in vitro. The phosphorylated MPR 300-CT was cross-linked by means of bis(sulfosuccinimidyl)suberate mainly to a cytosolic protein of 35 kDa (referred to as TIP 35...... with TIP 35 is phosphorylation-specific. Furthermore, TIP 35 was only cross-linked to the MPR 300-CT phosphorylated by casein kinase II whereas the MPR 300-CT phosphorylated by protein kinase A failed to cross-link to TIP 35. These results indicate that the cytoplasmic tail of the MPR 300 interacts...

Mitochondrial protein acetylation mediates nutrient sensing of mitochondrial protein synthesis and mitonuclear protein balance.

Science.gov (United States)

Di Domenico, Antonella; Hofer, Annette; Tundo, Federica; Wenz, Tina

2014-11-01

Changes in nutrient supply require global metabolic reprogramming to optimize the utilization of the nutrients. Mitochondria as a central component of the cellular metabolism play a key role in this adaptive process. Since mitochondria harbor their own genome, which encodes essential enzymes, mitochondrial protein synthesis is a determinant of metabolic adaptation. While regulation of cytoplasmic protein synthesis in response to metabolic challenges has been studied in great detail, mechanisms which adapt mitochondrial translation in response to metabolic challenges remain elusive. Our results suggest that the mitochondrial acetylation status controlled by Sirt3 and its proposed opponent GCN5L1 is an important regulator of the metabolic adaptation of mitochondrial translation. Moreover, both proteins modulate regulators of cytoplasmic protein synthesis as well as the mitonuclear protein balance making Sirt3 and GCN5L1 key players in synchronizing mitochondrial and cytoplasmic translation. Our results thereby highlight regulation of mitochondrial translation as a novel component in the cellular nutrient sensing scheme and identify mitochondrial acetylation as a new regulatory principle for the metabolic competence of mitochondrial protein synthesis. © 2014 International Union of Biochemistry and Molecular Biology.

Identification and characterization of vp7 gene in Bombyx mori cytoplasmic polyhedrosis virus.

Science.gov (United States)

He, Lei; Hu, Xiaolong; Zhu, Min; Liang, Zi; Chen, Fei; Zhu, Liyuan; Kuang, Sulan; Cao, Guangli; Xue, Renyu; Gong, Chengliang

2017-09-05

The genome of Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) contains 10 double stranded RNA segments (S1-S10). The segment 7 (S7) encodes 50kDa protein which is considered as a structural protein. The expression pattern and function of p50 in the virus life cycle are still unclear. In this study, the viral structural protein 7 (VP7) polyclonal antibody was prepared with immunized mouse to explore the presence of small VP7 gene-encoded proteins in Bombyx mori cytoplasmic polyhedrosis virus. The expression pattern of vp7 gene was investigated by its overexpression in BmN cells. In addition to VP7, supplementary band was identified with western blotting technique. The virion, BmCPV infected cells and midguts were also examined using western blotting technique. 4, 2 and 5 bands were detected in the corresponding samples, respectively. The replication of BmCPV genome in the cultured cells and midgut of silkworm was decreased by reducing the expression level of vp7 gene using RNA interference. In immunoprecipitation experiments, using a polyclonal antiserum directed against the VP7, one additional shorter band in BmCPV infected midguts was detected, and then the band was analyzed with mass spectrum (MS), the MS results showed thatone candidate interacted protein (VP7 voltage-dependent anion-selective channel-like isoform, VDAC) was identified from silkworm. We concluded that the novel viral product was generated with a leaky scanning mechanism and the VDAC may be an interacted protein with VP7. Copyright © 2017 Elsevier B.V. All rights reserved.

Members of the HCMV US12 family of predicted heptaspanning membrane proteins have unique intracellular distributions, including association with the cytoplasmic virion assembly complex

International Nuclear Information System (INIS)

Das, Subhendu; Pellett, Philip E.

2007-01-01

The human cytomegalovirus (HCMV) US12 gene family is a group of 10 predicted seven-transmembrane domain proteins that have some features in common with G-protein-coupled receptors. Little is known of their patterns of expression, localization, or functional interactions. Here, we studied the intracellular localization of three US12 family members, US14, US17, and US18, with respect to various intracellular markers and the cytoplasmic virion assembly compartment (AC). The three proteins have distinct patterns of expression, which include associations with the AC. US14 is often distributed in a uniform granular manner throughout the cytoplasm, concentrating in the AC in some cells. US17 is expressed in a segmented manner, with its N-terminal domain localizing to the periphery of what we show here to be the AC and the C-terminal domain localizing to nuclei and the cytoplasm [Das, S., Skomorovska-Prokvolit, Y., Wang, F. Z., Pellett, P.E., 2006. Infection-dependent nuclear localization of US17, a member of the US12 family of human cytomegalovirus-encoded seven-transmembrane proteins. J. Virol. 80, 1191-1203]. Here, we show that the C-terminal domain is present at the center of the AC, in close association with markers of early endosomes; the N-terminal staining corresponds to an area stained by markers for the Golgi and trans-Golgi. US18 is distributed throughout the cytoplasm, concentrating in the AC at later stages of infection; it is localized more to the periphery of the AC than are US14 and US17C, in association with markers of the trans-Golgi. Although not detected in virions, their structures and localization in various zones within the AC suggest possible roles for these proteins in the process of virion maturation and egress

The Protein Identifier Cross-Referencing (PICR service: reconciling protein identifiers across multiple source databases

Directory of Open Access Journals (Sweden)

Leinonen Rasko

2007-10-01

Full Text Available Abstract Background Each major protein database uses its own conventions when assigning protein identifiers. Resolving the various, potentially unstable, identifiers that refer to identical proteins is a major challenge. This is a common problem when attempting to unify datasets that have been annotated with proteins from multiple data sources or querying data providers with one flavour of protein identifiers when the source database uses another. Partial solutions for protein identifier mapping exist but they are limited to specific species or techniques and to a very small number of databases. As a result, we have not found a solution that is generic enough and broad enough in mapping scope to suit our needs. Results We have created the Protein Identifier Cross-Reference (PICR service, a web application that provides interactive and programmatic (SOAP and REST access to a mapping algorithm that uses the UniProt Archive (UniParc as a data warehouse to offer protein cross-references based on 100% sequence identity to proteins from over 70 distinct source databases loaded into UniParc. Mappings can be limited by source database, taxonomic ID and activity status in the source database. Users can copy/paste or upload files containing protein identifiers or sequences in FASTA format to obtain mappings using the interactive interface. Search results can be viewed in simple or detailed HTML tables or downloaded as comma-separated values (CSV or Microsoft Excel (XLS files suitable for use in a local database or a spreadsheet. Alternatively, a SOAP interface is available to integrate PICR functionality in other applications, as is a lightweight REST interface. Conclusion We offer a publicly available service that can interactively map protein identifiers and protein sequences to the majority of commonly used protein databases. Programmatic access is available through a standards-compliant SOAP interface or a lightweight REST interface. The PICR

Genetic analysis of the cytoplasmic dynein subunit families.

Science.gov (United States)

Pfister, K Kevin; Shah, Paresh R; Hummerich, Holger; Russ, Andreas; Cotton, James; Annuar, Azlina Ahmad; King, Stephen M; Fisher, Elizabeth M C

2006-01-01

Cytoplasmic dyneins, the principal microtubule minus-end-directed motor proteins of the cell, are involved in many essential cellular processes. The major form of this enzyme is a complex of at least six protein subunits, and in mammals all but one of the subunits are encoded by at least two genes. Here we review current knowledge concerning the subunits, their interactions, and their functional roles as derived from biochemical and genetic analyses. We also carried out extensive database searches to look for new genes and to clarify anomalies in the databases. Our analysis documents evolutionary relationships among the dynein subunits of mammals and other model organisms, and sheds new light on the role of this diverse group of proteins, highlighting the existence of two cytoplasmic dynein complexes with distinct cellular roles.

Genetic analysis of the cytoplasmic dynein subunit families.

Directory of Open Access Journals (Sweden)

K Kevin Pfister

2006-01-01

Full Text Available Cytoplasmic dyneins, the principal microtubule minus-end-directed motor proteins of the cell, are involved in many essential cellular processes. The major form of this enzyme is a complex of at least six protein subunits, and in mammals all but one of the subunits are encoded by at least two genes. Here we review current knowledge concerning the subunits, their interactions, and their functional roles as derived from biochemical and genetic analyses. We also carried out extensive database searches to look for new genes and to clarify anomalies in the databases. Our analysis documents evolutionary relationships among the dynein subunits of mammals and other model organisms, and sheds new light on the role of this diverse group of proteins, highlighting the existence of two cytoplasmic dynein complexes with distinct cellular roles.

Functional Architecture of the Cytoplasmic Entrance to the Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel Pore.

Science.gov (United States)

El Hiani, Yassine; Linsdell, Paul

2015-06-19

As an ion channel, the cystic fibrosis transmembrane conductance regulator must form a continuous pathway for the movement of Cl(-) and other anions between the cytoplasm and the extracellular solution. Both the structure and the function of the membrane-spanning part of this pathway are well defined. In contrast, the structure of the pathway that connects the cytoplasm to the membrane-spanning regions is unknown, and functional roles for different parts of the protein forming this pathway have not been described. We used patch clamp recording and substituted cysteine accessibility mutagenesis to identify positively charged amino acid side chains that attract cytoplasmic Cl(-) ions to the inner mouth of the pore. Our results indicate that the side chains of Lys-190, Arg-248, Arg-303, Lys-370, Lys-1041, and Arg-1048, located in different intracellular loops of the protein, play important roles in the electrostatic attraction of Cl(-) ions. Mutation and covalent modification of these residues have charge-dependent effects on the rate of Cl(-) permeation, demonstrating their functional role in maximization of Cl(-) flux. Other nearby positively charged side chains were not involved in electrostatic interactions with Cl(-). The location of these Cl(-)-attractive residues suggests that cytoplasmic Cl(-) ions enter the pore via a lateral portal located between the cytoplasmic extensions to the fourth and sixth transmembrane helices; a secondary, functionally less relevant portal might exist between the extensions to the 10th and 12th transmembrane helices. These results define the cytoplasmic mouth of the pore and show how it attracts Cl(-) ions from the cytoplasm. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Phase separation between nucleoid and cytoplasm in Escherichia coli as defined by immersive refractometry.

Science.gov (United States)

Valkenburg, J A; Woldringh, C L

1984-01-01

The refractive indices of nucleoid and cytoplasm in Escherichia coli were derived theoretically and experimentally. For the theoretical estimates, we made use of the known macromolecular composition of E. coli B/r (G. Churchward and H. Bremer, J. Theor. Biol. 94:651-670, 1982) and of estimates of cell and nucleoid volumes. These were obtained from micrographs of living bacteria made with a confocal scanning light microscope. The theoretical values were calculated, assuming that all DNA occurred in the nucleoid and that all protein and RNA occurred in the cytoplasm. Comparison with experimental refractive index values directly obtained by immersive refractometry showed that, besides its DNA, the nucleoid must contain an additional amount of solids equivalent to 8.6% (wt/vol) protein. With the nucleoid containing 6.8% (wt/vol) DNA and 8.6% (wt/vol) protein and the cytoplasm containing 21% (wt/vol) protein and 4% (wt/vol) RNA, a mass difference is obtained, which accounts for the phase separation observed between the nucleoid and cytoplasm in living cells by phase-contrast microscopy. The decrease in the refractive index of the nucleoid relative to that of the cytoplasm observed upon, for instance, OsO4 fixation was interpreted as being indicative of the loss of protein content in the nucleoid. Images PMID:6389508

Characterization of Elements Regulating the Nuclear-to-Cytoplasmic Translocation of ICP0 in Late Herpes Simplex Virus 1 Infection.

Science.gov (United States)

Samrat, Subodh Kumar; Ha, Binh L; Zheng, Yi; Gu, Haidong

2018-01-15

HSV-1 infection, we investigated the potential players involved in this nuclear-to-cytoplasmic translocation. We found that there is a nuclear retention force in an ICP0 E3 ubiquitin ligase-dependent manner. In addition, we identified the C terminus of ICP0 as a cis element cooperating with late viral proteins to overcome the nuclear retention and stimulate the nuclear-to-cytoplasmic translocation of ICP0. Copyright © 2018 American Society for Microbiology.

Nuclear translocation and regulation of intranuclear distribution of cytoplasmic poly(A-binding protein are distinct processes mediated by two Epstein Barr virus proteins.

Directory of Open Access Journals (Sweden)

Richard Park

Full Text Available Many viruses target cytoplasmic polyA binding protein (PABPC to effect widespread inhibition of host gene expression, a process termed viral host-shutoff (vhs. During lytic replication of Epstein Barr Virus (EBV we observed that PABPC was efficiently translocated from the cytoplasm to the nucleus. Translocated PABPC was diffusely distributed but was excluded from viral replication compartments. Vhs during EBV infection is regulated by the viral alkaline nuclease, BGLF5. Transfection of BGLF5 alone into BGLF5-KO cells or uninfected 293 cells promoted translocation of PAPBC that was distributed in clumps in the nucleus. ZEBRA, a viral bZIP protein, performs essential functions in the lytic program of EBV, including activation or repression of downstream viral genes. ZEBRA is also an essential replication protein that binds to viral oriLyt and interacts with other viral replication proteins. We report that ZEBRA also functions as a regulator of vhs. ZEBRA translocated PABPC to the nucleus, controlled the intranuclear distribution of PABPC, and caused global shutoff of host gene expression. Transfection of ZEBRA alone into 293 cells caused nuclear translocation of PABPC in the majority of cells in which ZEBRA was expressed. Co-transfection of ZEBRA with BGLF5 into BGLF5-KO cells or uninfected 293 cells rescued the diffuse intranuclear pattern of PABPC seen during lytic replication. ZEBRA mutants defective for DNA-binding were capable of regulating the intranuclear distribution of PABPC, and caused PABPC to co-localize with ZEBRA. One ZEBRA mutant, Z(S186E, was deficient in translocation yet was capable of altering the intranuclear distribution of PABPC. Therefore ZEBRA-mediated nuclear translocation of PABPC and regulation of intranuclear PABPC distribution are distinct events. Using a click chemistry-based assay for new protein synthesis, we show that ZEBRA and BGLF5 each function as viral host shutoff factors.

MapA, an iron-regulated, cytoplasmic membrane protein in the cyanobacterium Synechococcus sp. strain PCC7942.

Science.gov (United States)

Webb, R; Troyan, T; Sherman, D; Sherman, L A

1994-08-01

Growth of Synechococcus sp. strain PCC 7942 in iron-deficient media leads to the accumulation of an approximately 34-kDa protein. The gene encoding this protein, mapA (membrane-associated protein A), has been cloned and sequenced (GenBank accession number, L01621). The mapA transcript is not detectable in normally grown cultures but is stably accumulated by cells grown in iron-deficient media. However, the promoter sequence for this gene does not resemble other bacterial iron-regulated promoters described to date. The carboxyl-terminal region of the derived amino acid sequence of MapA resembles bacterial proteins involved in iron acquisition, whereas the amino-terminal end of MapA has a high degree of amino acid identity with the abundant, chloroplast envelope protein E37. An approach employing improved cellular fractionation techniques as well as electron microscopy and immunocytochemistry was essential in localizing MapA protein to the cytoplasmic membrane of Synechococcus sp. strain PCC 7942. When these cells were grown under iron-deficient conditions, a significant fraction of MapA could also be localized to the thylakoid membranes.

Genome-wide identification, sequence characterization, and protein-protein interaction properties of DDB1 (damaged DNA binding protein-1)-binding WD40-repeat family members in Solanum lycopersicum.

Science.gov (United States)

Zhu, Yunye; Huang, Shengxiong; Miao, Min; Tang, Xiaofeng; Yue, Junyang; Wang, Wenjie; Liu, Yongsheng

2015-06-01

One hundred DDB1 (damaged DNA binding protein-1)-binding WD40-repeat domain (DWD) family genes were identified in the S. lycopersicum genome. The DWD genes encode proteins presumably functioning as the substrate recognition subunits of the cullin4-ring ubiquitin E3 ligase complex. These findings provide candidate genes and a research platform for further gene functionality and molecular breeding study. A subclass of DDB1 (damaged DNA binding protein-1)-binding WD40-repeat domain (DWD) family proteins has been demonstrated to function as the substrate recognition subunits of the cullin4-ring ubiquitin E3 ligase complex. However, little information is available about the cognate subfamily genes in tomato (S. lycopersicum). In this study, based on the recently released tomato genome sequences, 100 tomato genes encoding DWD proteins that potentially interact with DDB1 were identified and characterized, including analyses of the detailed annotations, chromosome locations and compositions of conserved amino acid domains. In addition, a phylogenetic tree, which comprises of three main groups, of the subfamily genes was constructed. The physical interaction between tomato DDB1 and 14 representative DWD proteins was determined by yeast two-hybrid and co-immunoprecipitation assays. The subcellular localization of these 14 representative DWD proteins was determined. Six of them were localized in both nucleus and cytoplasm, seven proteins exclusively in cytoplasm, and one protein either in nucleus and cytoplasm, or exclusively in cytoplasm. Comparative genomic analysis demonstrated that the expansion of these subfamily members in tomato predominantly resulted from two whole-genome triplication events in the evolution history.

Glycoprotein cytoplasmic domain sequences required for rescue of a vesicular stomatitis virus glycoprotein mutant

International Nuclear Information System (INIS)

Whitt, M.A.; Chong, L.; Rose, J.K.

1989-01-01

The authors have used transient expression of the wild-type vesicular stomatitis virus (VSV) glycoprotein (G protein) from cloned cDNA to rescue a temperature-sensitive G protein mutant of VSV in cells at the nonpermissive temperature. Using cDNAs encoding G proteins with deletions in the normal 29-amino-acid cytoplasmic domain, they determined that the presence of either the membrane-proximal 9 amino acids or the membrane-distal 12 amino acids was sufficient for rescue of the temperature-sensitive mutant. G proteins with cytoplasmic domains derived from other cellular or viral G proteins did not rescue the mutant, nor did G proteins with one or three amino acids of the normal cytoplasmic domain. Rescue correlated directly with the ability of the G proteins to be incorporated into virus particles. This was shown by analysis of radiolabeled particles separated on sucrose gradients as well as by electron microscopy of rescued virus after immunogold labeling. Quantitation of surface expression showed that all of the mutated G proteins were expressed less efficiently on the cell surface than was wild-type G protein. However, they were able to correct for differences in rescue efficiency resulting from differences in the level of surface expression by reducing wild-type G protein expression to levels equivalent to those observed for the mutated G proteins. The results provide evidence that at least a portion of the cytoplasmic domain is required for efficient assembly of the VSV G protein into virions during virus budding

Identification of Proteins Involved in Carbohydrate Metabolism and Energy Metabolism Pathways and Their Regulation of Cytoplasmic Male Sterility in Wheat

Directory of Open Access Journals (Sweden)

Xingxia Geng

2018-01-01

Full Text Available Cytoplasmic male sterility (CMS where no functional pollen is produced has important roles in wheat breeding. The anther is a unique organ for male gametogenesis and its abnormal development can cause male sterility. However, the mechanisms and regulatory networks related to plant male sterility are poorly understood. In this study, we conducted comparative analyses using isobaric tags for relative and absolute quantification (iTRAQ of the pollen proteins in a CMS line and its wheat maintainer. Differentially abundant proteins (DAPs were analyzed based on Gene Ontology classifications, metabolic pathways and transcriptional regulation networks using Blast2GO. We identified 5570 proteins based on 23,277 peptides, which matched with 73,688 spectra, including proteins in key pathways such as glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase and 6-phosphofructokinase 1 in the glycolysis pathway, isocitrate dehydrogenase and citrate synthase in the tricarboxylic acid cycle and nicotinamide adenine dinucleotide (NADH-dehydrogenase and adenosine-triphosphate (ATP synthases in the oxidative phosphorylation pathway. These proteins may comprise a network that regulates male sterility in wheat. Quantitative real time polymerase chain reaction (qRT-PCR analysis, ATP assays and total sugar assays validated the iTRAQ results. These DAPs could be associated with abnormal pollen grain formation and male sterility. Our findings provide insights into the molecular mechanism related to male sterility in wheat.

Identification of Proteins Involved in Carbohydrate Metabolism and Energy Metabolism Pathways and Their Regulation of Cytoplasmic Male Sterility in Wheat.

Science.gov (United States)

Geng, Xingxia; Ye, Jiali; Yang, Xuetong; Li, Sha; Zhang, Lingli; Song, Xiyue

2018-01-23

Cytoplasmic male sterility (CMS) where no functional pollen is produced has important roles in wheat breeding. The anther is a unique organ for male gametogenesis and its abnormal development can cause male sterility. However, the mechanisms and regulatory networks related to plant male sterility are poorly understood. In this study, we conducted comparative analyses using isobaric tags for relative and absolute quantification (iTRAQ) of the pollen proteins in a CMS line and its wheat maintainer. Differentially abundant proteins (DAPs) were analyzed based on Gene Ontology classifications, metabolic pathways and transcriptional regulation networks using Blast2GO. We identified 5570 proteins based on 23,277 peptides, which matched with 73,688 spectra, including proteins in key pathways such as glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase and 6-phosphofructokinase 1 in the glycolysis pathway, isocitrate dehydrogenase and citrate synthase in the tricarboxylic acid cycle and nicotinamide adenine dinucleotide (NADH)-dehydrogenase and adenosine-triphosphate (ATP) synthases in the oxidative phosphorylation pathway. These proteins may comprise a network that regulates male sterility in wheat. Quantitative real time polymerase chain reaction (qRT-PCR) analysis, ATP assays and total sugar assays validated the iTRAQ results. These DAPs could be associated with abnormal pollen grain formation and male sterility. Our findings provide insights into the molecular mechanism related to male sterility in wheat.

Cytoplasmic transduction peptide (CTP): New approach for the delivery of biomolecules into cytoplasm in vitro and in vivo

International Nuclear Information System (INIS)

Kim, Daeyou; Jeon, Choonju; Kim, Jeong-Hwan; Kim, Mi-Seon; Yoon, Cheol-Hee; Choi, In-Soo; Kim, Sung-Hoon; Bae, Yong-Soo

2006-01-01

The protein transduction domain (PTD) of HIV-1 TAT has been extensively documented with regard to its membrane transduction potential, as well as its efficient delivery of biomolecules in vivo. However, the majority of PTD and PTD-conjugated molecules translocate to the nucleus rather than to the cytoplasm after transduction, due to the functional nuclear localization sequence (NLS). Here, we report a cytoplasmic transduction peptide (CTP), which was deliberately designed to ensure the efficient cytoplasmic delivery of the CTP-fused biomolecules. In comparison with PTD, CTP and its fusion partners exhibited a clear preference for cytoplasmic localization, and also markedly enhanced membrane transduction potential. Unlike the mechanism underlying PTD-mediated transduction, CTP-mediated transduction occurs independently of the lipid raft-dependent macropinocytosis pathway. The CTP-conjugated Smac/DIABLO peptide (Smac-CTP) was also shown to be much more efficient than Smac-PTD in the blockage of the antiapoptotic properties of XIAP, suggesting that cytoplasmic functional molecules can be more efficiently targeted by CTP-mediated delivery. In in vivo trafficking studies, CTP-fused β-gal exhibited unique organ tropisms to the liver and lymph nodes when systemically injected into mice, whereas PTD-β-gal exhibited no such tropisms. Taken together, our findings implicate CTP as a novel delivery peptide appropriate for (i) molecular targeting to cytoplasmic compartments in vitro, (ii) the development of class I-associated CTL vaccines, and (iii) special drug delivery in vivo, without causing any untoward effects on nuclear genetic material

Heterogeneous nuclear ribonuclear protein K interacts with Sindbis virus nonstructural proteins and viral subgenomic mRNA

International Nuclear Information System (INIS)

Burnham, Andrew J.; Gong, Lei; Hardy, Richard W.

2007-01-01

Alphaviruses are a group of arthropod-borne human and animal pathogens that can cause epidemics of significant public health and economic consequence. Alphavirus RNA synthesis requires four virally encoded nonstructural proteins and probably a number of cellular proteins. Using comparative two-dimensional electrophoresis we were able to identify proteins enriched in cytoplasmic membrane fractions containing viral RNA synthetic complexes following infection with Sindbis virus. Our studies demonstrated the following: (i) the host protein hnRNP K is enriched in cytoplasmic membrane fractions following Sindbis virus infection, (ii) viral nonstructural proteins co-immunoprecipitate with hnRNP K, (iii) nsP2 and hnRNP K co-localize in the cytoplasm of Sindbis virus infected cells, (iv) Sindbis virus subgenomic mRNA, but not genomic RNA co-immunoprecipitates with hnRNP K, (v) viral RNA does not appear to be required for the interaction of hnRNP K with the nonstructural proteins. Potential functions of hnRNP K during virus replication are discussed

Absence of hydrocortisone from cytoplasmic hormone-protein complexes formed in vivo after administration of biologically active doses of [3H] hydrocortisone

International Nuclear Information System (INIS)

Voigt, J.; Grote, H.; Sekeris, C.E.

1981-01-01

After administration of [ 3 H] hydrocortisone to adrenalectomized rats, hormone-protein complexes were isolated from liver cytosol by DEAE-cellulose chromatography. After application of biologically active and inactive doses of hydrocortisone five binding components were detected eluting at the same salt concentrations as the hormone-protein complexes observed after incubation of cytosol with [ 3 H] hydrocortisone in vitro. The isolated hormone-protein fractions were acidified and extracted with ethylacetate and the steroids were analyzed by thin-layer chromatography. No significant amount of hydrocortisone could be detected in any of the complexes formed in vivo 5-60 min after administration of biologically active doses of hydrocortisone. 3xi,11β,17α,20xi, 21-Pentahydroxypregnane, steroidal carboxy acids, glucuronides and a very polar conjugate of hydrocortisone were found in the different fractions. After an in vivo dose of hydrocortisone of about 1/5000th of the minimal dose required for enzyme induction, hydrocortisone could be found in all the cytoplasmic hormone-protein complexes formed. In contrast to the cytoplasmic hormone-protein complexes, hydrocortisone could be readily demonstrated in nuclei isolated after the administration of biologically active doses of hormone, although acid metabolites were found to represent the main part of the radioactive compounds present in the nuclei. These acid metabolites were located in the nuclear envelope. (orig.)

Marker-assisted identification of restorer gene(s) in iso-cytoplasmic restorer lines of WA cytoplasm in rice and assessment of their fertility restoration potential across environments.

Science.gov (United States)

Kumar, Amit; Bhowmick, Prolay Kumar; Singh, Vikram Jeet; Malik, Manoj; Gupta, Ashish Kumar; Seth, R; Nagarajan, M; Krishnan, S Gopala; Singh, Ashok Kumar

2017-10-01

Iso-cytoplasmic restorers possess the same male sterile cytoplasm as the cytoplasmic male sterile (CMS) lines, thereby minimizing the potential cyto-nuclear conflict in the hybrids. Restoration of fertility of the wild abortive CMS is governed by two major genes namely, Rf3 and Rf4 . Therefore, assessing the allelic status of these restorer genes in the iso-cytoplasmic restorers using molecular markers will not only help in estimating the efficiency of these genes either alone or in combination, in fertility restoration in the hybrids in different environments, but will also be useful in determining the efficacy of these markers. In the present study, the efficiency of molecular markers in identifying genotypes carrying restorer allele of the gene(s) Rf3 and Rf4, restoring male fertility of WA cytoplasm in rice was assessed in a set of 100 iso-cytoplasmic rice restorers using gene linked as well as candidate gene based markers. In order to validate the efficacy of markers in identifying the restorers, a sub-set of selected 25 iso-cytoplasmic rice restorers were crossed with four different cytoplasmic male sterile lines namely, IR 79156A, IR 58025A, Pusa 6A and RTN 12A, and the pollen and spikelet fertility of the F 1 s were evaluated at three different locations. Marker analysis showed that Rf4 was the predominant fertility restorer gene in the iso-cytoplasmic restorers and Rf3 had a synergistic effect on fertility restoration. The efficiency of gene based markers, DRCG-RF4-14 and DRRM-RF3-10 for Rf4 (87%) and Rf3 (84%) genes was higher than respective gene-linked SSR markers RM6100 (80%) and RM3873 (82%). It is concluded that the gene based markers can be effectively used in identifying fertility restorer lines obviating the need for making crosses and evaluating the F 1 s. Though gene based markers are more efficient, there is a need to identify functional polymorphisms which can provide 100% efficiency. Three iso-cytoplasmic restorers namely, PRR 300, PRR 363

Proteomic analysis reveals strong mitochondrial involvement in cytoplasmic male sterility of pepper (Capsicum annuum L.).

Science.gov (United States)

Guo, Jinju; Wang, Peng; Cheng, Qing; Sun, Limin; Wang, Hongyu; Wang, Yutong; Kao, Lina; Li, Yanan; Qiu, Tuoyu; Yang, Wencai; Shen, Huolin

2017-09-25

Although cytoplasmic male sterility (CMS) is widely used for developing pepper hybrids, its molecular mechanism remains unclear. In this study, we used a high-throughput proteomics method called label-free to compare protein abundance across a pepper CMS line (A-line) and its isogenic maintainer line (B-line). Data are available via ProteomeXchange with identifier PXD006104. Approximately 324 differentially abundant protein species were identified and quantified; among which, 47 were up-accumulated and 140 were down-accumulated in the A-line; additionally, 75 and 62 protein species were specifically accumulated in the A-line and B-line, respectively. Protein species involved in pollen exine formation, pyruvate metabolic processes, the tricarboxylic acid cycle, the mitochondrial electron transport chain, and oxidative stress response were observed to be differentially accumulated between A-line and B-line, suggesting their potential roles in the regulation of pepper pollen abortion. Based on our data, we proposed a potential regulatory network for pepper CMS that unifies these processes. Artificial emasculation is a major obstacle in pepper hybrid breeding for its high labor cost and poor seed purity. While the use of cytoplasmic male sterility (CMS) in hybrid system is seriously frustrated because a long time is needed to cultivate male sterility line and its isogenic restore line. Transgenic technology is an effective and rapid method to obtain male sterility lines and its widely application has very important significance in speeding up breeding process in pepper. Although numerous studies have been conducted to select the genes related to male sterility, the molecular mechanism of cytoplasmic male sterility in pepper remains unknown. In this study, we used the high-throughput proteomic method called "label-free", coupled with liquid chromatography-quadrupole mass spectrometry (LC-MS/MS), to perform a novel comparison of expression profiles in a CMS pepper line

Epidermal Growth Factor Cytoplasmic Domain Affects ErbB Protein Degradation by the Lysosomal and Ubiquitin-Proteasome Pathway in Human Cancer Cells

Directory of Open Access Journals (Sweden)

Aleksandra Glogowska

2012-05-01

Full Text Available The cytoplasmic domains of EGF-like ligands, including EGF cytoplasmic domain (EGFcyt, have important biological functions. Using specific constructs and peptides of human EGF cytoplasmic domain, we demonstrate that EGFcyt facilitates lysosomal and proteasomal protein degradation, and this coincided with growth inhibition of human thyroid and glioma carcinoma cells. EGFcyt and exon 22–23-encoded peptide (EGF22.23 enhanced procathepsin B (procathB expression and procathB-mediated lysosomal degradation of EGFR/ErbB1 as determined by inhibitors for procathB and the lysosomal ATPase inhibitor BafA1. Presence of mbEGFctF, EGFcyt, EGF22.23, and exon 23-encoded peptides suppressed the expression of the deubiqitinating enzyme ubiquitin C-terminal hydrolase-L1 (UCH-L1. This coincided with hyperubiquitination of total cellular proteins and ErbB1/2 and reduced proteasome activity. Upon small interfering RNA-mediated silencing of endogenously expressed UCH-L1, a similar hyperubiquitinylation phenotype, reduced ErbB1/2 content, and attenuated growth was observed. The exon 23-encoded peptide region of EGFcyt was important for these biologic actions. Structural homology modeling of human EGFcyt showed that this molecular region formed an exposed surface loop. Peptides derived from this EGFcyt loop structure may aid in the design of novel peptide therapeutics aimed at inhibiting growth of cancer cells.

Microtubule–microtubule sliding by kinesin-1 is essential for normal cytoplasmic streaming in Drosophila oocytes

Science.gov (United States)

Lu, Wen; Winding, Michael; Lakonishok, Margot; Wildonger, Jill

2016-01-01

Cytoplasmic streaming in Drosophila oocytes is a microtubule-based bulk cytoplasmic movement. Streaming efficiently circulates and localizes mRNAs and proteins deposited by the nurse cells across the oocyte. This movement is driven by kinesin-1, a major microtubule motor. Recently, we have shown that kinesin-1 heavy chain (KHC) can transport one microtubule on another microtubule, thus driving microtubule–microtubule sliding in multiple cell types. To study the role of microtubule sliding in oocyte cytoplasmic streaming, we used a Khc mutant that is deficient in microtubule sliding but able to transport a majority of cargoes. We demonstrated that streaming is reduced by genomic replacement of wild-type Khc with this sliding-deficient mutant. Streaming can be fully rescued by wild-type KHC and partially rescued by a chimeric motor that cannot move organelles but is active in microtubule sliding. Consistent with these data, we identified two populations of microtubules in fast-streaming oocytes: a network of stable microtubules anchored to the actin cortex and free cytoplasmic microtubules that moved in the ooplasm. We further demonstrated that the reduced streaming in sliding-deficient oocytes resulted in posterior determination defects. Together, we propose that kinesin-1 slides free cytoplasmic microtubules against cortically immobilized microtubules, generating forces that contribute to cytoplasmic streaming and are essential for the refinement of posterior determinants. PMID:27512034

G2E3 is a nucleo-cytoplasmic shuttling protein with DNA damage responsive localization

International Nuclear Information System (INIS)

Brooks, William S.; Banerjee, Sami; Crawford, David F.

2007-01-01

G2E3 was originally described as a G2/M-specific gene with DNA damage responsive expression. The presence of a conserved HECT domain within the carboxy-terminus of the protein indicated that it likely functions as a ubiquitin ligase or E3. Although HECT domains are known to function in this capacity for many proteins, we demonstrate that a portion of the HECT domain from G2E3 plays an important role in the dynamic subcellular localization of the protein. We have shown that G2E3 is a nucleo-cytoplasmic shuttling protein with nuclear export mediated by a novel nuclear export domain that functions independently of CRM1. In full-length G2E3, a separate region of the HECT domain suppresses the function of the NES. Additionally, G2E3 contains a nucleolar localization signal (NoLS) in its amino terminus. Localization of G2E3 to the nucleolus is a dynamic process, and the protein delocalizes from the nucleolus rapidly after DNA damage. Cell cycle phase-specific expression and highly regulated subcellular localization of G2E3 suggest a possible role in cell cycle regulation and the cellular response to DNA damage

Cytoplasmic male sterility of rice with boro II cytoplasm is caused by a cytotoxic peptide and is restored by two related PPR motif genes via distinct modes of mRNA silencing.

Science.gov (United States)

Wang, Zhonghua; Zou, Yanjiao; Li, Xiaoyu; Zhang, Qunyu; Chen, Letian; Wu, Hao; Su, Dihua; Chen, Yuanling; Guo, Jingxin; Luo, Da; Long, Yunming; Zhong, Yang; Liu, Yao-Guang

2006-03-01

Cytoplasmic male sterility (CMS) and nucleus-controlled fertility restoration are widespread plant reproductive features that provide useful tools to exploit heterosis in crops. However, the molecular mechanism underlying this kind of cytoplasmic-nuclear interaction remains unclear. Here, we show in rice (Oryza sativa) with Boro II cytoplasm that an abnormal mitochondrial open reading frame, orf79, is cotranscribed with a duplicated atp6 (B-atp6) gene and encodes a cytotoxic peptide. Expression of orf79 in CMS lines and transgenic rice plants caused gametophytic male sterility. Immunoblot analysis showed that the ORF79 protein accumulates specifically in microspores. Two fertility restorer genes, Rf1a and Rf1b, were identified at the classical locus Rf-1 as members of a multigene cluster that encode pentatricopeptide repeat proteins. RF1A and RF1B are both targeted to mitochondria and can restore male fertility by blocking ORF79 production via endonucleolytic cleavage (RF1A) or degradation (RF1B) of dicistronic B-atp6/orf79 mRNA. In the presence of both restorers, RF1A was epistatic over RF1B in the mRNA processing. We have also shown that RF1A plays an additional role in promoting the editing of atp6 mRNAs, independent of its cleavage function.

Proteomics - a novel approach to the identification and characterisation of plasmodesmatal proteins

International Nuclear Information System (INIS)

Faulkner, C.R.; Blackman, L.M.; Lyon, B.R.; Overall, R.L.

2001-01-01

The development of proteomic methods, such as 2-dimensional gel electrophoresis (2-DE), has established a high resolution means of identifying and characterising proteins from a given protein mixture. The biochemical composition of plasmodesmata, the intercellular channels between plant cells, is poorly described despite extensive attempts to identify protemaceous plasmodesmatal components. These attempts have been confounded by the large number of proteins in the cell wall. We have exploited the anatomy of the alga Chara corallina to separate tissues with (nodal cells) and tissues without (internodal cells) plasmodesmata. Proteins specific to the cytoplasmic and wall protein extracts of nodal and internodal tissue were identified by comparison of 2-DE gels of these extracts. In particular, a 95 kDa protein was identified as specific to the nodal cells in both 1-dimensional and 2-dimensional comparisons of cytoplasmic nodal and internodal protein extracts. This protein was analysed by electron spray ionization time of flight tandem mass spectroscopy (ESI-TOF MS/MS) and the sequence obtained showed similarity to plant lipoxygenases. Further proteins of interest were identified in 2-DE resolution of extracts from the nodal cytoplasm, including two 49 kDa proteins and two 46 kDa proteins, and from the nodal cell walls, including a cluster of proteins around 30 kDa. Thus, a proteomic strategy for the identification and characterisation of proteins specific to different cell types in Chara corallina has been developed, with potential application to the identification and characterisation of plasmodesmatal proteins

Activation of chromatin degradation by a protein factor of thymocyte cytoplasm of irradiated mice

International Nuclear Information System (INIS)

Soldatenkov, V.A.; Filippovich, I.V.

1986-01-01

A cytoplasmic thymocyte fraction isolated 1 h after irradiation of mice accelerates chromatin degradation in isolated nuclei. Treatment of the cytoplasmic fraction by heat and injection of cycloheximide to mice prevent the acceleration of DNA degradation. The analysis of the chromatin degradation products and the kinetics of this process at acid and alkaline pH shows that activation of DNA degradation in thymocytes by a factor obtained from the irradiated cell cytoplasm is specific for a Ca 2+ , Mg 2+ -dependent enzyme. The time- and dose-dependent parameters of the appearance in the thymocyte cytoplasm of the factor influencing degradation of chromatin are in a good agreement with both the time of the onset of its postirradiation degradation and the dose dependence of this process

Association of nad7a Gene with Cytoplasmic Male Sterility in Pigeonpea

Directory of Open Access Journals (Sweden)

Pallavi Sinha

2015-07-01

Full Text Available Cytoplasmic male sterility (CMS has been exploited in the commercial pigeonpea [ (L. Millsp.] hybrid breeding system; however, the molecular mechanism behind this system is unknown. To understand the underlying molecular mechanism involved in A CMS system derived from (Haines Maesen, 34 mitochondrial genes were analyzed for expression profiling and structural variation analysis between CMS line (ICRISAT Pigeonpea A line, ICPA 2039 and its cognate maintainer (ICPB 2039. Expression profiling of 34 mitochondrial genes revealed nine genes with significant fold differential gene expression at ≤ 0.01, including one gene, , with 1366-fold higher expression in CMS line as compared with the maintainer. Structural variation analysis of these mitochondrial genes identified length variation between ICPA 2039 and ICPB 2039 for (subunit of gene. Sanger sequencing of and genes in the CMS and the maintainer lines identified two single nucleotide polymorphisms (SNPs in upstream region of and a deletion of 10 bp in in the CMS line. Protein structure evaluation showed conformational changes in predicted protein structures for between ICPA 2039 and ICPB 2039 lines. All above analyses indicate association of gene with the CMS for A cytoplasm in pigeonpea. Additionally, one polymerase chain reaction (PCR based Indel marker ( has been developed and validated for testing genetic purity of A derived CMS lines to strengthen the commercial hybrid breeding program in pigeonpea.

HuR represses Wnt/β-catenin-mediated transcriptional activity by promoting cytoplasmic localization of β-catenin

Energy Technology Data Exchange (ETDEWEB)

Kim, Inae; Hur, Jung; Jeong, Sunjoo, E-mail: sjsj@dankook.ac.kr

2015-01-30

Highlights: • Wnt signaling as well as β-catenin overexpression enhance HuR cytoplasmic export. • HuR overexpression promotes cytoplasmic localization of β-catenin from the perinuclear fraction. • Wnt/β-catenin-mediated transcriptional activity is repressesed by HuR. - Abstract: β-Catenin is the key transcriptional activator of canonical Wnt signaling in the nucleus; thus, nuclear accumulation of β-catenin is a critical step for expressing target genes. β-Catenin accumulates in the nucleus of cancer cells where it activates oncogenic target genes. Hu antigen R (HuR) is a RNA binding protein that regulates multiple post-transcriptional processes including RNA stability. Thus, cytoplasmic HuR protein may be involved in tumorigenesis by stabilizing oncogenic transcripts, but the molecular mechanism remains unclear. Here, we observed that Wnt/β-catenin signaling induced export of the HuR protein, whereas HuR overexpression promoted accumulation of the β-catenin protein in the cytoplasm. Thus, Wnt/β-catenin-mediated transcriptional activity in the nucleus was reduced by overexpressing HuR. These results suggest novel and uncharacterized cytoplasmic β-catenin functions related to HuR-mediated RNA metabolism in cancer cells.

HuR represses Wnt/β-catenin-mediated transcriptional activity by promoting cytoplasmic localization of β-catenin

International Nuclear Information System (INIS)

Kim, Inae; Hur, Jung; Jeong, Sunjoo

2015-01-01

Highlights: • Wnt signaling as well as β-catenin overexpression enhance HuR cytoplasmic export. • HuR overexpression promotes cytoplasmic localization of β-catenin from the perinuclear fraction. • Wnt/β-catenin-mediated transcriptional activity is repressesed by HuR. - Abstract: β-Catenin is the key transcriptional activator of canonical Wnt signaling in the nucleus; thus, nuclear accumulation of β-catenin is a critical step for expressing target genes. β-Catenin accumulates in the nucleus of cancer cells where it activates oncogenic target genes. Hu antigen R (HuR) is a RNA binding protein that regulates multiple post-transcriptional processes including RNA stability. Thus, cytoplasmic HuR protein may be involved in tumorigenesis by stabilizing oncogenic transcripts, but the molecular mechanism remains unclear. Here, we observed that Wnt/β-catenin signaling induced export of the HuR protein, whereas HuR overexpression promoted accumulation of the β-catenin protein in the cytoplasm. Thus, Wnt/β-catenin-mediated transcriptional activity in the nucleus was reduced by overexpressing HuR. These results suggest novel and uncharacterized cytoplasmic β-catenin functions related to HuR-mediated RNA metabolism in cancer cells

Protein profiling in potato (Solanum tuberosum L.) leaf tissues by differential centrifugation.

Science.gov (United States)

Lim, Sanghyun; Chisholm, Kenneth; Coffin, Robert H; Peters, Rick D; Al-Mughrabi, Khalil I; Wang-Pruski, Gefu; Pinto, Devanand M

2012-04-06

Foliar diseases, such as late blight, result in serious threats to potato production. As such, potato leaf tissue becomes an important substrate to study biological processes, such as plant defense responses to infection. Nonetheless, the potato leaf proteome remains poorly characterized. Here, we report protein profiling of potato leaf tissues using a modified differential centrifugation approach to separate the leaf tissues into cell wall and cytoplasmic fractions. This method helps to increase the number of identified proteins, including targeted putative cell wall proteins. The method allowed for the identification of 1484 nonredundant potato leaf proteins, of which 364 and 447 were reproducibly identified proteins in the cell wall and cytoplasmic fractions, respectively. Reproducibly identified proteins corresponded to over 70% of proteins identified in each replicate. A diverse range of proteins was identified based on their theoretical pI values, molecular masses, functional classification, and biological processes. Such a protein extraction method is effective for the establishment of a highly qualified proteome profile.

The molecular mechanism and physiological role of cytoplasmic streaming.

Science.gov (United States)

Tominaga, Motoki; Ito, Kohji

2015-10-01

Cytoplasmic streaming occurs widely in plants ranging from algae to angiosperms. However, the molecular mechanism and physiological role of cytoplasmic streaming have long remained unelucidated. Recent molecular genetic approaches have identified specific myosin members (XI-2 and XI-K as major and XI-1, XI-B, and XI-I as minor motive forces) for the generation of cytoplasmic streaming among 13 myosin XIs in Arabidopsis thaliana. Simultaneous knockout of these myosin XI members led to a reduced velocity of cytoplasmic streaming and marked defects of plant development. Furthermore, the artificial modifications of myosin XI-2 velocity changed plant and cell sizes along with the velocity of cytoplasmic streaming. Therefore, we assume that cytoplasmic streaming is one of the key regulators in determining plant size. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Chimeric Proton-Pumping Rhodopsins Containing the Cytoplasmic Loop of Bovine Rhodopsin

Science.gov (United States)

Sasaki, Kengo; Yamashita, Takahiro; Yoshida, Kazuho; Inoue, Keiichi; Shichida, Yoshinori; Kandori, Hideki

2014-01-01

G-protein-coupled receptors (GPCRs) transmit stimuli to intracellular signaling systems. Rhodopsin (Rh), which is a prototypical GPCR, possesses an 11-cis retinal. Photoisomerization of 11-cis to all-trans leads to structural changes in the protein of cytoplasmic loops, activating G-protein. Microbial rhodopsins are similar heptahelical membrane proteins that function as bacterial sensors, light-driven ion-pumps, or light-gated channels. They possess an all-trans retinal, and photoisomerization to 13-cis triggers structural changes in protein. Despite these similarities, there is no sequence homology between visual and microbial rhodopsins, and microbial rhodopsins do not activate G-proteins. In this study, new chimeric proton-pumping rhodopsins, proteorhodopsin (PR) and Gloeobacter rhodopsin (GR) were designed by replacing cytoplasmic loops with bovine Rh loops. Although G-protein was not activated by the PR chimeras, all 12 GR chimeras activated G-protein. The GR chimera containing the second cytoplasmic loop of bovine Rh did not activate G-protein. However, the chimera with a second and third double-loop further enhanced G-protein activation. Introduction of an E132Q mutation slowed the photocycle 30-fold and enhanced activation. The highest catalytic activity of the GR chimera was still 3,200 times lower than bovine Rh but only 64 times lower than amphioxus Go-rhodopsin. This GR chimera showed a strong absorption change of the amide-I band on a light-minus-dark difference FTIR spectrum which could represent a larger helical opening, important for G-protein activation. The light-dependent catalytic activity of this GR chimera makes it a potential optogenetic tool for enzymatic activation by light. PMID:24621599

Interactions of cullin3/KCTD5 complexes with both cytoplasmic and nuclear proteins: Evidence for a role in protein stabilization

Energy Technology Data Exchange (ETDEWEB)

Rutz, Natalja; Heilbronn, Regine; Weger, Stefan, E-mail: stefan.weger@charite.de

2015-08-28

Based on its specific interaction with cullin3 mediated by an N-terminal BTB/POZ homologous domain, KCTD5 has been proposed to function as substrate adapter for cullin3 based ubiquitin E3 ligases. In the present study we tried to validate this hypothesis through identification and characterization of additional KCTD5 interaction partners. For the replication protein MCM7, the zinc finger protein ZNF711 and FAM193B, a yet poorly characterized cytoplasmic protein, we could demonstrate specific interaction with KCTD5 both in yeast two-hybrid and co-precipitation studies in mammalian cells. Whereas trimeric complexes of cullin3 and KCTD5 with the respective KCTD5 binding partner were formed, KCTD5/cullin3 induced polyubiquitylation and/or proteasome-dependent degradation of these binding partners could not be demonstrated. On the contrary, KCTD5 or Cullin3 overexpression increased ZNF711 protein stability. - Highlights: • KCTD5 nuclear translocation depends upon M phase and protein oligomerization. • Identification of MCM7, ZNF711 and FAM193 as KCTD5 interaction partners. • Formation of trimeric complexes of KCTD5/cullin3 with MCM7, ZNF711 and FAM193B. • KCTD5 is not involved in polyubiquitylation of MCM7 replication factor. • The KCTD5/cullin3 complex stabilizes ZNF711 transcription factor.

Differential proteomics analysis to identify proteins and pathways associated with male sterility of soybean using iTRAQ-based strategy.

Science.gov (United States)

Li, Jiajia; Ding, Xianlong; Han, Shaohuai; He, Tingting; Zhang, Hao; Yang, Longshu; Yang, Shouping; Gai, Junyi

2016-04-14

To further elucidate the molecular mechanism of cytoplasmic male sterility (CMS) in soybean, a differential proteomic analysis was completed between the CMS line NJCMS1A and its maintainer NJCMS1B using iTRAQ-based strategy. As a result, 180 differential abundance proteins (DAPs) were identified, of which, 60 were down-regulated and 120 were up-regulated in NJCMS1A compared with NJCMS1B. Bioinformatic analysis showed that 167 DAPs were annotated in 41 Gene Ontology functional groups, 106 DAPs were classified into 20 clusters of orthologous groups of protein categories, and 128 DAPs were enrichment in 53 KEGG pathways. Fifteen differential level proteins/genes with the same expression pattern were identified in the further conjoint analysis of DAPs and the previously reported differential expression genes. Moreover, multiple reaction monitoring test, qRT-PCR analysis and enzyme activity assay validated that the iTRAQ results were reliable. Based on functional analysis of DAPs, we concluded that male sterility in NJCMS1A might be related to insufficiencies in energy supply, unbalance of protein synthesis and degradation, disruption of flavonoid synthesis, programmed cell death, abnormalities of substance metabolism, etc. These results might facilitate our understanding of the molecular mechanisms behind CMS in soybean. Soybean is an important global crop that provides protein and oil. Heterosis is a significantly potential approach to increase the yield of soybean. Cytoplasmic male sterility (CMS) plays a vital role in the production of hybrid seeds. However, the genetic and molecular mechanisms of male sterility in soybean still need to be further elucidated. In the present paper, a differential proteomic analysis was carried out and the results showed that several key proteins involved in key pathways were associated with male sterility in soybean. This work provides a new insight to understand the genetic and molecular mechanisms underlying CMS in soybean

Cytoplasmic protein binding to highly conserved sequences in the 3' untranslated region of mouse protamine 2 mRNA, a translationally regulated transcript of male germ cells

International Nuclear Information System (INIS)

Kwon, Y.K.; Hecht, N.B.

1991-01-01

The expression of the protamines, the predominant nuclear proteins of mammalian spermatozoa, is regulated translationally during male germ-cell development. The 3' untranslated region (UTR) of protamine 1 mRNA has been reported to control its time of translation. To understand the mechanisms controlling translation of the protamine mRNAs, we have sought to identify cis elements of the 3' UTR of protamine 2 mRNA that are recognized by cytoplasmic factors. From gel retardation assays, two sequence elements are shown to form specific RNA-protein complexes. Protein binding sites of the two complexes were determined by RNase T1 mapping, by blocking the putative binding sites with antisense oligonucleotides, and by competition assays. The sequences of these elements, located between nucleotides + 537 and + 572 in protamine 2 mRNA, are highly conserved among postmeiotic translationally regulated nuclear proteins of the mammalian testis. Two closely linked protein binding sites were detected. UV-crosslinking studies revealed that a protein of about 18 kDa binds to one of the conserved sequences. These data demonstrate specific protein binding to a highly conserved 3' UTR of translationally regulated testicular mRNA

CTP synthase forms cytoophidia in the cytoplasm and nucleus

International Nuclear Information System (INIS)

Gou, Ke-Mian; Chang, Chia-Chun; Shen, Qing-Ji; Sung, Li-Ying; Liu, Ji-Long

2014-01-01

CTP synthase is an essential metabolic enzyme responsible for the de novo synthesis of CTP. Multiple studies have recently showed that CTP synthase protein molecules form filamentous structures termed cytoophidia or CTP synthase filaments in the cytoplasm of eukaryotic cells, as well as in bacteria. Here we report that CTP synthase can form cytoophidia not only in the cytoplasm, but also in the nucleus of eukaryotic cells. Both glutamine deprivation and glutamine analog treatment promote formation of cytoplasmic cytoophidia (C-cytoophidia) and nuclear cytoophidia (N-cytoophidia). N-cytoophidia are generally shorter and thinner than their cytoplasmic counterparts. In mammalian cells, both CTP synthase 1 and CTP synthase 2 can form cytoophidia. Using live imaging, we have observed that both C-cytoophidia and N-cytoophidia undergo multiple rounds of fusion upon glutamine analog treatment. Our study reveals the coexistence of cytoophidia in the cytoplasm and nucleus, therefore providing a good opportunity to investigate the intracellular compartmentation of CTP synthase. - Highlights: • CTP synthase forms cytoophidia not only in the cytoplasm but also in the nucleus. • Glutamine deprivation and Glutamine analogs promotes cytoophidium formation. • N-cytoophidia exhibit distinct morphology when compared to C-cytoophidia. • Both CTP synthase 1 and CTP synthase 2 form cytoophidia in mammalian cells. • Fusions of cytoophidia occur in the cytoplasm and nucleus

CTP synthase forms cytoophidia in the cytoplasm and nucleus

Energy Technology Data Exchange (ETDEWEB)

Gou, Ke-Mian [MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT (United Kingdom); State Key Laboratory for Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193 (China); Chang, Chia-Chun [Institute of Biotechnology, National Taiwan University, Taipei, Taiwan, ROC (China); Shen, Qing-Ji [MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT (United Kingdom); Sung, Li-Ying, E-mail: liyingsung@ntu.edu.tw [Institute of Biotechnology, National Taiwan University, Taipei, Taiwan, ROC (China); Agricultural Biotechnology Research Center, Academia Sinica, Taipei 115, Taiwan, ROC (China); Liu, Ji-Long, E-mail: jilong.liu@dpag.ox.ac.uk [MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT (United Kingdom)

2014-04-15

CTP synthase is an essential metabolic enzyme responsible for the de novo synthesis of CTP. Multiple studies have recently showed that CTP synthase protein molecules form filamentous structures termed cytoophidia or CTP synthase filaments in the cytoplasm of eukaryotic cells, as well as in bacteria. Here we report that CTP synthase can form cytoophidia not only in the cytoplasm, but also in the nucleus of eukaryotic cells. Both glutamine deprivation and glutamine analog treatment promote formation of cytoplasmic cytoophidia (C-cytoophidia) and nuclear cytoophidia (N-cytoophidia). N-cytoophidia are generally shorter and thinner than their cytoplasmic counterparts. In mammalian cells, both CTP synthase 1 and CTP synthase 2 can form cytoophidia. Using live imaging, we have observed that both C-cytoophidia and N-cytoophidia undergo multiple rounds of fusion upon glutamine analog treatment. Our study reveals the coexistence of cytoophidia in the cytoplasm and nucleus, therefore providing a good opportunity to investigate the intracellular compartmentation of CTP synthase. - Highlights: • CTP synthase forms cytoophidia not only in the cytoplasm but also in the nucleus. • Glutamine deprivation and Glutamine analogs promotes cytoophidium formation. • N-cytoophidia exhibit distinct morphology when compared to C-cytoophidia. • Both CTP synthase 1 and CTP synthase 2 form cytoophidia in mammalian cells. • Fusions of cytoophidia occur in the cytoplasm and nucleus.

Novel mitochondria-targeted heat-soluble proteins identified in the anhydrobiotic Tardigrade improve osmotic tolerance of human cells.

Directory of Open Access Journals (Sweden)

Sae Tanaka

Full Text Available Tardigrades are able to tolerate almost complete dehydration through transition to a metabolically inactive state, called "anhydrobiosis". Late Embryogenesis Abundant (LEA proteins are heat-soluble proteins involved in the desiccation tolerance of many anhydrobiotic organisms. Tardigrades, Ramazzottius varieornatus, however, express predominantly tardigrade-unique heat-soluble proteins: CAHS (Cytoplasmic Abundant Heat Soluble and SAHS (Secretory Abundant Heat Soluble proteins, which are secreted or localized in most intracellular compartments, except the mitochondria. Although mitochondrial integrity is crucial to ensure cellular survival, protective molecules for mitochondria have remained elusive. Here, we identified two novel mitochondrial heat-soluble proteins, RvLEAM and MAHS (Mitochondrial Abundant Heat Soluble, as potent mitochondrial protectants from Ramazzottius varieornatus. RvLEAM is a group3 LEA protein and immunohistochemistry confirmed its mitochondrial localization in tardigrade cells. MAHS-green fluorescent protein fusion protein localized in human mitochondria and was heat-soluble in vitro, though no sequence similarity with other known proteins was found, and one region was conserved among tardigrades. Furthermore, we demonstrated that RvLEAM protein as well as MAHS protein improved the hyperosmotic tolerance of human cells. The findings of the present study revealed that tardigrade mitochondria contain at least two types of heat-soluble proteins that might have protective roles in water-deficient environments.

Microtubule plus end-tracking proteins play critical roles in directional growth of hyphae by regulating the dynamics of cytoplasmic microtubules in Aspergillus nidulans.

Science.gov (United States)

Zeng, Cui J Tracy; Kim, Hye-Ryun; Vargas Arispuro, Irasema; Kim, Jung-Mi; Huang, An-Chi; Liu, Bo

2014-11-01

Cytoplasmic microtubules (MTs) serve as a rate-limiting factor for hyphal tip growth in the filamentous fungus Aspergillus nidulans. We hypothesized that this function depended on the MT plus end-tracking proteins (+TIPs) including the EB1 family protein EBA that decorated the MT plus ends undergoing polymerization. The ebAΔ mutation reduced colony growth and the mutant hyphae appeared in an undulating pattern instead of exhibiting unidirectional growth in the control. These phenotypes were enhanced by a mutation in another +TIP gene clipA. EBA was required for plus end-tracking of CLIPA, the Kinesin-7 motor KipA, and the XMAP215 homologue AlpA. In addition, cytoplasmic dynein also depended on EBA to track on most polymerizing MT plus ends, but not for its conspicuous appearance at the MT ends near the hyphal apex. The loss of EBA reduced the number of cytoplasmic MTs and prolonged dwelling times for MTs after reaching the hyphal apex. Finally, we found that colonies were formed in the absence of EBA, CLIPA, and NUDA together, suggesting that they were dispensable for fundamental functions of MTs. This study provided a comprehensive delineation of the relationship among different +TIPs and their contributions to MT dynamics and unidirectional hyphal expansion in filamentous fungi. © 2014 John Wiley & Sons Ltd.

Promising SINEs for embargoing nuclear-cytoplasmic export as an anticancer strategy.

Science.gov (United States)

Tan, David S P; Bedard, Philippe L; Kuruvilla, John; Siu, Lillian L; Razak, Albiruni R Abdul

2014-05-01

In cancer cells, the nuclear-cytoplasmic transport machinery is frequently disrupted, resulting in mislocalization and loss of function for many key regulatory proteins. In this review, the mechanisms by which tumor cells co-opt the nuclear transport machinery to facilitate carcinogenesis, cell survival, drug resistance, and tumor progression will be elucidated, with a particular focus on the role of the nuclear-cytoplasmic export protein. The recent development of a new generation of selective inhibitors of nuclear export (XPO1 antagonists) and how these novel anticancer drugs may bring us closer to the implementation of this therapeutic strategy in the clinic will be discussed.

A genome-wide screen identifies conserved protein hubs required for cadherin-mediated cell–cell adhesion

Science.gov (United States)

Toret, Christopher P.; D’Ambrosio, Michael V.; Vale, Ronald D.; Simon, Michael A.

2014-01-01

Cadherins and associated catenins provide an important structural interface between neighboring cells, the actin cytoskeleton, and intracellular signaling pathways in a variety of cell types throughout the Metazoa. However, the full inventory of the proteins and pathways required for cadherin-mediated adhesion has not been established. To this end, we completed a genome-wide (∼14,000 genes) ribonucleic acid interference (RNAi) screen that targeted Ca2+-dependent adhesion in DE-cadherin–expressing Drosophila melanogaster S2 cells in suspension culture. This novel screen eliminated Ca2+-independent cell–cell adhesion, integrin-based adhesion, cell spreading, and cell migration. We identified 17 interconnected regulatory hubs, based on protein functions and protein–protein interactions that regulate the levels of the core cadherin–catenin complex and coordinate cadherin-mediated cell–cell adhesion. Representative proteins from these hubs were analyzed further in Drosophila oogenesis, using targeted germline RNAi, and adhesion was analyzed in Madin–Darby canine kidney mammalian epithelial cell–cell adhesion. These experiments reveal roles for a diversity of cellular pathways that are required for cadherin function in Metazoa, including cytoskeleton organization, cell–substrate interactions, and nuclear and cytoplasmic signaling. PMID:24446484

iTRAQ-Based Proteomics Analyses of Sterile/Fertile Anthers from a Thermo-Sensitive Cytoplasmic Male-Sterile Wheat with Aegilops kotschyi Cytoplasm

Directory of Open Access Journals (Sweden)

Gaoming Zhang

2018-05-01

Full Text Available A “two-line hybrid system” was developed, previously based on thermo-sensitive cytoplasmic male sterility in Aegilops kotschyi (K-TCMS, which can be used in wheat breeding. The K-TCMS line exhibits complete male sterility and it can be used to produce hybrid wheat seeds during the normal wheat-growing season; it propagates via self-pollination at high temperatures. Isobaric tags for relative and absolute quantification-based quantitative proteome and bioinformatics analyses of the TCMS line KTM3315A were conducted under different fertility conditions to understand the mechanisms of fertility conversion in the pollen development stages. In total, 4639 proteins were identified, the differentially abundant proteins that increased/decreased in plants with differences in fertility were mainly involved with energy metabolism, starch and sucrose metabolism, phenylpropanoid biosynthesis, protein synthesis, translation, folding, and degradation. Compared with the sterile condition, many of the proteins that related to energy and phenylpropanoid metabolism increased during the anther development stage. Thus, we suggest that energy and phenylpropanoid metabolism pathways are important for fertility conversion in K-TCMS wheat. These findings provide valuable insights into the proteins involved with anther and pollen development, thereby, helping to further understand the mechanism of TCMS in wheat.

Protein Correlation Profiles Identify Lipid Droplet Proteins with High Confidence*

Science.gov (United States)

Krahmer, Natalie; Hilger, Maximiliane; Kory, Nora; Wilfling, Florian; Stoehr, Gabriele; Mann, Matthias; Farese, Robert V.; Walther, Tobias C.

2013-01-01

Lipid droplets (LDs) are important organelles in energy metabolism and lipid storage. Their cores are composed of neutral lipids that form a hydrophobic phase and are surrounded by a phospholipid monolayer that harbors specific proteins. Most well-established LD proteins perform important functions, particularly in cellular lipid metabolism. Morphological studies show LDs in close proximity to and interacting with membrane-bound cellular organelles, including the endoplasmic reticulum, mitochondria, peroxisomes, and endosomes. Because of these close associations, it is difficult to purify LDs to homogeneity. Consequently, the confident identification of bona fide LD proteins via proteomics has been challenging. Here, we report a methodology for LD protein identification based on mass spectrometry and protein correlation profiles. Using LD purification and quantitative, high-resolution mass spectrometry, we identified LD proteins by correlating their purification profiles to those of known LD proteins. Application of the protein correlation profile strategy to LDs isolated from Drosophila S2 cells led to the identification of 111 LD proteins in a cellular LD fraction in which 1481 proteins were detected. LD localization was confirmed in a subset of identified proteins via microscopy of the expressed proteins, thereby validating the approach. Among the identified LD proteins were both well-characterized LD proteins and proteins not previously known to be localized to LDs. Our method provides a high-confidence LD proteome of Drosophila cells and a novel approach that can be applied to identify LD proteins of other cell types and tissues. PMID:23319140

Hydrodynamic flow in the cytoplasm of plant cells.

NARCIS (Netherlands)

Esseling-Ozdoba, A.; Houtman, D.; Lammeren, A.A. van; Eiser, E.; Emons, A.M.C.

2008-01-01

Plant cells show myosin-driven organelle movement, called cytoplasmic streaming. Soluble molecules, such as metabolites do not move with motor proteins but by diffusion. However, is all of this streaming active motor-driven organelle transport? Our recent simulation study (Houtman et al., 2007)

Hydrodynamic flow in the cytoplasm of plant cells

NARCIS (Netherlands)

Esseling-Ozdoba, A.; Houtman, D.; van Lammeren, A.A.M.; Eiser, E.; Emons, A.M.C.

2008-01-01

Plant cells show myosin-driven organelle movement, called cytoplasmic streaming. Soluble molecules, such as metabolites do not move with motor proteins but by diffusion. However, is all of this streaming active motor-driven organelle transport? Our recent simulation study (Houtman et al., 2007)

Hydrodynamic flow in the cytoplasm of plant cells

NARCIS (Netherlands)

Esseling-Ozdoba, A.; Houtman, D.; Lammeren, van A.A.M.; Eiser, E.; Emons, A.M.C.

2008-01-01

Plant cells show myosin-driven organelle movement, called cytoplasmic streaming. Soluble molecules, such as metabolites do not move with motor proteins but by diffusion. However, is all of this streaming active motor-driven organelle transport? Our recent simulation study ( Houtman et al., 2007 )

Characterization of cytoplasmic male sterility of rice with Lead Rice cytoplasm in comparison with that with Chinsurah Boro II cytoplasm.

Science.gov (United States)

Itabashi, Etsuko; Kazama, Tomohiko; Toriyama, Kinya

2009-02-01

Rice with LD-type cytoplasmic male sterility (CMS) possesses the cytoplasm of 'Lead Rice' and its fertility is recovered by a nuclear fertility restorer gene Rf1. Rf1 promotes processing of a CMS-associated mitochondrial RNA of atp6-orf79, which consists of atp6 and orf79, in BT-CMS with the cytoplasm of 'Chinsurah Boro II'. In this study, we found that LD-cytoplasm contained a sequence variant of orf79 downstream of atp6. Northern blot analysis showed that atp6-orf79 RNA of LD-cytoplasm was co-transcribed and was processed in the presence of Rf1 in the same manner as in BT-cytoplasm. Western blot analysis showed that the ORF79 peptide did not accumulate in an LD-CMS line, while ORF79 accumulated in a BT-CMS line and was diminished by Rf1. These results suggest that accumulation of ORF79 is not the cause of CMS in LD-cytoplasm and the mechanism of male-sterility induction/fertility restoration in LD-CMS is different from that in BT-CMS.

Comparative genomic analysis identified a mutation related to enhanced heterologous protein production in the filamentous fungus Aspergillus oryzae.

Science.gov (United States)

Jin, Feng-Jie; Katayama, Takuya; Maruyama, Jun-Ichi; Kitamoto, Katsuhiko

2016-11-01

Genomic mapping of mutations using next-generation sequencing technologies has facilitated the identification of genes contributing to fundamental biological processes, including human diseases. However, few studies have used this approach to identify mutations contributing to heterologous protein production in industrial strains of filamentous fungi, such as Aspergillus oryzae. In a screening of A. oryzae strains that hyper-produce human lysozyme (HLY), we previously isolated an AUT1 mutant that showed higher production of various heterologous proteins; however, the underlying factors contributing to the increased heterologous protein production remained unclear. Here, using a comparative genomic approach performed with whole-genome sequences, we attempted to identify the genes responsible for the high-level production of heterologous proteins in the AUT1 mutant. The comparative sequence analysis led to the detection of a gene (AO090120000003), designated autA, which was predicted to encode an unknown cytoplasmic protein containing an alpha/beta-hydrolase fold domain. Mutation or deletion of autA was associated with higher production levels of HLY. Specifically, the HLY yields of the autA mutant and deletion strains were twofold higher than that of the control strain during the early stages of cultivation. Taken together, these results indicate that combining classical mutagenesis approaches with comparative genomic analysis facilitates the identification of novel genes involved in heterologous protein production in filamentous fungi.

Susceptibility to virus-cell fusion at the plasma membrane is reduced through expression of HIV gp41 cytoplasmic domains

International Nuclear Information System (INIS)

Malinowsky, Katharina; Luksza, Julia; Dittmar, Matthias T.

2008-01-01

The cytoplasmic tail of the HIV transmembrane protein plays an important role in viral infection. In this study we analyzed the role of retroviral cytoplasmic tails in modulating the cytoskeleton and interfering with virus-cell fusion. HeLaP4 cells expressing different HIV cytoplasmic tail constructs showed reduced acetylated tubulin levels whereas the cytoplasmic tail of MLV did not alter microtubule stability indicating a unique function for the lentiviral cytoplasmic tail. The effect on tubulin is mediated through the membrane proximal region of the HIV cytoplasmic tail and was independent of membrane localization. Site-directed mutagenesis identified three motifs in the HIV-2 cytoplasmic tail required to effect the reduction in acetylated tubulin. Both the YxxΦ domain and amino acids 21 to 45 of the HIV-2 cytoplasmic tail need to be present to change the level of acetylated tubulin in transfected cells. T-cells stably expressing one HIV-2 cytoplasmic tail derived construct showed also a reduction in acetylated tubulin thus confirming the importance of this effect not only for HeLaP4 and 293T cells. Challenge experiments using transiently transfected HeLaP4 cells and T cells stably expressing an HIV cytoplasmic tail construct revealed both reduced virus-cell fusion and replication of HIV-1 NL4.3 compared to control cells. In the virus-cell fusion assay only virions pseudotyped with either HIV or MLV envelopes showed reduced fusion efficiency, whereas VSV-G pseudotyped virions where not affected by the expression of HIV derived cytoplasmic tail constructs, indicating that fusion at the plasma but not endosomal membrane is affected. Overexpression of human histone-deacetylase 6 (HDAC6) and constitutively active RhoA resulted in a reduction of acetylated tubulin and reduced virus-cell fusion as significant as that observed following expression of HIV cytoplasmic tail constructs. Inhibition of HDAC6 showed a strong increase in acetylated tubulin and increase of

Human regulator of telomere elongation helicase 1 (RTEL1) is required for the nuclear and cytoplasmic trafficking of pre-U2 RNA.

Science.gov (United States)

Schertzer, Michael; Jouravleva, Karina; Perderiset, Mylene; Dingli, Florent; Loew, Damarys; Le Guen, Tangui; Bardoni, Barbara; de Villartay, Jean-Pierre; Revy, Patrick; Londoño-Vallejo, Arturo

2015-02-18

Hoyeraal-Hreidarsson syndrome (HHS) is a severe form of Dyskeratosis congenita characterized by developmental defects, bone marrow failure and immunodeficiency and has been associated with telomere dysfunction. Recently, mutations in Regulator of Telomere ELongation helicase 1 (RTEL1), a helicase first identified in Mus musculus as being responsible for the maintenance of long telomeres, have been identified in several HHS patients. Here we show that RTEL1 is required for the export and the correct cytoplasmic trafficking of the small nuclear (sn) RNA pre-U2, a component of the major spliceosome complex. RTEL1-HHS cells show abnormal subcellular partitioning of pre-U2, defects in the recycling of ribonucleotide proteins (RNP) in the cytoplasm and splicing defects. While most of these phenotypes can be suppressed by re-expressing the wild-type protein in RTEL1-HHS cells, expression of RTEL1 mutated variants in immortalized cells provokes cytoplasmic mislocalizations of pre-U2 and other RNP components, as well as splicing defects, thus phenocopying RTEL1-HHS cellular defects. Strikingly, expression of a cytoplasmic form of RTEL1 is sufficient to correct RNP mislocalizations both in RTEL1-HHS cells and in cells expressing nuclear mutated forms of RTEL1. This work unravels completely unanticipated roles for RTEL1 in RNP trafficking and strongly suggests that defects in RNP biogenesis pathways contribute to the pathology of HHS. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Cytoplasmic proteasomes are not indispensable for cell growth in Saccharomyces cerevisiae

Energy Technology Data Exchange (ETDEWEB)

Tsuchiya, Hikaru; Arai, Naoko; Tanaka, Keiji, E-mail: tanaka-kj@igakuken.or.jp; Saeki, Yasushi, E-mail: saeki-ys@igakuken.or.jp

2013-07-05

Highlights: •We succeeded to control the proteasome localization by the anchor-away technique. •Nuclear proteasome-depleted cells showed a lethal phenotype. •Cytoplasmic proteasomes are not indispensable for cell growth in dividing cells. -- Abstract: The 26S proteasome is an essential protease complex responsible for the degradation of ubiquitinated proteins in eukaryotic cells. In rapidly proliferating yeast cells, proteasomes are mainly localized in the nucleus, but the biological significance of the proteasome localization is still unclear. In this study, we investigated the relationship between the proteasome localization and the functions by the anchor-away technique, a ligand-dependent sequestration of a target protein into specific compartment(s). Anchoring of the proteasome to the plasma membrane or the ribosome resulted in conditional depletion of the nuclear proteasomes, whereas anchoring to histone resulted in the proteasome sequestration into the nucleus. We observed that the accumulation of ubiquitinated proteins in all the proteasome-targeted cells, suggesting that both the nuclear and cytoplasmic proteasomes have proteolytic functions and that the ubiquitinated proteins are produced and degraded in each compartment. Consistent with previous studies, the nuclear proteasome-depleted cells exhibited a lethal phenotype. In contrast, the nuclear sequestration of the proteasome resulted only in a mild growth defect, suggesting that the cytoplasmic proteasomes are not basically indispensable for cell growth in rapidly growing yeast cells.

Phorbol ester tumor promoter induced the synthesis of two major cytoplasmic proteins: identity with two proteins induced under heat-shocked and glucose-starved conditions

International Nuclear Information System (INIS)

Zhang, H.; Chen, K.Y.; Liu, A.Y.C.

1987-01-01

The regulation of specific protein synthesis by the phorbol ester tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), was evaluated using the L-8 and C-2 myoblast and the 3T3-L1 fibroblast cell cultures. TPA increased, by 2-4 fold, the synthesis rates of two cytoplasmic proteins with apparent molecular weights of 89,000 and 74,000 as determined by SDS-polyacrylamide gel electrophoresis and autoradiography. The concentration of TPA and the time of incubation needed to elicit this induction was determined to be 10 μg/ml and 20 hrs, respectively. Increasing the concentration of TPA to 100, 200, and 500 ng/ml did not result in a greater magnitude of induction. The possibility that these two TPA-induced proteins may be identical to proteins with similar molecular weights induced under heat-shocked or glucose-starved conditions was evaluated by 1-D and 2-D gel electrophoresis and autoradiography. Results provided evidence that the TPA-induced 89,000- and 74,000-dalton proteins were identical to hsp 89 and hsp 74, 2 out of a set of 8-9 proteins induced under heat shocked conditions. Furthermore, they are identical to two of the set of glucose-regulated proteins induced under a glucose-starved condition

Protein synthesis controls phosphate homeostasis.

Science.gov (United States)

Pontes, Mauricio H; Groisman, Eduardo A

2018-01-01

Phosphorus is an essential element assimilated largely as orthophosphate (Pi). Cells respond to Pi starvation by importing Pi from their surroundings. We now report that impaired protein synthesis alone triggers a Pi starvation response even when Pi is plentiful in the extracellular milieu. In the bacterium Salmonella enterica serovar Typhimurium , this response entails phosphorylation of the regulatory protein PhoB and transcription of PhoB-dependent Pi transporter genes and is eliminated upon stimulation of adenosine triphosphate (ATP) hydrolysis. When protein synthesis is impaired due to low cytoplasmic magnesium (Mg 2+ ), Salmonella triggers the Pi starvation response because ribosomes are destabilized, which reduces ATP consumption and thus free cytoplasmic Pi. This response is transient because low cytoplasmic Mg 2+ promotes an uptake in Mg 2+ and a decrease in ATP levels, which stabilizes ribosomes, resulting in ATP consumption and Pi increase, thus ending the response. Notably, pharmacological inhibition of protein synthesis also elicited a Pi starvation response in the bacterium Escherichia coli and the yeast Saccharomyces cerevisiae Our findings identify a regulatory connection between protein synthesis and Pi homeostasis that is widespread in nature. © 2018 Pontes and Groisman; Published by Cold Spring Harbor Laboratory Press.

Water diffusion in cytoplasmic streaming in Elodea internodal cells under the effect of antimitotic agents.

Science.gov (United States)

Vorob'ev, Vladimir N; Anisimov, Alexander V; Dautova, Nailya R

2008-07-01

The translational displacement of the cytoplasmic water in Elodea stem cells resulting from protein motor activity was measured using the NMR method. A 24-h treatment with vincristine results in a reduction of the translational displacement of the cytoplasmic water. With a constant cytoplasmic streaming velocity, the dynamics of the translational displacement of the cytoplasmic water under the effect of taxol are characterized by a continuous increase at a concentration of 0.05 mM, and reaching a plateau at a concentration of 0.5 mM.

GLYCINE-RICH RNA-BINDING PROTEIN1 interacts with RECEPTOR-LIKE CYTOPLASMIC PROTEIN KINASE1 and suppresses cell death and defense responses in pepper (Capsicum annuum).

Science.gov (United States)

Kim, Dae Sung; Kim, Nak Hyun; Hwang, Byung Kook

2015-01-01

Plants use a variety of innate immune regulators to trigger cell death and defense responses against pathogen attack. We identified pepper (Capsicum annuum) GLYCINE-RICH RNA-BINDING PROTEIN1 (CaGRP1) as a RECEPTOR-LIKE CYTOPLASMIC PROTEIN KINASE1 (CaPIK1)-interacting partner, based on bimolecular fluorescence complementation and coimmunoprecipitation analyses as well as gene silencing and transient expression analysis. CaGRP1 contains an N-terminal RNA recognition motif and a glycine-rich region at the C-terminus. The CaGRP1 protein had DNA- and RNA-binding activity in vitro. CaGRP1 interacted with CaPIK1 in planta. CaGRP1 and CaGRP1-CaPIK1 complexes were localized to the nucleus in plant cells. CaPIK1 phosphorylated CaGRP1 in vitro and in planta. Transient coexpression of CaGRP1 with CaPIK1 suppressed the CaPIK1-triggered cell death response, accompanied by a reduced CaPIK1-triggered reactive oxygen species (ROS) burst. The RNA recognition motif region of CaGRP1 was responsible for the nuclear localization of CaGRP1 as well as the suppression of the CaPIK1-triggered cell death response. CaGRP1 silencing in pepper conferred enhanced resistance to Xanthomonas campestris pv vesicatoria (Xcv) infection; however, CaPIK1-silenced plants were more susceptible to Xcv. CaGRP1 interacts with CaPIK1 and negatively regulates CaPIK1-triggered cell death and defense responses by suppressing ROS accumulation. © 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.

Actin polymerisation at the cytoplasmic face of eukaryotic nuclei

Directory of Open Access Journals (Sweden)

David-Watine Brigitte

2006-05-01

Full Text Available Abstract Background There exists abundant molecular and ultra-structural evidence to suggest that cytoplasmic actin can physically interact with the nuclear envelope (NE membrane system. However, this interaction has yet to be characterised in living interphase cells. Results Using a fluorescent conjugate of the actin binding drug cytochalasin D (CD-BODIPY we provide evidence that polymerising actin accumulates in vicinity to the NE. In addition, both transiently expressed fluorescent actin and cytoplasmic micro-injection of fluorescent actin resulted in accumulation of actin at the NE-membrane. Consistent with the idea that the cytoplasmic phase of NE-membranes can support this novel pool of perinuclear actin polymerisation we show that isolated, intact, differentiated primary hepatocyte nuclei support actin polymerisation in vitro. Further this phenomenon was inhibited by treatments hindering steric access to outer-nuclear-membrane proteins (e.g. wheat germ agglutinin, anti-nesprin and anti-nucleoporin antibodies. Conclusion We conclude that actin polymerisation occurs around interphase nuclei of living cells at the cytoplasmic phase of NE-membranes.

Adducin family proteins possess different nuclear export potentials.

Science.gov (United States)

Liu, Chia-Mei; Hsu, Wen-Hsin; Lin, Wan-Yi; Chen, Hong-Chen

2017-05-10

The adducin (ADD) family proteins, namely ADD1, ADD2, and ADD3, are actin-binding proteins that play important roles in the stabilization of membrane cytoskeleton and cell-cell junctions. All the ADD proteins contain a highly conserved bipartite nuclear localization signal (NLS) at the carboxyl termini, but only ADD1 can localize to the nucleus. The reason for this discrepancy is not clear. To avoid the potential effect of cell-cell junctions on the distribution of ADD proteins, HA epitope-tagged ADD proteins and mutants were transiently expressed in NIH3T3 fibroblasts and their distribution in the cytoplasm and nucleus was examined by immunofluorescence staining. Several nuclear proteins were identified to interact with ADD1 by mass spectrometry, which were further verified by co-immunoprecipitation. In this study, we found that ADD1 was detectable both in the cytoplasm and nucleus, whereas ADD2 and ADD3 were detected only in the cytoplasm. However, ADD2 and ADD3 were partially (~40%) sequestered in the nucleus by leptomycin B, a CRM1/exportin1 inhibitor. Upon the removal of leptomycin B, ADD2 and ADD3 re-distributed to the cytoplasm. These results indicate that ADD2 and ADD3 possess functional NLS and are quickly transported to the cytoplasm upon entering the nucleus. Indeed, we found that ADD2 and ADD3 possess much higher potential to counteract the activity of the NLS derived from Simian virus 40 large T-antigen than ADD1. All the ADD proteins appear to contain multiple nuclear export signals mainly in their head and neck domains. However, except for the leucine-rich motif ( 377 FEALMRMLDWLGYRT 391 ) in the neck domain of ADD1, no other classic nuclear export signal was identified in the ADD proteins. In addition, the nuclear retention of ADD1 facilitates its interaction with RNA polymerase II and zinc-finger protein 331. Our results suggest that ADD2 and ADD3 possess functional NLS and shuttle between the cytoplasm and nucleus. The discrepancy in the

Membrane-Dependent Effects of a Cytoplasmic Helix on the Structure and Drug Binding of the Influenza Virus M2 Protein

Science.gov (United States)

Cady, Sarah; Wang, Tuo; Hong, Mei

2011-01-01

The influenza A M2 protein forms a proton channel for virus infection and also mediates virus assembly and budding. The minimum protein length that encodes both functions contains the transmembrane (TM) domain (roughly residues 22 to 46) for the amantadine-sensitive proton-channel activity and an amphipathic cytoplasmic helix (roughly residues 45 to 62) for curvature induction and virus budding. However, structural studies involving the TM domain with or without the amphipathic helix differed on the drug-binding site. Here we use solid-state NMR spectroscopy to determine the amantadine binding site in the cytoplasmic-helix-containing M2(21–61). 13C-2H distance measurements of 13C-labeled protein and 2H-labeled amantadine showed that in DMPC bilayers, the first equivalent of drug bound S31 inside the M2(21–61) pore, similar to the behavior of M2TM in DMPC bilayers. The non-specific surface site of D44 observed in M2TM is disfavored in the longer peptide. Thus, the pharmacologically relevant drug-binding site in the fully functional M2(21–61) is S31 in the TM pore. Interestingly, when M2(21–61) was reconstituted into a virus-mimetic membrane containing 30% cholesterol, no chemical shift perturbation was observed for pore-lining residues, while M2TM in the same membrane exhibited drug-induced chemical shift changes. Reduction of the cholesterol level and the use of unsaturated phospholipids shifted the conformational equilibrium of M2TM fully to the bound state, but did not rescue drug binding to M2(21–61). These results suggest that the amphipathic helix, together with cholesterol, modulates the ability of the TM helices to bind amantadine. Thus, the M2 protein interacts with the lipid membrane and small-molecule inhibitors in a complex fashion, and a careful examination of the environmental dependence of the protein conformation is required to fully understand the structure-function relation of this protein. PMID:21661724

Mutational analysis of the EMCV 2A protein identifies a nuclear localization signal and an eIF4E binding site

International Nuclear Information System (INIS)

Groppo, Rachel; Brown, Bradley A.; Palmenberg, Ann C.

2011-01-01

Cardioviruses have a unique 2A protein (143 aa). During genome translation, the encephalomyocarditis virus (EMCV) 2A is released through a ribosome skipping event mitigated through C-terminal 2A sequences and by subsequent N-terminal reaction with viral 3C pro . Although viral replication is cytoplasmic, mature 2A accumulates in nucleoli shortly after infection. Some protein also transiently associates with cytoplasmic 40S ribosomal subunits, an activity contributing to inhibition of cellular cap-dependent translation. Cardiovirus sequences predict an eIF4E binding site (aa 126-134) and a nuclear localization signal (NLS, aa 91-102), within 2A, both of which are functional during EMCV infection. Point mutations preventing eIF4E:2A interactions gave small-plaque phenotype viruses, but still inhibited cellular cap-dependent translation. Deletions within the NLS motif relocalized 2A to the cytoplasm and abrogated the inhibition of cap-dependent translation. A fusion protein linking the 2A NLS to eGFP was sufficient to redirect the reporter to the nucleus but not into nucleoli.

In vivo evidence of TonB shuttling between the cytoplasmic and outer membrane in Escherichia coli.

Science.gov (United States)

Larsen, Ray A; Letain, Tracy E; Postle, Kathleen

2003-07-01

Gram-negative bacteria are able to convert potential energy inherent in the proton gradient of the cytoplasmic membrane into active nutrient transport across the outer membrane. The transduction of energy is mediated by TonB protein. Previous studies suggest a model in which TonB makes sequential and cyclic contact with proteins in each membrane, a process called shuttling. A key feature of shuttling is that the amino-terminal signal anchor must quit its association with the cytoplasmic membrane, and TonB becomes associated solely with the outer membrane. However, the initial studies did not exclude the possibility that TonB was artifactually pulled from the cytoplasmic membrane by the fractionation process. To resolve this ambiguity, we devised a method to test whether the extreme TonB amino-terminus, located in the cytoplasm, ever became accessible to the cys-specific, cytoplasmic membrane-impermeant molecule, Oregon Green(R) 488 maleimide (OGM) in vivo. A full-length TonB and a truncated TonB were modified to carry a sole cysteine at position 3. Both full-length TonB and truncated TonB (consisting of the amino-terminal two-thirds) achieved identical conformations in the cytoplasmic membrane, as determined by their abilities to cross-link to the cytoplasmic membrane protein ExbB and their abilities to respond conformationally to the presence or absence of proton motive force. Full-length TonB could be amino-terminally labelled in vivo, suggesting that it was periplasmically exposed. In contrast, truncated TonB, which did not associate with the outer membrane, was not specifically labelled in vivo. The truncated TonB also acted as a control for leakage of OGM across the cytoplasmic membrane. Further, the extent of labelling for full-length TonB correlated roughly with the proportion of TonB found at the outer membrane. These findings suggest that TonB does indeed disengage from the cytoplasmic membrane during energy transduction and shuttle to the outer membrane.

Secretory TAT-peptide-mediated protein transduction of LIF receptor α-chain distal cytoplasmic motifs into human myeloid HL-60 cells

Directory of Open Access Journals (Sweden)

Q. Sun

2012-10-01

Full Text Available The distal cytoplasmic motifs of leukemia inhibitory factor receptor α-chain (LIFRα-CT3 can independently induce intracellular myeloid differentiation in acute myeloid leukemia (AML cells by gene transfection; however, there are significant limitations in the potential clinical use of these motifs due to liposome-derived genetic modifications. To produce a potentially therapeutic LIFRα-CT3 with cell-permeable activity, we constructed a eukaryotic expression pcDNA3.0-TAT-CT3-cMyc plasmid with a signal peptide (ss inserted into the N-terminal that codes for an ss-TAT-CT3-cMyc fusion protein. The stable transfection of Chinese hamster ovary (CHO cells via this vector and subsequent selection by Geneticin resulted in cell lines that express and secrete TAT-CT3-cMyc. The spent medium of pcDNA3.0-TAT-CT3-cMyc-transfected CHO cells could be purified using a cMyc-epitope-tag agarose affinity chromatography column and could be detected via SDS-PAGE, with antibodies against cMyc-tag. The direct administration of TAT-CT3-cMyc to HL-60 cell culture media caused the enrichment of CT3-cMyc in the cytoplasm and nucleus within 30 min and led to a significant reduction of viable cells (P < 0.05 8 h after exposure. The advantages of using this mammalian expression system include the ease of generating TAT fusion proteins that are adequately transcripted and the potential for a sustained production of such proteins in vitro for future AML therapy.

Secretory TAT-peptide-mediated protein transduction of LIF receptor α-chain distal cytoplasmic motifs into human myeloid HL-60 cells

International Nuclear Information System (INIS)

Sun, Q.; Xiong, J.; Lu, J.; Xu, S.; Li, Y.; Zhong, X.P.; Gao, G.K.; Liu, H.Q.

2012-01-01

The distal cytoplasmic motifs of leukemia inhibitory factor receptor α-chain (LIFRα-CT3) can independently induce intracellular myeloid differentiation in acute myeloid leukemia (AML) cells by gene transfection; however, there are significant limitations in the potential clinical use of these motifs due to liposome-derived genetic modifications. To produce a potentially therapeutic LIFRα-CT3 with cell-permeable activity, we constructed a eukaryotic expression pcDNA3.0-TAT-CT3-cMyc plasmid with a signal peptide (ss) inserted into the N-terminal that codes for an ss-TAT-CT3-cMyc fusion protein. The stable transfection of Chinese hamster ovary (CHO) cells via this vector and subsequent selection by Geneticin resulted in cell lines that express and secrete TAT-CT3-cMyc. The spent medium of pcDNA3.0-TAT-CT3-cMyc-transfected CHO cells could be purified using a cMyc-epitope-tag agarose affinity chromatography column and could be detected via SDS-PAGE, with antibodies against cMyc-tag. The direct administration of TAT-CT3-cMyc to HL-60 cell culture media caused the enrichment of CT3-cMyc in the cytoplasm and nucleus within 30 min and led to a significant reduction of viable cells (P < 0.05) 8 h after exposure. The advantages of using this mammalian expression system include the ease of generating TAT fusion proteins that are adequately transcripted and the potential for a sustained production of such proteins in vitro for future AML therapy

Secretory TAT-peptide-mediated protein transduction of LIF receptor α-chain distal cytoplasmic motifs into human myeloid HL-60 cells

Energy Technology Data Exchange (ETDEWEB)

Sun, Q. [Department of Hyperbaric Medicine, No. 401 Hospital of PLA, Qingdao (China); Department of Histology and Embryology, Faculty of Basic Medical Sciences, Second Military Medical University, Shanghai (China); Xiong, J. [Department of Histology and Embryology, Faculty of Basic Medical Sciences, Second Military Medical University, Shanghai (China); Lu, J. [Office of Medical Education, Training Department, Second Military Medical University, Shanghai (China); Xu, S. [Department of Histology and Embryology, Faculty of Basic Medical Sciences, Second Military Medical University, Shanghai (China); Li, Y. [State Food and Drug Administration of China,Huangdao Branch, Qingdao (China); Zhong, X.P.; Gao, G.K. [Department of Hyperbaric Medicine, No. 401 Hospital of PLA, Qingdao (China); Liu, H.Q. [2Department of Histology and Embryology, Faculty of Basic Medical Sciences, Second Military Medical University, Shanghai (China)

2012-06-22

The distal cytoplasmic motifs of leukemia inhibitory factor receptor α-chain (LIFRα-CT3) can independently induce intracellular myeloid differentiation in acute myeloid leukemia (AML) cells by gene transfection; however, there are significant limitations in the potential clinical use of these motifs due to liposome-derived genetic modifications. To produce a potentially therapeutic LIFRα-CT3 with cell-permeable activity, we constructed a eukaryotic expression pcDNA3.0-TAT-CT3-cMyc plasmid with a signal peptide (ss) inserted into the N-terminal that codes for an ss-TAT-CT3-cMyc fusion protein. The stable transfection of Chinese hamster ovary (CHO) cells via this vector and subsequent selection by Geneticin resulted in cell lines that express and secrete TAT-CT3-cMyc. The spent medium of pcDNA3.0-TAT-CT3-cMyc-transfected CHO cells could be purified using a cMyc-epitope-tag agarose affinity chromatography column and could be detected via SDS-PAGE, with antibodies against cMyc-tag. The direct administration of TAT-CT3-cMyc to HL-60 cell culture media caused the enrichment of CT3-cMyc in the cytoplasm and nucleus within 30 min and led to a significant reduction of viable cells (P < 0.05) 8 h after exposure. The advantages of using this mammalian expression system include the ease of generating TAT fusion proteins that are adequately transcripted and the potential for a sustained production of such proteins in vitro for future AML therapy.

Cytoplasm localization of aminopeptidase M1 and its functional activity in root hair cells and BY-2 cells.

Science.gov (United States)

Lee, Ok Ran; Cho, Hyung-Taeg

2012-12-01

Aminopeptidase M1 (APM1) was the first M1 metallopeptidase family member identified in Arabidopsis, isolated by its affinity for the auxin transport inhibitor N-1-naphthylphthalamic acid (NPA). A loss-of-function mutation showed various developmental defects in cell division and auxin transport. APM1 was shown to be localized in endomembrane structures, the cytoplasm, and the plasma membrane. These previous results suggested that APM1 has diverse functional roles in different cell and tissue types. Here we report that APM1 localized to the cytoplasm, and its over-expression in the root hair cell caused longer root hair phenotypes. Treatment of aminopeptidase inhibitors caused internalization of auxin efflux PIN-FORMED proteins in root hair cells and suppressed short root hair phenotype of PIN3 overexpression line (PIN3ox). APM1 also localized to the cytoplasm in tobacco BY-2 cells, its over-expression had little effect on auxin transport in these cells.

Extensive proteomic screening identifies the obesity-related NYGGF4 protein as a novel LRP1-interactor, showing reduced expression in early Alzheimer's disease

Directory of Open Access Journals (Sweden)

Taddei Kevin

2010-01-01

Full Text Available Abstract Background The low-density lipoprotein receptor related protein 1 (LRP1 has been implicated in Alzheimer's disease (AD but its signalling has not been fully evaluated. There is good evidence that the cytoplasmic domain of LRP1 is involved in protein-protein interactions, important in the cell biology of LRP1. Results We carried out three yeast two-hybrid screens to identify proteins that interact with the cytoplasmic domain of LRP1. The screens included both conventional screens as well as a novel, split-ubiquitin-based screen in which an LRP1 construct was expressed and screened as a transmembrane protein. The split-ubiquitin screen was validated in a screen using full-length amyloid protein precursor (APP, which successfully identified FE65 and FE65L2, as well as novel interactors (Rab3a, Napg, and ubiquitin b. Using both a conventional screen as well as the split-ubiquitin screen, we identified NYGGF4 as a novel LRP1 interactor. The interaction between LRP1 and NYGGF4 was validated using two-hybrid assays, coprecipitation and colocalization in mammalian cells. Mutation analysis demonstrated a specific interaction of NYGGF4 with an NPXY motif that required an intact tyrosine residue. Interestingly, while we confirmed that other LRP1 interactors we identified, including JIP1B and EB-1, were also able to bind to APP, NYGGF4 was unique in that it showed specific binding with LRP1. Expression of NYGGF4 decreased significantly in patients with AD as compared to age-matched controls, and showed decreasing expression with AD disease progression. Examination of Nyggf4 expression in mice with different alleles of the human APOE4 gene showed significant differences in Nyggf4 expression. Conclusions These results implicate NYGGF4 as a novel and specific interactor of LRP1. Decreased expression of LRP1 and NYGGF4 over disease, evident with the presence of even moderate numbers of neuritic plaques, suggests that LRP1-NYGGF4 is a system altered

Pleckstrin Homology Domain Diffusion in Dictyostelium Cytoplasm Studied Using Fluorescence Correlation Spectroscopy

NARCIS (Netherlands)

Engel, Ruchira; Hink, Mark A.; Bosgraaf, Leonard; Haastert, Peter J.M. van; Visser, Antonie J.W.G.

2004-01-01

The translocation of pleckstrin homology (PH) domain-containing proteins from the cytoplasm to the plasma membrane plays an important role in the chemotaxis mechanism of Dictyostelium cells. The diffusion of three PH domain-green fluorescent protein (GFP) fusions (PH2-GFP, PH10-GFP, and PH-CRAC

Pleckstrin homology domain diffusion in Dictyostelium cytoplasm studied using fluorescence correlation spectroscopy

NARCIS (Netherlands)

Ruchira, A.; Hink, M.A.; Bosgraaf, L.; Haastert, van P.J.M.; Visser, A.J.W.G.

2004-01-01

The translocation of pleckstrin homology (PH) domain-containing proteins from the cytoplasm to the plasma membrane plays an important role in the chemotaxis mechanism of Dictyostelium cells. The diffusion of three PH domain-green fluorescent protein (GFP) fusions (PH2-GFP, PH10-GFP, and PH-CRAC

Identifying Key Attributes for Protein Beverages.

Science.gov (United States)

Oltman, A E; Lopetcharat, K; Bastian, E; Drake, M A

2015-06-01

This study identified key attributes of protein beverages and evaluated effects of priming on liking of protein beverages. An adaptive choice-based conjoint study was conducted along with Kano analysis to gain insight on protein beverage consumers (n = 432). Attributes evaluated included label claim, protein type, amount of protein, carbohydrates, sweeteners, and metabolic benefits. Utility scores for levels and importance scores for attributes were determined. Subsequently, two pairs of clear acidic whey protein beverages were manufactured that differed by age of protein source or the amount of whey protein per serving. Beverages were evaluated by 151 consumers on two occasions with or without priming statements. One priming statement declared "great flavor," the other priming statement declared 20 g protein per serving. A two way analysis of variance was applied to discern the role of each priming statement. The most important attribute for protein beverages was sweetener type, followed by amount of protein, followed by type of protein followed by label claim. Beverages with whey protein, naturally sweetened, reduced sugar and ≥15 g protein per serving were most desired. Three consumer clusters were identified, differentiated by their preferences for protein type, sweetener and amount of protein. Priming statements positively impacted concept liking (P 0.05). Consistent with trained panel profiles of increased cardboard flavor with higher protein content, consumers liked beverages with 10 g protein more than beverages with 20 g protein (6.8 compared with 5.7, P appeal. © 2015 Institute of Food Technologists®

Nonsecreted cytoplasmic alpha-fetoprotein: a newly discovered role in intracellular signaling and regulation. An update and commentary.

Science.gov (United States)

Mizejewski, G J

2015-12-01

The concept of a non-secreted cytoplasmic-bound form of alpha-fetoprotein is not a new notion in AFP biological activities. Cytoplasmic AFP (CyAFP) is a long known but forgotten protein in search of a function other than a histochemical biomarker. In this report, CyAFP is presented as an "old" protein with a newly described intracellular function. In 1976, CyAFP was shown to be a product of hepatoma cells utilizing 14Cleucine incorporation and demonstrated by autoradiographic procedures. The synthesis of CyAFP without secretion was demonstrated to occur in both malignant and non-malignant cells encompassing hepatomas, ascite fluid cells, immature rodent uterus, MCF-7 breast cancers, and cytosols from human breast cancer patients. Using computer protein matching and alignments in AFP versus members of the nuclear receptor superfamily, a consecutive series of leucine zipper (heptad) repeats in AFP was previously reported, suggesting the possibility for protein-to-protein interactions. The potential for heptad heterodimerization between protein-binding partners provided the rationale for proposing that CyAFP might have the capability to form molecular hetero-complexes with cytoplasmic based transcription factors. More recent investigations have now provided experimental evidence that CyAFP is capable of colocalizing and interacting with transcription-associated factors. Such proteins can modulate intracellular signaling leading to regulation of transcription factors and initiation of growth in human cancer cells. Although circulating serum AFP is known as a growth-enhancing factor during development, cytoplasmic AFP has a lethal role in the oncogenesis, growth, and metastasis of adult liver cancer.

Cytoplasmic tail domain of glycoprotein B is essential for HHV-6 infection

Energy Technology Data Exchange (ETDEWEB)

Mahmoud, Nora F. [Division of Clinical Virology, Center for Infectious Diseases, Kobe University Graduate School of Medicine, Kobe (Japan); Faculty of Pharmacy, Suez Canal University, Ismailia (Egypt); Jasirwan, Chyntia [Division of Clinical Virology, Center for Infectious Diseases, Kobe University Graduate School of Medicine, Kobe (Japan); Division of Hepatobiliary, Department of Internal Medicine, Faculty of Medicine, University of Indonesia (Indonesia); Kanemoto, Satoshi; Wakata, Aika; Wang, Bochao; Hata, Yuuki [Division of Clinical Virology, Center for Infectious Diseases, Kobe University Graduate School of Medicine, Kobe (Japan); Nagamata, Satoshi [Division of Clinical Virology, Center for Infectious Diseases, Kobe University Graduate School of Medicine, Kobe (Japan); Department of Obstetrics and Gynecology, Kobe University Graduate School of Medicine, Kobe (Japan); Kawabata, Akiko [Division of Clinical Virology, Center for Infectious Diseases, Kobe University Graduate School of Medicine, Kobe (Japan); Tang, Huamin [Division of Clinical Virology, Center for Infectious Diseases, Kobe University Graduate School of Medicine, Kobe (Japan); Department of Immunology, Nanjing Medical University, Nanjing (China); Mori, Yasuko, E-mail: ymori@med.kobe-u.ac.jp [Division of Clinical Virology, Center for Infectious Diseases, Kobe University Graduate School of Medicine, Kobe (Japan)

2016-03-15

Human herpesvirus 6 (HHV-6) glycoprotein B (gB) is an abundantly expressed viral glycoprotein required for viral entry and cell fusion, and is highly conserved among herpesviruses. The present study examined the function of HHV-6 gB cytoplasmic tail domain (CTD). A gB CTD deletion mutant was constructed which, in contrast to its revertant, could not be reconstituted. Moreover, deletion of gB cytoplasmic tail impaired the intracellular transport of gB protein to the trans-Golgi network (TGN). Taken together, these results suggest that gB CTD is critical for HHV-6 propagation and important for intracellular transportation. - Highlights: • Glycoprotein B (gB) is highly conserved among herpesviruses. • HHV-6 gB is also abundantly expressed in virions. • In the present study, we showed the function of HHV-6 gB cytoplasmic tail domain (CTD). • We found that deletion of gB CTD impairs the intracellular transport of gB protein to the trans-Golgi network (TGN), and CTD of gB is critical for HHV-6 propagation.

Cytoplasmic tail domain of glycoprotein B is essential for HHV-6 infection

International Nuclear Information System (INIS)

Mahmoud, Nora F.; Jasirwan, Chyntia; Kanemoto, Satoshi; Wakata, Aika; Wang, Bochao; Hata, Yuuki; Nagamata, Satoshi; Kawabata, Akiko; Tang, Huamin; Mori, Yasuko

2016-01-01

Human herpesvirus 6 (HHV-6) glycoprotein B (gB) is an abundantly expressed viral glycoprotein required for viral entry and cell fusion, and is highly conserved among herpesviruses. The present study examined the function of HHV-6 gB cytoplasmic tail domain (CTD). A gB CTD deletion mutant was constructed which, in contrast to its revertant, could not be reconstituted. Moreover, deletion of gB cytoplasmic tail impaired the intracellular transport of gB protein to the trans-Golgi network (TGN). Taken together, these results suggest that gB CTD is critical for HHV-6 propagation and important for intracellular transportation. - Highlights: • Glycoprotein B (gB) is highly conserved among herpesviruses. • HHV-6 gB is also abundantly expressed in virions. • In the present study, we showed the function of HHV-6 gB cytoplasmic tail domain (CTD). • We found that deletion of gB CTD impairs the intracellular transport of gB protein to the trans-Golgi network (TGN), and CTD of gB is critical for HHV-6 propagation.

Monoacylated Cellular Prion Proteins Reduce Amyloid-β-Induced Activation of Cytoplasmic Phospholipase A2 and Synapse Damage

Directory of Open Access Journals (Sweden)

Ewan West

2015-06-01

Full Text Available Alzheimer’s disease (AD is a progressive neurodegenerative disease characterized by the accumulation of amyloid-β (Aβ and the loss of synapses. Aggregation of the cellular prion protein (PrPC by Aβ oligomers induced synapse damage in cultured neurons. PrPC is attached to membranes via a glycosylphosphatidylinositol (GPI anchor, the composition of which affects protein targeting and cell signaling. Monoacylated PrPC incorporated into neurons bound “natural Aβ”, sequestering Aβ outside lipid rafts and preventing its accumulation at synapses. The presence of monoacylated PrPC reduced the Aβ-induced activation of cytoplasmic phospholipase A2 (cPLA2 and Aβ-induced synapse damage. This protective effect was stimulus specific, as treated neurons remained sensitive to α-synuclein, a protein associated with synapse damage in Parkinson’s disease. In synaptosomes, the aggregation of PrPC by Aβ oligomers triggered the formation of a signaling complex containing the cPLA2.a process, disrupted by monoacylated PrPC. We propose that monoacylated PrPC acts as a molecular sponge, binding Aβ oligomers at the neuronal perikarya without activating cPLA2 or triggering synapse damage.

Nucleoporin Nup98 mediates galectin-3 nuclear-cytoplasmic trafficking

Energy Technology Data Exchange (ETDEWEB)

Funasaka, Tatsuyoshi, E-mail: funasaka@staff.kanazawa-u.ac.jp [Laboratory of Molecular and Cellular Biology, Department of Biology, Faculty of Natural Systems, Institute of Science and Engineering, Kanazawa University, Ishikawa (Japan); Balan, Vitaly; Raz, Avraham [Department of Oncology and Pathology, Karmanos Cancer Institute, Wayne State University, School of Medicine, Detroit, MI (United States); Wong, Richard W., E-mail: rwong@staff.kanazawa-u.ac.jp [Laboratory of Molecular and Cellular Biology, Department of Biology, Faculty of Natural Systems, Institute of Science and Engineering, Kanazawa University, Ishikawa (Japan); Bio-AFM Frontier Research Center, Kanazawa Kanazawa University, Ishikawa (Japan)

2013-04-26

Highlights: •Nuclear pore protein Nup98 is a novel binding partner of galectin-3. •Nup98 transports galectin-3 into cytoplasm. •Nup98 depletion leads to galectin-3 nuclear transport and induces growth retardation. •Nup98 may involve in ß-catenin pathway through interaction with galectin-3. -- Abstract: Nucleoporin Nup98 is a component of the nuclear pore complex, and is important in transport across the nuclear pore. Many studies implicate nucleoporin in cancer progression, but no direct mechanistic studies of its effect in cancer have been reported. We show here that Nup98 specifically regulates nucleus–cytoplasm transport of galectin-3, which is a ß-galactoside-binding protein that affects adhesion, migration, and cancer progression, and controls cell growth through the ß-catenin signaling pathway in cancer cells. Nup98 interacted with galectin-3 on the nuclear membrane, and promoted galectin-3 cytoplasmic translocation whereas other nucleoporins did not show these functions. Inversely, silencing of Nup98 expression by siRNA technique localized galectin-3 to the nucleus and retarded cell growth, which was rescued by Nup98 transfection. In addition, Nup98 RNA interference significantly suppressed downstream mRNA expression in the ß-catenin pathway, such as cyclin D1 and FRA-1, while nuclear galectin-3 binds to ß-catenin to inhibit transcriptional activity. Reduced expression of ß-catenin target genes is consistent with the Nup98 reduction and the galectin-3–nucleus translocation rate. Overall, the results show Nup98’s involvement in nuclear–cytoplasm translocation of galectin-3 and ß-catenin signaling pathway in regulating cell proliferation, and the results depicted here suggest a novel therapeutic target/modality for cancers.

Brain-specific interaction of a 91-kDa membrane-bound protein with the cytoplasmic tail of the 300-kDa mannose 6-phosphate receptor

DEFF Research Database (Denmark)

Rosorius, O; Issinger, O G; Braulke, T

1996-01-01

The cytoplasmic tail of the 300 kDa mannose 6-phosphate receptor (MPR 300-CT) is thought to play an important role in sorting and targeting of lysosomal enzymes and the insulin-like growth factor II along the biosynthetic and endocytic pathway. In this study a brain specific 91 kDa protein and a ...... in neuronal cells....

Multiple-Localization and Hub Proteins

Science.gov (United States)

Ota, Motonori; Gonja, Hideki; Koike, Ryotaro; Fukuchi, Satoshi

2016-01-01

Protein-protein interactions are fundamental for all biological phenomena, and protein-protein interaction networks provide a global view of the interactions. The hub proteins, with many interaction partners, play vital roles in the networks. We investigated the subcellular localizations of proteins in the human network, and found that the ones localized in multiple subcellular compartments, especially the nucleus/cytoplasm proteins (NCP), the cytoplasm/cell membrane proteins (CMP), and the nucleus/cytoplasm/cell membrane proteins (NCMP), tend to be hubs. Examinations of keywords suggested that among NCP, those related to post-translational modifications and transcription functions are the major contributors to the large number of interactions. These types of proteins are characterized by a multi-domain architecture and intrinsic disorder. A survey of the typical hub proteins with prominent numbers of interaction partners in the type revealed that most are either transcription factors or co-regulators involved in signaling pathways. They translocate from the cytoplasm to the nucleus, triggered by the phosphorylation and/or ubiquitination of intrinsically disordered regions. Among CMP and NCMP, the contributors to the numerous interactions are related to either kinase or ubiquitin ligase activity. Many of them reside on the cytoplasmic side of the cell membrane, and act as the upstream regulators of signaling pathways. Overall, these hub proteins function to transfer external signals to the nucleus, through the cell membrane and the cytoplasm. Our analysis suggests that multiple-localization is a crucial concept to characterize groups of hub proteins and their biological functions in cellular information processing. PMID:27285823

Cytoplasmic translocation of polypyrimidine tract-binding protein and its binding to viral RNA during Japanese encephalitis virus infection inhibits virus replication.

Directory of Open Access Journals (Sweden)

Deepika Bhullar

Full Text Available Japanese encephalitis virus (JEV has a single-stranded, positive-sense RNA genome containing a single open reading frame flanked by the 5'- and 3'-non-coding regions (NCRs. The virus genome replicates via a negative-sense RNA intermediate. The NCRs and their complementary sequences in the negative-sense RNA are the sites for assembly of the RNA replicase complex thereby regulating the RNA synthesis and virus replication. In this study, we show that the 55-kDa polypyrimidine tract-binding protein (PTB interacts in vitro with both the 5'-NCR of the positive-sense genomic RNA--5NCR(+, and its complementary sequence in the negative-sense replication intermediate RNA--3NCR(-. The interaction of viral RNA with PTB was validated in infected cells by JEV RNA co-immunoprecipitation and JEV RNA-PTB colocalization experiments. Interestingly, we observed phosphorylation-coupled translocation of nuclear PTB to cytoplasmic foci that co-localized with JEV RNA early during JEV infection. Our studies employing the PTB silencing and over-expression in cultured cells established an inhibitory role of PTB in JEV replication. Using RNA-protein binding assay we show that PTB competitively inhibits association of JEV 3NCR(- RNA with viral RNA-dependent RNA polymerase (NS5 protein, an event required for the synthesis of the plus-sense genomic RNA. cAMP is known to promote the Protein kinase A (PKA-mediated PTB phosphorylation. We show that cells treated with a cAMP analogue had an enhanced level of phosphorylated PTB in the cytoplasm and a significantly suppressed JEV replication. Data presented here show a novel, cAMP-induced, PTB-mediated, innate host response that could effectively suppress JEV replication in mammalian cells.

mRNA decay proteins are targeted to poly(A+ RNA and dsRNA-containing cytoplasmic foci that resemble P-bodies in Entamoeba histolytica.

Directory of Open Access Journals (Sweden)

Itzel López-Rosas

Full Text Available In higher eukaryotes, mRNA degradation and RNA-based gene silencing occur in cytoplasmic foci referred to as processing bodies (P-bodies. In protozoan parasites, the presence of P-bodies and their putative role in mRNA decay have yet to be comprehensively addressed. Identification of P-bodies might provide information on how mRNA degradation machineries evolved in lower eukaryotes. Here, we used immunofluorescence and confocal microscopy assays to investigate the cellular localization of mRNA degradation proteins in the human intestinal parasite Entamoeba histolytica and found evidence of the existence of P-bodies. Two mRNA decay factors, namely the EhXRN2 exoribonuclease and the EhDCP2 decapping enzyme, were localized in cytoplasmic foci in a pattern resembling P-body organization. Given that amoebic foci appear to be smaller and less rounded than those described in higher eukaryotes, we have named them "P-body-like structures". These foci contain additional mRNA degradation factors, including the EhCAF1 deadenylase and the EhAGO2-2 protein involved in RNA interference. Biochemical analysis revealed that EhCAF1 co-immunoprecipitated with EhXRN2 but not with EhDCP2 or EhAGO2-2, thus linking deadenylation to 5'-to-3' mRNA decay. The number of EhCAF1-containing foci significantly decreased after inhibition of transcription and translation with actinomycin D and cycloheximide, respectively. Furthermore, results of RNA-FISH assays showed that (i EhCAF1 colocalized with poly(A(+ RNA and (ii during silencing of the Ehpc4 gene by RNA interference, EhAGO2-2 colocalized with small interfering RNAs in cytoplasmic foci. Our observation of decapping, deadenylation and RNA interference proteins within P-body-like foci suggests that these structures have been conserved after originating in the early evolution of eukaryotic lineages. To the best of our knowledge, this is the first study to report on the localization of mRNA decay proteins within P

Quantitative proteomics identifies Gemin5, a scaffolding protein involved in ribonucleoprotein assembly, as a novel partner for eukaryotic initiation factor 4E

DEFF Research Database (Denmark)

Fierro-Monti, Ivo; Mohammed, Shabaz; Matthiesen, Rune

2006-01-01

Protein complexes are dynamic entities; identification and quantitation of their components is critical in elucidating functional roles under specific cellular conditions. We report the first quantitative proteomic analysis of the human cap-binding protein complex. Components and proteins......-starved tumorigenic human mesenchymal stromal cells, attested to their activated translational states. The WD-repeat, scaffolding-protein Gemin5 was identified as a novel eIF4E binding partner, which interacted directly with eIF4E through a motif (YXXXXLPhi) present in a number of eIF4E-interacting partners. Elevated...... levels of Gemin5:eIF4E complexes were found in phorbol ester treated HEK293 cells. Gemin5 and eIF4E co-localized to cytoplasmic P-bodies in human osteosarcoma U2OS cells. Interaction between eIF4E and Gemin5 and their co-localization to the P-bodies, may serve to recruit capped mRNAs to these RNP...

Cytoplasmic Skp2 expression is associated with p-Akt1 and predicts poor prognosis in human breast carcinomas.

Directory of Open Access Journals (Sweden)

Jing Liu

Full Text Available BACKGROUND: S-phase kinase protein 2 (Skp2, an oncogenic protein, is a key regulator in different cellular and molecular processes, through ubiquitin-proteasome degradation pathway. Increased levels of Skp2 are observed in various types of cancer and associated with poor prognosis. However, in human breast carcinomas, the underlying mechanism and prognostic significance of cytoplasmic Skp2 is still undefined. METHODS: To investigate the role of cytoplasmic Skp2 expression in human breast carcinomas, we immnohistochemically assessed cytoplasmic Skp2, p-Akt1, and p27 expression in 251 patients with invasive ductal carcinomas of the breast. Association of cytoplasmic Skp2 expression with p-Akt1 and p27 was analyzed as well as correspondence with other clinicopathological parameters. Disease-free survival and overall survival were determined based on the Kaplan-Meier method and Cox regression models. RESULTS: Cytoplasmic of Skp2 was detected in 165 out of 251 (65.7% patients. Cytoplasmic Skp2 expression was associated with larger tumor size, more advanced histological grade, and positive HER2 expression. Increased cytoplasmic Skp2 expression correlated with p-Akt1 expression, with 54.2% (51/94 of low p-Akt1-expressing breast carcinomas, but 72.6% (114/157 of high p-Akt1-expressing breast carcinomas exhibiting cytoplasmic Skp2 expression. Elevated cytoplasmic Skp2 expression with low p-Akt1 expression was associated with poor disease-free and overall survival (DFS and OS, and Cox regression models demonstrated that cytoplasmic Skp2 expression was an independent prognostic marker for invasive breast carcinomas. CONCLUSION: Cytoplasmic Skp2 expression is associated with aggressive prognostic factors, such as larger tumor size, and advanced histological grade of the breast cancers. Results demonstrate that combined cytoplasmic Skp2 and p-Akt1 expression may be prognostic for patients with invasive breast carcinomas, and cytoplasmic Skp2 may serve as a

Simultaneous cytoplasmic and nuclear protein expression of melanoma antigen-A family and NY-ESO-1 cancer-testis antigens represents an independent marker for poor survival in head and neck cancer.

Science.gov (United States)

Laban, Simon; Atanackovic, Djordje; Luetkens, Tim; Knecht, Rainald; Busch, Chia-Jung; Freytag, Marcus; Spagnoli, Giulio; Ritter, Gerd; Hoffmann, Thomas K; Knuth, Alexander; Sauter, Guido; Wilczak, Waldemar; Blessmann, Marco; Borgmann, Kerstin; Muenscher, Adrian; Clauditz, Till S

2014-09-01

The prognosis of head and neck squamous cell carcinoma (HNSCC) patients remains poor. The identification of high-risk subgroups is needed for the development of custom-tailored therapies. The expression of cancer-testis antigens (CTAs) has been linked to a worse prognosis in other cancer types; however, their prognostic value in HNSCC is unclear because only few patients have been examined and data on CTA protein expression are sparse. A tissue microarray consisting of tumor samples from 453 HNSCC patients was evaluated for the expression of CTA proteins using immunohistochemistry. Frequency of expression and the subcellular expression pattern (nuclear, cytoplasmic, or both) was recorded. Protein expression of melanoma antigen (MAGE)-A family CTA, MAGE-C family CTA and NY-ESO-1 was found in approximately 30, 7 and 4% of tumors, respectively. The subcellular expression pattern in particular had a marked impact on the patients' prognosis. Median overall survival (OS) of patients with (i) simultaneous cytoplasmic and nuclear expression compared to (ii) either cytoplasmic or nuclear expression and (iii) negative patients was 23.0 versus 109.0 versus 102.5 months, for pan-MAGE (p family members or NY-ESO-1 represent a subgroup with an extraordinarily poor survival. The development of immunotherapeutic strategies targeting these CTA may, therefore, be a promising approach to improve the outcome of HNSCC patients. © 2014 UICC.

Mechanisms for cytoplasmic organization: an overview.

Science.gov (United States)

Pagliaro, L

2000-01-01

One of the basic characteristics of life is the intrinsic organization of cytoplasm, yet we know surprisingly little about the manner in which cytoplasmic macromolecules are arranged. It is clear that cytoplasm is not the homogeneous "soup" it was once envisioned to be, but a comprehensive model for cytoplasmic organization is not available in modern cell biology. The premise of this volume is that phase separation in cytoplasm may play a role in organization at the subcellular level. Other mechanisms for non-membrane-bounded intracellular organization have previously been proposed. Some of these will be reviewed in this chapter. Multiple mechanisms, involving phase separation, specific intracellular targeting, formation of macromolecular complexes, and channeling, all could well contribute to cytoplasmic organization. Temporal and spatial organization, as well as composition, are likely to be important in defining the characteristics of cytoplasm.

Replacement of the cytoplasmic domain alters sorting of a viral glycoprotein in polarized cells.

OpenAIRE

Puddington, L; Woodgett, C; Rose, J K

1987-01-01

The envelope glycoprotein (G protein) of vesicular stomatitis virus (VSV) is transported to the basolateral plasma membrane of polarized epithelial cells, whereas the hemagglutinin glycoprotein (HA protein) of influenza virus is transported to the apical plasma membrane. To determine if the cytoplasmic domain of VSV G protein might be important in directing G protein to the basolateral membrane, we derived polarized Madin-Darby canine kidney cell lines expressing G protein or G protein with i...

pH-Responsive Micelle-Based Cytoplasmic Delivery System for Induction of Cellular Immunity

Directory of Open Access Journals (Sweden)

Eiji Yuba

2017-11-01

Full Text Available (1 Background: Cytoplasmic delivery of antigens is crucial for the induction of cellular immunity, which is an important immune response for the treatment of cancer and infectious diseases. To date, fusogenic protein-incorporated liposomes and pH-responsive polymer-modified liposomes have been used to achieve cytoplasmic delivery of antigen via membrane rupture or fusion with endosomes. However, a more versatile cytoplasmic delivery system is desired for practical use. For this study, we developed pH-responsive micelles composed of dilauroyl phosphatidylcholine (DLPC and deoxycholic acid and investigated their cytoplasmic delivery performance and immunity-inducing capability. (2 Methods: Interaction of micelles with fluorescence dye-loaded liposomes, intracellular distribution of micelles, and antigenic proteins were observed. Finally, antigen-specific cellular immune response was evaluated in vivo using ELIspot assay. (3 Results: Micelles induced leakage of contents from liposomes via lipid mixing at low pH. Micelles were taken up by dendritic cells mainly via macropinocytosis and delivered ovalbumin (OVA into the cytosol. After intradermal injection of micelles and OVA, OVA-specific cellular immunity was induced in the spleen. (4 Conclusions: pH-responsive micelles composed of DLPC and deoxycholic acid are promising as enhancers of cytosol delivery of antigens and the induction capability of cellular immunity for the treatment of cancer immunotherapy and infectious diseases.

Cytoplasmic Male Sterility of Rice with Boro II Cytoplasm Is Caused by a Cytotoxic Peptide and Is Restored by Two Related PPR Motif Genes via Distinct Modes of mRNA Silencing[W

Science.gov (United States)

Wang, Zhonghua; Zou, Yanjiao; Li, Xiaoyu; Zhang, Qunyu; Chen, Letian; Wu, Hao; Su, Dihua; Chen, Yuanling; Guo, Jingxin; Luo, Da; Long, Yunming; Zhong, Yang; Liu, Yao-Guang

2006-01-01

Cytoplasmic male sterility (CMS) and nucleus-controlled fertility restoration are widespread plant reproductive features that provide useful tools to exploit heterosis in crops. However, the molecular mechanism underlying this kind of cytoplasmic–nuclear interaction remains unclear. Here, we show in rice (Oryza sativa) with Boro II cytoplasm that an abnormal mitochondrial open reading frame, orf79, is cotranscribed with a duplicated atp6 (B-atp6) gene and encodes a cytotoxic peptide. Expression of orf79 in CMS lines and transgenic rice plants caused gametophytic male sterility. Immunoblot analysis showed that the ORF79 protein accumulates specifically in microspores. Two fertility restorer genes, Rf1a and Rf1b, were identified at the classical locus Rf-1 as members of a multigene cluster that encode pentatricopeptide repeat proteins. RF1A and RF1B are both targeted to mitochondria and can restore male fertility by blocking ORF79 production via endonucleolytic cleavage (RF1A) or degradation (RF1B) of dicistronic B-atp6/orf79 mRNA. In the presence of both restorers, RF1A was epistatic over RF1B in the mRNA processing. We have also shown that RF1A plays an additional role in promoting the editing of atp6 mRNAs, independent of its cleavage function. PMID:16489123

Plant cytoplasmic GAPDH: redox post-translational modifications and moonlighting properties

Directory of Open Access Journals (Sweden)

Mirko eZaffagnini

2013-11-01

Full Text Available Glyceraldehyde-3-phosphate dehydrogenase (GAPDH is a ubiquitous enzyme involved in glycolysis and shown, particularly in animal cells, to play additional roles in several unrelated non-metabolic processes such as control of gene expression and apoptosis. This functional versatility is regulated, in part at least, by redox post-translational modifications that alter GAPDH catalytic activity and influence the subcellular localization of the enzyme. In spite of the well established moonlighting (multifunctional properties of animal GAPDH, little is known about non-metabolic roles of GAPDH in plants. Plant cells contain several GAPDH isoforms with different catalytic and regulatory properties, located both in the cytoplasm and in plastids, and participating in glycolysis and the Calvin-Benson cycle. A general feature of all GAPDH proteins is the presence of an acidic catalytic cysteine in the active site that is overly sensitive to oxidative modifications, including glutathionylation and S-nitrosylation. In Arabidopsis, oxidatively-modified cytoplasmic GAPDH has been successfully used as a tool to investigate the role of reduced glutathione, thioredoxins and glutaredoxins in the control of different types of redox post-translational modifications. Oxidative modifications inhibit GAPDH activity, but might enable additional functions in plant cells. Mounting evidence support the concept that plant cytoplasmic GAPDH may fulfill alternative, non-metabolic functions that are triggered by redox post-translational modifications of the protein under stress conditions. The aim of this review is to detail the molecular mechanisms underlying the redox regulation of plant cytoplasmic GAPDH in the light of its crystal structure, and to provide a brief inventory of the well known redox-dependent multi-facetted properties of animal GAPDH, together with the emerging roles of oxidatively-modified GAPDH in stress signaling pathways in plants.

Nuclear pore complex protein mediated nuclear localization of dicer protein in human cells.

Directory of Open Access Journals (Sweden)

Yoshinari Ando

Full Text Available Human DICER1 protein cleaves double-stranded RNA into small sizes, a crucial step in production of single-stranded RNAs which are mediating factors of cytoplasmic RNA interference. Here, we clearly demonstrate that human DICER1 protein localizes not only to the cytoplasm but also to the nucleoplasm. We also find that human DICER1 protein associates with the NUP153 protein, one component of the nuclear pore complex. This association is detected predominantly in the cytoplasm but is also clearly distinguishable at the nuclear periphery. Additional characterization of the NUP153-DICER1 association suggests NUP153 plays a crucial role in the nuclear localization of the DICER1 protein.

Protein 4.1 and its interaction with other cytoskeletal proteins in Xenopus laevis oogenesis.

Science.gov (United States)

Carotenuto, Rosa; Petrucci, Tamara C; Correas, Isabel; Vaccaro, Maria C; De Marco, Nadia; Dale, Brian; Wilding, Martin

2009-06-01

In human red blood cells, protein 4.1 (4.1R) is an 80-kDa polypeptide that stabilizes the spectrin-actin network and anchors it to the plasma membrane. In non-erythroid cells there is a great variety of 4.1R isoforms, mainly generated by alternative pre-mRNA splicing, which localize at various intracellular sites, including the nucleus. We studied protein 4.1R distribution in relation to beta-spectrin, actin and cytokeratin during Xenopus oogenesis. Immunoprecipitation experiments indicate that at least two isoforms of protein 4.1R are present in Xenopus laevis oocytes: a 56-kDa form in the cytoplasm and a 37-kDa form in the germinal vesicle (GV). Antibodies to beta-spectrin reveal two bands of 239 and 100 kDa in the cytoplasm. Coimmunoprecipitation experiments indicate that both the 37- and 56-kDa isoforms of protein 4.1R associate with the 100-kDa isoform of beta-spectrin. Moreover, the 56-kDa form coimmunoprecipitates with a cytokeratin of the same molecular weight. Confocal immunolocalization shows that protein 4.1R distribution is in the peripheral cytoplasm, in the mitochondrial cloud (MC) and in the GV of previtellogenic oocytes. In the cytoplasm of vitellogenic oocytes, a loose network of fibers stained by the anti-protein 4.1R antibody spreads across the cytoplasm. beta-Spectrin has a similar distribution. Protein 4.1R was found to colocalize with actin in the cortex of oocytes in the form of fluorescent dots. Double immunolocalization of protein 4.1R and cytokeratin depicts two separate networks that overlap throughout the whole cytoplasm. Protein 4.1R filaments partially colocalize with cytokeratin in both the animal and vegetal hemispheres. We hypothesize that protein 4.1R could function as a linker protein between cytokeratin and the actin-based cytoskeleton.

Yeast Interacting Proteins Database: YOL006C, YMR233W [Yeast Interacting Proteins Database

Lifescience Database Archive (English)

Full Text Available fusion protein localizes to the cytoplasm, nucleus and nucleolus Rows with this prey as prey (1) Rows with t...on protein localizes to the cytoplasm, nucleus and nucleolus Rows with this prey

High Expression of Cry1Ac Protein in Cotton (Gossypium hirsutum by Combining Independent Transgenic Events that Target the Protein to Cytoplasm and Plastids.

Directory of Open Access Journals (Sweden)

Amarjeet Kumar Singh

Full Text Available Transgenic cotton was developed using two constructs containing a truncated and codon-modified cry1Ac gene (1,848 bp, which was originally characterized from Bacillus thuringiensis subspecies kurstaki strain HD73 that encodes a toxin highly effective against many lepidopteran pests. In Construct I, the cry1Ac gene was cloned under FMVde, a strong constitutively expressing promoter, to express the encoded protein in the cytoplasm. In Construct II, the encoded protein was directed to the plastids using a transit peptide taken from the cotton rbcSIb gene. Genetic transformation experiments with Construct I resulted in a single copy insertion event in which the Cry1Ac protein expression level was 2-2.5 times greater than in the Bacillus thuringiensis cotton event Mon 531, which is currently used in varieties and hybrids grown extensively in India and elsewhere. Another high expression event was selected from transgenics developed with Construct II. The Cry protein expression resulting from this event was observed only in the green plant parts. No transgenic protein expression was observed in the non-green parts, including roots, seeds and non-green floral tissues. Thus, leucoplasts may lack the mechanism to allow entry of a protein tagged with the transit peptide from a protein that is only synthesized in tissues containing mature plastids. Combining the two events through sexual crossing led to near additive levels of the toxin at 4-5 times the level currently used in the field. The two high expression events and their combination will allow for effective resistance management against lepidopteran insect pests, particularly Helicoverpa armigera, using a high dosage strategy.

Accessory factors of cytoplasmic viral RNA sensors required for antiviral innate immune response

Directory of Open Access Journals (Sweden)

Hiroyuki eOshiumi

2016-05-01

Full Text Available Type I interferon (IFN induces many antiviral factors in host cells. RIG-I-like receptors (RLRs are cytoplasmic viral RNA sensors that trigger the signal to induce the innate immune response that includes type I IFN production. RIG-I and MDA5 are RLRs that form nucleoprotein filaments along viral double-stranded RNA, resulting in the activation of MAVS adaptor molecule. The MAVS protein forms a prion-like aggregation structure, leading to type I IFN production. RIG-I and MDA5 undergo post-translational modification. TRIM25 and Riplet ubiquitin ligases deliver a K63-linked polyubiquitin moiety to the RIG-I N-terminal caspase activation and recruitment domains (CARDs and C-terminal region; the polyubiquitin chain then stabilizes the two-CARD tetramer structure required for MAVS assembly. MDA5 activation is regulated by phosphorylation. RIOK3 is a protein kinase that phosphorylates the MDA5 protein in a steady state, and PP1α/γ dephosphorylate this protein, resulting in its activation. RIG-I and MDA5 require cytoplasmic RNA helicases for their efficient activation. LGP2, another RLR, is an RNA helicase involved in RLR signaling. This protein does not possess N-terminal CARDs and thus cannot trigger downstream signaling by itself. Recent studies have revealed that this protein modulates MDA5 filament formation, resulting in enhanced type I IFN production. Several other cytoplasmic RNA helicases are involved in RLR signaling. DDX3, DHX29, DHX36, and DDX60 RNA helicases have been reported to be involved in RLR-mediated type I IFN production after viral infection. However, the underlying mechanism is largely unknown. Future studies are required to reveal the role of RNA helicases in the RLR signaling pathway.

Identification of a functional, CRM-1-dependent nuclear export signal in hepatitis C virus core protein.

Directory of Open Access Journals (Sweden)

Andrea Cerutti

Full Text Available Hepatitis C virus (HCV infection is a major cause of chronic liver disease worldwide. HCV core protein is involved in nucleocapsid formation, but it also interacts with multiple cytoplasmic and nuclear molecules and plays a crucial role in the development of liver disease and hepatocarcinogenesis. The core protein is found mostly in the cytoplasm during HCV infection, but also in the nucleus in patients with hepatocarcinoma and in core-transgenic mice. HCV core contains nuclear localization signals (NLS, but no nuclear export signal (NES has yet been identified.We show here that the aa(109-133 region directs the translocation of core from the nucleus to the cytoplasm by the CRM-1-mediated nuclear export pathway. Mutagenesis of the three hydrophobic residues (L119, I123 and L126 in the identified NES or in the sequence encoding the mature core aa(1-173 significantly enhanced the nuclear localisation of the corresponding proteins in transfected Huh7 cells. Both the NES and the adjacent hydrophobic sequence in domain II of core were required to maintain the core protein or its fragments in the cytoplasmic compartment. Electron microscopy studies of the JFH1 replication model demonstrated that core was translocated into the nucleus a few minutes after the virus entered the cell. The blockade of nucleocytoplasmic export by leptomycin B treatment early in infection led to the detection of core protein in the nucleus by confocal microscopy and coincided with a decrease in virus replication.Our data suggest that the functional NLS and NES direct HCV core protein shuttling between the cytoplasmic and nuclear compartments, with at least some core protein transported to the nucleus. These new properties of HCV core may be essential for virus multiplication and interaction with nuclear molecules, influence cell signaling and the pathogenesis of HCV infection.

Identification of a functional, CRM-1-dependent nuclear export signal in hepatitis C virus core protein.

Science.gov (United States)

Cerutti, Andrea; Maillard, Patrick; Minisini, Rosalba; Vidalain, Pierre-Olivier; Roohvand, Farzin; Pecheur, Eve-Isabelle; Pirisi, Mario; Budkowska, Agata

2011-01-01

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease worldwide. HCV core protein is involved in nucleocapsid formation, but it also interacts with multiple cytoplasmic and nuclear molecules and plays a crucial role in the development of liver disease and hepatocarcinogenesis. The core protein is found mostly in the cytoplasm during HCV infection, but also in the nucleus in patients with hepatocarcinoma and in core-transgenic mice. HCV core contains nuclear localization signals (NLS), but no nuclear export signal (NES) has yet been identified.We show here that the aa(109-133) region directs the translocation of core from the nucleus to the cytoplasm by the CRM-1-mediated nuclear export pathway. Mutagenesis of the three hydrophobic residues (L119, I123 and L126) in the identified NES or in the sequence encoding the mature core aa(1-173) significantly enhanced the nuclear localisation of the corresponding proteins in transfected Huh7 cells. Both the NES and the adjacent hydrophobic sequence in domain II of core were required to maintain the core protein or its fragments in the cytoplasmic compartment. Electron microscopy studies of the JFH1 replication model demonstrated that core was translocated into the nucleus a few minutes after the virus entered the cell. The blockade of nucleocytoplasmic export by leptomycin B treatment early in infection led to the detection of core protein in the nucleus by confocal microscopy and coincided with a decrease in virus replication.Our data suggest that the functional NLS and NES direct HCV core protein shuttling between the cytoplasmic and nuclear compartments, with at least some core protein transported to the nucleus. These new properties of HCV core may be essential for virus multiplication and interaction with nuclear molecules, influence cell signaling and the pathogenesis of HCV infection.

Transcriptome analysis of cytoplasmic male sterility and restoration in CMS-D8 cotton.

Science.gov (United States)

Suzuki, Hideaki; Rodriguez-Uribe, Laura; Xu, Jiannong; Zhang, Jinfa

2013-10-01

A global view of differential expression of genes in CMS-D8 of cotton was presented in this study which will facilitate the understanding of cytoplasmic male sterility in cotton. Cytoplasmic male sterility (CMS) is a maternally inherited trait in higher plants which is incapable of producing functional pollen. However, the male fertility can be restored by one or more nuclear-encoded restorer genes. A genome-wide transcriptome analysis of CMS and restoration in cotton is currently lacking. In this study, Affymetrix GeneChips© Cotton Genome Array containing 24,132 transcripts was used to compare differentially expressed (DE) genes of flower buds at the meiosis stage between CMS and its restorer cotton plants conditioned by the D8 cytoplasm. A total of 458 (1.9 %) of DE genes including 127 up-regulated and 331 down-regulated ones were identified in the CMS-D8 line. Quantitative RT-PCR was used to validate 10 DE genes selected from seven functional categories. The most frequent DE gene group was found to encode putative proteins involved in cell wall expansion, such as pectinesterase, pectate lyase, pectin methylesterase, glyoxal oxidase, polygalacturonase, indole-3-acetic acid-amino synthetase, and xyloglucan endo-transglycosylase. Genes in cytoskeleton category including actin, which plays a key role in cell wall expansion, cell elongation and cell division, were also highly differentially expressed between the fertile and CMS plants. This work represents the first study in utilizing microarray to identify CMS-related genes by comparing overall DE genes between fertile and CMS plants in cotton. The results provide evidence that many CMS-associated genes are mainly involved in cell wall expansion. Further analysis will be required to elucidate the molecular mechanisms of male sterility which will facilitate the development of new hybrid cultivars in cotton.

Antibody-mediated enzyme replacement therapy targeting both lysosomal and cytoplasmic glycogen in Pompe disease.

Science.gov (United States)

Yi, Haiqing; Sun, Tao; Armstrong, Dustin; Borneman, Scott; Yang, Chunyu; Austin, Stephanie; Kishnani, Priya S; Sun, Baodong

2017-05-01

Pompe disease is characterized by accumulation of both lysosomal and cytoplasmic glycogen primarily in skeletal and cardiac muscles. Mannose-6-phosphate receptor-mediated enzyme replacement therapy (ERT) with recombinant human acid α-glucosidase (rhGAA) targets the enzyme to lysosomes and thus is unable to digest cytoplasmic glycogen. Studies have shown that anti-DNA antibody 3E10 penetrates living cells and delivers "cargo" proteins to the cytosol or nucleus via equilibrative nucleoside transporter ENT2. We speculate that 3E10-mediated ERT with GAA will target both lysosomal and cytoplasmic glycogen in Pompe disease. A fusion protein (FabGAA) containing a humanized Fab fragment derived from the murine 3E10 antibody and the 110 kDa human GAA precursor was constructed and produced in CHO cells. Immunostaining with an anti-Fab antibody revealed that the Fab signals did not co-localize with the lysosomal marker LAMP2 in cultured L6 myoblasts or Pompe patient fibroblasts after incubation with FabGAA. Western blot with an anti-GAA antibody showed presence of the 150 kDa full-length FabGAA in the cell lysates, in addition to the 95- and 76 kDa processed forms of GAA that were also seen in the rhGAA-treated cells. Blocking of mannose-6-phosphate receptor with mannose-6-phosphate markedly reduced the 95- and the 76 kDa forms but not the 150 kDa form. In GAA-KO mice, FabGAA achieved similar treatment efficacy as rhGAA at an equal molar dose in reducing tissue glycogen contents. Our data suggest that FabGAA retains the ability of rhGAA to treat lysosomal glycogen accumulation and has the beneficial potential over rhGAA to reduce cytoplasmic glycogen storage in Pompe disease. FabGAA can be delivered to both the cytoplasm and lysosomes in cultured cells. FabGAA equally reduced lysosomal glycogen accumulation as rhGAA in GAA-KO mice. FabGAA has the beneficial potential over rhGAA to clear cytoplasmic glycogen. This study suggests a novel antibody-enzyme fusion protein therapy

NRIP is newly identified as a Z-disc protein, activating calmodulin signaling for skeletal muscle contraction and regeneration.

Science.gov (United States)

Chen, Hsin-Hsiung; Chen, Wen-Pin; Yan, Wan-Lun; Huang, Yuan-Chun; Chang, Szu-Wei; Fu, Wen-Mei; Su, Ming-Jai; Yu, I-Shing; Tsai, Tzung-Chieh; Yan, Yu-Ting; Tsao, Yeou-Ping; Chen, Show-Li

2015-11-15

Nuclear receptor interaction protein (NRIP, also known as DCAF6 and IQWD1) is a Ca(2+)-dependent calmodulin-binding protein. In this study, we newly identify NRIP as a Z-disc protein in skeletal muscle. NRIP-knockout mice were generated and found to have reduced muscle strength, susceptibility to fatigue and impaired adaptive exercise performance. The mechanisms of NRIP-regulated muscle contraction depend on NRIP being downstream of Ca(2+) signaling, where it stimulates activation of both 'calcineurin-nuclear factor of activated T-cells, cytoplasmic 1' (CaN-NFATc1; also known as NFATC1) and calmodulin-dependent protein kinase II (CaMKII) through interaction with calmodulin (CaM), resulting in the induction of mitochondrial activity and the expression of genes encoding the slow class of myosin, and in the regulation of Ca(2+) homeostasis through the internal Ca(2+) stores of the sarcoplasmic reticulum. Moreover, NRIP-knockout mice have a delayed regenerative capacity. The amount of NRIP can be enhanced after muscle injury and is responsible for muscle regeneration, which is associated with the increased expression of myogenin, desmin and embryonic myosin heavy chain during myogenesis, as well as for myotube formation. In conclusion, NRIP is a novel Z-disc protein that is important for skeletal muscle strength and regenerative capacity. © 2015. Published by The Company of Biologists Ltd.

Integral and peripheral association of proteins and protein complexes with Yersinia pestis inner and outer membranes

Directory of Open Access Journals (Sweden)

Bunai Christine L

2009-02-01

Full Text Available Abstract Yersinia pestis proteins were sequentially extracted from crude membranes with a high salt buffer (2.5 M NaBr, an alkaline solution (180 mM Na2CO3, pH 11.3 and membrane denaturants (8 M urea, 2 M thiourea and 1% amidosulfobetaine-14. Separation of proteins by 2D gel electrophoresis was followed by identification of more than 600 gene products by MS. Data from differential 2D gel display experiments, comparing protein abundances in cytoplasmic, periplasmic and all three membrane fractions, were used to assign proteins found in the membrane fractions to three protein categories: (i integral membrane proteins and peripheral membrane proteins with low solubility in aqueous solutions (220 entries; (ii peripheral membrane proteins with moderate to high solubility in aqueous solutions (127 entries; (iii cytoplasmic or ribosomal membrane-contaminating proteins (80 entries. Thirty-one proteins were experimentally associated with the outer membrane (OM. Circa 50 proteins thought to be part of membrane-localized, multi-subunit complexes were identified in high Mr fractions of membrane extracts via size exclusion chromatography. This data supported biologically meaningful assignments of many proteins to the membrane periphery. Since only 32 inner membrane (IM proteins with two or more predicted transmembrane domains (TMDs were profiled in 2D gels, we resorted to a proteomic analysis by 2D-LC-MS/MS. Ninety-four additional IM proteins with two or more TMDs were identified. The total number of proteins associated with Y. pestis membranes increased to 456 and included representatives of all six β-barrel OM protein families and 25 distinct IM transporter families.

A role for complexes of survival of motor neurons (SMN) protein with gemins and profilin in neurite-like cytoplasmic extensions of cultured nerve cells

International Nuclear Information System (INIS)

Sharma, Aarti; Lambrechts, Anja; Le thi Hao; Le, Thanh T.; Sewry, Caroline A.; Ampe, Christophe; Burghes, Arthur H.M.; Morris, Glenn E.

2005-01-01

Spinal muscular atrophy (SMA) is caused by reduced levels of SMN (survival of motor neurons protein) and consequent loss of motor neurons. SMN is involved in snRNP transport and nuclear RNA splicing, but axonal transport of SMN has also been shown to occur in motor neurons. SMN also binds to the small actin-binding protein, profilin. We now show that SMN and profilin II co-localise in the cytoplasm of differentiating rat PC12 cells and in neurite-like extensions, especially at their growth cones. Many components of known SMN complexes were also found in these extensions, including gemin2 (SIP-1), gemin6, gemin7 and unrip (unr-interacting protein). Coilin p80 and Sm core protein immunoreactivity, however, were seen only in the nucleus. SMN is known to associate with β-actin mRNA and specific hnRNPs in axons and in neurite extensions of cultured nerve cells, and SMN also stimulates neurite outgrowth in cultures. Our results are therefore consistent with SMN complexes, rather than SMN alone, being involved in the transport of actin mRNPs along the axon as in the transport of snRNPs into the nucleus by similar SMN complexes. Antisense knockdown of profilin I and II isoforms inhibited neurite outgrowth of PC12 cells and caused accumulation of SMN and its associated proteins in cytoplasmic aggregates. BIAcore studies demonstrated a high affinity interaction of SMN with profilin IIa, the isoform present in developing neurons. Pathogenic missense mutations in SMN, or deletion of exons 5 and 7, prevented this interaction. The interaction is functional in that SMN can modulate actin polymerisation in vitro by reducing the inhibitory effect of profilin IIa. This suggests that reduced SMN in SMA might cause axonal pathfinding defects by disturbing the normal regulation of microfilament growth by profilins

A Proteomics Approach to Identify New Putative Cardiac Intercalated Disk Proteins.

Directory of Open Access Journals (Sweden)

Siddarth Soni

Full Text Available Synchronous beating of the heart is dependent on the efficient functioning of the cardiac intercalated disk (ID. The ID is composed of a complex protein network enabling electrical continuity and chemical communication between individual cardiomyocytes. Recently, several different studies have shed light on increasingly prevalent cardiac diseases involving the ID. Insufficient knowledge of its composition makes it difficult to study these disease mechanisms in more detail and therefore here we aim expand the ID proteome. Here, using a combination of general membrane enrichment, in-depth quantitative proteomics and an intracellular location driven bioinformatics approach, we aim to discover new putative ID proteins in rat ventricular tissue.General membrane isolation, enriched amongst others also with ID proteins as based on presence of the established markers connexin-43 and n-cadherin, was performed using centrifugation. By mass spectrometry, we quantitatively evaluated the level of 3455 proteins in the enriched membrane fraction (EMF and its counterpart, the soluble cytoplasmic fraction. These data were stringently filtered to generate a final set of 97 enriched, putative ID proteins. These included Cx43 and n-cadherin, but also many interesting novel candidates. We selected 4 candidates (Flotillin-2 (FLOT2, Nexilin (NEXN, Popeye-domain-containg-protein 2 (POPDC2 and thioredoxin-related-transmembrane-protein 2 (TMX2 and confirmed their co-localization with n-cadherin in the ID of human and rat heart cryo-sections, and isolated dog cardiomyocytes.The presented proteomics dataset of putative new ID proteins is a valuable resource for future research into this important molecular intersection of the heart.

Histone titration against the genome sets the DNA-to-cytoplasm threshold for the Xenopus midblastula transition

Science.gov (United States)

Amodeo, Amanda A.; Jukam, David; Straight, Aaron F.; Skotheim, Jan M.

2015-01-01

During early development, animal embryos depend on maternally deposited RNA until zygotic genes become transcriptionally active. Before this maternal-to-zygotic transition, many species execute rapid and synchronous cell divisions without growth phases or cell cycle checkpoints. The coordinated onset of transcription, cell cycle lengthening, and cell cycle checkpoints comprise the midblastula transition (MBT). A long-standing model in the frog, Xenopus laevis, posits that MBT timing is controlled by a maternally loaded inhibitory factor that is titrated against the exponentially increasing amount of DNA. To identify MBT regulators, we developed an assay using Xenopus egg extract that recapitulates the activation of transcription only above the DNA-to-cytoplasm ratio found in embryos at the MBT. We used this system to biochemically purify factors responsible for inhibiting transcription below the threshold DNA-to-cytoplasm ratio. This unbiased approach identified histones H3 and H4 as concentration-dependent inhibitory factors. Addition or depletion of H3/H4 from the extract quantitatively shifted the amount of DNA required for transcriptional activation in vitro. Moreover, reduction of H3 protein in embryos induced premature transcriptional activation and cell cycle lengthening, and the addition of H3/H4 shortened post-MBT cell cycles. Our observations support a model for MBT regulation by DNA-based titration and suggest that depletion of free histones regulates the MBT. More broadly, our work shows how a constant concentration DNA binding molecule can effectively measure the amount of cytoplasm per genome to coordinate division, growth, and development. PMID:25713373

Ubiquitination of MBNL1 Is Required for Its Cytoplasmic Localization and Function in Promoting Neurite Outgrowth

Directory of Open Access Journals (Sweden)

Pei-Ying Wang

2018-02-01

Full Text Available The Muscleblind-like protein family (MBNL plays an important role in regulating the transition between differentiation and pluripotency and in the pathogenesis of myotonic dystrophy type 1 (DM1, a CTG expansion disorder. How different MBNL isoforms contribute to the differentiation and are affected in DM1 has not been investigated. Here, we show that the MBNL1 cytoplasmic, but not nuclear, isoform promotes neurite morphogenesis and reverses the morphological defects caused by expanded CUG RNA. Cytoplasmic MBNL1 is polyubiquitinated by lysine 63 (K63. Reduced cytoplasmic MBNL1 in the DM1 mouse brain is consistent with the reduced extent of K63 ubiquitination. Expanded CUG RNA induced the deubiqutination of cytoplasmic MBNL1, which resulted in nuclear translocation and morphological impairment that could be ameliorated by inhibiting K63-linked polyubiquitin chain degradation. Our results suggest that K63-linked ubiquitination of MBNL1 is required for its cytoplasmic localization and that deubiquitination of cytoplasmic MBNL1 is pathogenic in the DM1 brain.

Evidence for catabolite degradation in the glucose-dependent inactivation of yeast cytoplasmic malate dehydrogenase

International Nuclear Information System (INIS)

Neeff, J.; Haegele, E.; Nauhaus, J.; Heer, U.; Mecke, D.

1978-01-01

The cytoplasmic malate dehydrogenase of Saccharomyces cerevisiae was radioactively labeled during its synthesis on a glucose-free derepression medium. After purification a sensitive radioimmunoassay for this enzyme could be developed. The assay showed that after the physiological, glucose-dependent 'catabolite inactivation' of cytoplasmic malate dehydrogenase an inactive enzyme protein is immunologically not detectable. Together with the irreversibility of this reaction in vivo this finding strongly suggests a proteolytic mechanism of enzyme inactivation. For this process the term 'catabolite degradation' is used. (orig.) [de

Biofilm-specific extracellular matrix proteins of non-typeable Haemophilus influenzae

Science.gov (United States)

Wu, Siva; Baum, Marc M.; Kerwin, James; Guerrero-Given, Debbie; Webster, Simon; Schaudinn, Christoph; VanderVelde, David; Webster, Paul

2014-01-01

Non-typeable Haemophilus influenzae (NTHi), a human respiratory tract pathogen can form colony biofilms in vitro. Bacterial cells and the amorphous extracellular matrix (ECM) constituting the biofilm can be separated using sonication. The ECM from 24 hr and 96 hr NTHi biofilms contained polysaccharides and proteinaceous components as detected by NMR and FTIR spectroscopy. More conventional chemical assays on the biofilm ECM confirmed the presence of these components and also DNA. Proteomics revealed eighteen proteins present in biofilm ECM that were not detected in planktonic bacteria. One ECM protein was unique to 24 hr biofilms, two were found only in 96 hr biofilms, and fifteen were present in the ECM of both 24 hr and 96 hr NTHi biofilms. All proteins identified were either associated with bacterial membranes or were cytoplasmic proteins. Immunocytochemistry showed two of the identified proteins, a DNA-directed RNA polymerase and the outer membrane protein OMP P2, associated with bacteria and biofilm ECM. Identification of biofilm-specific proteins present in immature biofilms is an important step in understanding the in vitro process of NTHi biofilm formation. The presence of a cytoplasmic protein and a membrane protein in the biofilm ECM of immature NTHi biofilms suggests that bacterial cell lysis may be a feature of early biofilm formation. PMID:24942343

Human B cells fail to secrete type I interferons upon cytoplasmic DNA exposure.

Science.gov (United States)

Gram, Anna M; Sun, Chenglong; Landman, Sanne L; Oosenbrug, Timo; Koppejan, Hester J; Kwakkenbos, Mark J; Hoeben, Rob C; Paludan, Søren R; Ressing, Maaike E

2017-11-01

Most cells are believed to be capable of producing type I interferons (IFN I) as part of an innate immune response against, for instance, viral infections. In macrophages, IFN I is potently induced upon cytoplasmic exposure to foreign nucleic acids. Infection of these cells with herpesviruses leads to triggering of the DNA sensors interferon-inducible protein 16 (IFI16) and cyclic GMP-AMP (cGAMP) synthase (cGAS). Thereby, the stimulator of interferon genes (STING) and the downstream molecules TANK-binding kinase 1 (TBK1) and interferon regulatory factor 3 (IRF3) are sequentially activated culminating in IFN I secretion. Human gamma-herpesviruses, such as Epstein-Barr virus (EBV), exploit B cells as a reservoir for persistent infection. In this study, we investigated whether human B cells, similar to macrophages, engage the cytoplasmic DNA sensing pathway to induce an innate immune response. We found that the B cells fail to secrete IFN I upon cytoplasmic DNA exposure, although they express the DNA sensors cGAS and IFI16 and the signaling components TBK1 and IRF3. In primary human B lymphocytes and EBV-negative B cell lines, this deficiency is explained by a lack of detectable levels of the central adaptor protein STING. In contrast, EBV-transformed B cell lines did express STING, yet both these lines as well as STING-reconstituted EBV-negative B cells did not produce IFN I upon dsDNA or cGAMP stimulation. Our combined data show that the cytoplasmic DNA sensing pathway is dysfunctional in human B cells. This exemplifies that certain cell types cannot induce IFN I in response to cytoplasmic DNA exposure providing a potential niche for viral persistence. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Crystallographic characterization of the radixin FERM domain bound to the cytoplasmic tail of membrane-type 1 matrix metalloproteinase (MT1-MMP)

International Nuclear Information System (INIS)

Terawaki, Shin-ichi; Kitano, Ken; Aoyama, Miki; Hakoshima, Toshio

2008-01-01

The radixin FERM domain was shown to bind the MT1-MMP cytoplasmic peptide and crystals of the complex were obtained. ERM proteins play a role in the cross-linking found between plasma membranes and actin filaments. The N-terminal FERM domains of ERM proteins are responsible for membrane association through direct interaction with the cytoplasmic tails of integral membrane proteins. During cell migration and movement, membrane-type 1 matrix metalloproteinase (MT1-MMP) on plasma membranes sheds adhesion molecule CD44 in addition to degrading the extracellular matrix. Here, the interaction between the radixin FERM domain and the MT1-MMP cytoplasmic tail is reported and preliminary crystallographic characterization of crystals of the radixin FERM domain bound to the cytoplasmic tail of MT1-MMP is presented. The crystals belong to space group P6 1 22, with unit-cell parameters a = b = 122.7, c = 128.3 Å, and contain one complex in the crystallographic asymmetric unit. The diffraction data were collected to a resolution of 2.4 Å

Mena/VASP and αII-Spectrin complexes regulate cytoplasmic actin networks in cardiomyocytes and protect from conduction abnormalities and dilated cardiomyopathy.

Science.gov (United States)

Benz, Peter M; Merkel, Carla J; Offner, Kristin; Abeßer, Marco; Ullrich, Melanie; Fischer, Tobias; Bayer, Barbara; Wagner, Helga; Gambaryan, Stepan; Ursitti, Jeanine A; Adham, Ibrahim M; Linke, Wolfgang A; Feller, Stephan M; Fleming, Ingrid; Renné, Thomas; Frantz, Stefan; Unger, Andreas; Schuh, Kai

2013-08-12

In the heart, cytoplasmic actin networks are thought to have important roles in mechanical support, myofibrillogenesis, and ion channel function. However, subcellular localization of cytoplasmic actin isoforms and proteins involved in the modulation of the cytoplasmic actin networks are elusive. Mena and VASP are important regulators of actin dynamics. Due to the lethal phenotype of mice with combined deficiency in Mena and VASP, however, distinct cardiac roles of the proteins remain speculative. In the present study, we analyzed the physiological functions of Mena and VASP in the heart and also investigated the role of the proteins in the organization of cytoplasmic actin networks. We generated a mouse model, which simultaneously lacks Mena and VASP in the heart. Mena/VASP double-deficiency induced dilated cardiomyopathy and conduction abnormalities. In wild-type mice, Mena and VASP specifically interacted with a distinct αII-Spectrin splice variant (SH3i), which is in cardiomyocytes exclusively localized at Z- and intercalated discs. At Z- and intercalated discs, Mena and β-actin localized to the edges of the sarcomeres, where the thin filaments are anchored. In Mena/VASP double-deficient mice, β-actin networks were disrupted and the integrity of Z- and intercalated discs was markedly impaired. Together, our data suggest that Mena, VASP, and αII-Spectrin assemble cardiac multi-protein complexes, which regulate cytoplasmic actin networks. Conversely, Mena/VASP deficiency results in disrupted β-actin assembly, Z- and intercalated disc malformation, and induces dilated cardiomyopathy and conduction abnormalities.

Cytoplasmic Z-RNA

International Nuclear Information System (INIS)

Zarling, D.A.; Calhoun, C.J.; Hardin, C.C.; Zarling, A.H.

1987-01-01

Specific immunochemical probes for Z-RNA were generated and characterized to search for possible Z-RNA-like double helices in cells. Z-RNA was detected in the cytoplasm of fixed protozoan cells by immunofluorescence microscopy using these anti-Z-RNA IgCs. In contrast, autoimmune or experimentally elicited anti-DNA antibodies, specifically reactive with B-DNA or Z-DNA, stained the nuclei. Pre-or nonimmune IgGs did not bind to the cells. RNase A or T1 digestion eliminated anti-Z-RNA IgG binding to cytoplasmic determinants; however, DNase I or mung bean nuclease had no effect. Doxorubicin and ethidium bromide prevented anti-Z-RNA antibody binding; however, actinomycin D, which does not bind double-stranded RNA, did not. Anti-Z-RNA immunofluorescence was specifically blocked in competition assays by synthetic Z-RNA but not Z-DNA, A-RNA, or single-stranded RNAs. Thus, some cytoplasmic sequences in fixed cells exist in the left-handed Z-RNA conformation

AtTZF gene family localizes to cytoplasmic foci

OpenAIRE

Pomeranz, Marcelo; Lin, Pei-Chi; Finer, John; Jang, Jyan-Chyun

2010-01-01

In eukaryotes, mRNA turnover and translational repression represent important regulatory steps in gene expression. Curiously, when under cellular stresses, factors involved in these processes aggregate into cytoplasmic foci known as Processing bodies (P-bodies) and Stress Granules (SGs). In animals, CCCH Tandem Zinc Finger (TZF) proteins play important roles in mRNA decay within P-bodies. TTP, a P-body localized mammalian TZF, can bind to the 3'UTRs of mRNAs containing AU-rich elements (AREs)...

Cell cycle-dependent microtubule-based dynamic transport of cytoplasmic dynein in mammalian cells.

Directory of Open Access Journals (Sweden)

Takuya Kobayashi

Full Text Available BACKGROUND: Cytoplasmic dynein complex is a large multi-subunit microtubule (MT-associated molecular motor involved in various cellular functions including organelle positioning, vesicle transport and cell division. However, regulatory mechanism of the cell-cycle dependent distribution of dynein has not fully been understood. METHODOLOGY/PRINCIPAL FINDINGS: Here we report live-cell imaging of cytoplasmic dynein in HeLa cells, by expressing multifunctional green fluorescent protein (mfGFP-tagged 74-kDa intermediate chain (IC74. IC74-mfGFP was successfully incorporated into functional dynein complex. In interphase, dynein moved bi-directionally along with MTs, which might carry cargos such as transport vesicles. A substantial fraction of dynein moved toward cell periphery together with EB1, a member of MT plus end-tracking proteins (+TIPs, suggesting +TIPs-mediated transport of dynein. In late-interphase and prophase, dynein was localized at the centrosomes and the radial MT array. In prometaphase and metaphase, dynein was localized at spindle MTs where it frequently moved from spindle poles toward chromosomes or cell cortex. +TIPs may be involved in the transport of spindle dyneins. Possible kinetochore and cortical dyneins were also observed. CONCLUSIONS AND SIGNIFICANCE: These findings suggest that cytoplasmic dynein is transported to the site of action in preparation for the following cellular events, primarily by the MT-based transport. The MT-based transport may have greater advantage than simple diffusion of soluble dynein in rapid and efficient transport of the limited concentration of the protein.

Cytoplasmic- and extracellular-proteome analysis of Diplodia seriata: a phytopathogenic fungus involved in grapevine decline

Directory of Open Access Journals (Sweden)

Cobos Rebeca

2010-09-01

Full Text Available Abstract Background The phytopathogenic fungus Diplodia seriata, whose genome remains unsequenced, produces severe infections in fruit trees (fruit blight and grapevines. In this crop is recognized as one of the most prominent pathogens involved in grapevine trunk disease (or grapevine decline. This pathology can result in the death of adult plants and therefore it produces severe economical losses all around the world. To date no genes or proteins have been characterized in D. seriata that are involved in the pathogenicity process. In an effort to help identify potential gene products associated with pathogenicity and to gain a better understanding of the biology of D. seriata, we initiated a proteome-level study of the fungal mycelia and secretome. Results Intracellular and secreted proteins from D. seriata collected from liquid cultures were separated using two-dimensional gel electrophoresis. About 550 cytoplasmic proteins were reproducibly present in 3 independent extractions, being 53 identified by peptide mass fingerprinting and tandem mass spectrometry. The secretome analysis showed 75 secreted proteins reproducibly present in 3 biological replicates, being 16 identified. Several of the proteins had been previously identified as virulence factors in other fungal strains, although their contribution to pathogenicity in D. seriata remained to be analyzed. When D. seriata was grown in a medium supplemented with carboxymethylcellulose, 3 proteins were up-regulated and 30 down-regulated. Within the up-regulated proteins, two were identified as alcohol dehydrogenase and mitochondrial peroxyrredoxin-1, suggesting that they could play a significant role in the pathogenicity process. As for the 30 down-regulated proteins, 9 were identified being several of them involved in carbohydrate metabolism. Conclusions This study is the first report on proteomics on D. seriata. The proteomic data obtained will be important to understand the pathogenicity

Macrophage fusion is controlled by the cytoplasmic protein tyrosine phosphatase PTP-PEST/PTPN12.

Science.gov (United States)

Rhee, Inmoo; Davidson, Dominique; Souza, Cleiton Martins; Vacher, Jean; Veillette, André

2013-06-01

Macrophages can undergo cell-cell fusion, leading to the formation of multinucleated giant cells and osteoclasts. This process is believed to promote the proteolytic activity of macrophages toward pathogens, foreign bodies, and extracellular matrices. Here, we examined the role of PTP-PEST (PTPN12), a cytoplasmic protein tyrosine phosphatase, in macrophage fusion. Using a macrophage-targeted PTP-PEST-deficient mouse, we determined that PTP-PEST was not needed for macrophage differentiation or cytokine production. However, it was necessary for interleukin-4-induced macrophage fusion into multinucleated giant cells in vitro. It was also needed for macrophage fusion following implantation of a foreign body in vivo. Moreover, in the RAW264.7 macrophage cell line, PTP-PEST was required for receptor activator of nuclear factor kappa-B ligand (RANKL)-triggered macrophage fusion into osteoclasts. PTP-PEST had no impact on expression of fusion mediators such as β-integrins, E-cadherin, and CD47, which enable macrophages to become fusion competent. However, it was needed for polarization of macrophages, migration induced by the chemokine CC chemokine ligand 2 (CCL2), and integrin-induced spreading, three key events in the fusion process. PTP-PEST deficiency resulted in specific hyperphosphorylation of the protein tyrosine kinase Pyk2 and the adaptor paxillin. Moreover, a fusion defect was induced upon treatment of normal macrophages with a Pyk2 inhibitor. Together, these data argue that macrophage fusion is critically dependent on PTP-PEST. This function is seemingly due to the ability of PTP-PEST to control phosphorylation of Pyk2 and paxillin, thereby regulating cell polarization, migration, and spreading.

Detection of Antibodies to Brucella Cytoplasmic Proteins in the Cerebrospinal Fluid of Patients with Neurobrucellosis

Science.gov (United States)

Baldi, Pablo C.; Araj, George F.; Racaro, Graciela C.; Wallach, Jorge C.; Fossati, Carlos A.

1999-01-01

The diagnosis of human neurobrucellosis usually relies on the detection of antibodies to Brucella lipopolysaccharide (LPS) in cerebrospinal fluid (CSF) by agglutination tests or enzyme-linked immunosorbent assay (ELISA). Here we describe the detection of immunoglobulin G (IgG) to cytoplasmic proteins (CP) of Brucella spp. by ELISA and Western blotting in seven CSF samples from five patients with neurobrucellosis. While IgG to CP (titers of 200 to 12,800) and IgG to LPS (800 to 6,400) were found in the CSF of these patients, these antibodies were not detected in CSF samples from two patients who had systemic brucellosis without neurological involvement. The latter, however, had serum IgG and IgM to both LPS and CP. No reactivity to these antigens was found in CSF samples from 14 and 20 patients suffering from nonbrucellar meningitis and noninfectious diseases, respectively. These findings suggest that, in addition to its usefulness in the serological diagnosis of human systemic brucellosis, the ELISA with CP antigen can be used for the specific diagnosis of human neurobrucellosis. PMID:10473531

Human cytoplasmic copper chaperones Atox1 and CCS exchange copper ions in vitro.

Science.gov (United States)

Petzoldt, Svenja; Kahra, Dana; Kovermann, Michael; Dingeldein, Artur P G; Niemiec, Moritz S; Ådén, Jörgen; Wittung-Stafshede, Pernilla

2015-06-01

After Ctr1-mediated copper ion (Cu) entry into the human cytoplasm, chaperones Atox1 and CCS deliver Cu to P1B-type ATPases and to superoxide dismutase, respectively, via direct protein-protein interactions. Although the two Cu chaperones are presumed to work along independent pathways, we here assessed cross-reactivity between Atox1 and the first domain of CCS (CCS1) using biochemical and biophysical methods in vitro. By NMR we show that CCS1 is monomeric although it elutes differently from Atox1 in size exclusion chromatography (SEC). This property allows separation of Atox1 and CCS1 by SEC and, combined with the 254/280 nm ratio as an indicator of Cu loading, we demonstrate that Cu can be transferred from one protein to the other. Cu exchange also occurs with full-length CCS and, as expected, the interaction involves the metal binding sites since mutation of Cu-binding cysteine in Atox1 eliminates Cu transfer from CCS1. Cross-reactivity between CCS and Atox1 may aid in regulation of Cu distribution in the cytoplasm.

Interaction of Proteins Identified in Human Thyroid Cells

Science.gov (United States)

Pietsch, Jessica; Riwaldt, Stefan; Bauer, Johann; Sickmann, Albert; Weber, Gerhard; Grosse, Jirka; Infanger, Manfred; Eilles, Christoph; Grimm, Daniela

2013-01-01

Influence of gravity forces on the regulation of protein expression by healthy and malignant thyroid cells was studied with the aim to identify protein interactions. Western blot analyses of a limited number of proteins suggested a time-dependent regulation of protein expression by simulated microgravity. After applying free flow isoelectric focusing and mass spectrometry to search for differently expressed proteins by thyroid cells exposed to simulated microgravity for three days, a considerable number of candidates for gravi-sensitive proteins were detected. In order to show how proteins sensitive to microgravity could directly influence other proteins, we investigated all polypeptide chains identified with Mascot scores above 100, looking for groups of interacting proteins. Hence, UniProtKB entry numbers of all detected proteins were entered into the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) and processed. The program indicated that we had detected various groups of interacting proteins in each of the three cell lines studied. The major groups of interacting proteins play a role in pathways of carbohydrate and protein metabolism, regulation of cell growth and cell membrane structuring. Analyzing these groups, networks of interaction could be established which show how a punctual influence of simulated microgravity may propagate via various members of interaction chains. PMID:23303277

Interaction of Proteins Identified in Human Thyroid Cells

Directory of Open Access Journals (Sweden)

Jessica Pietsch

2013-01-01

Full Text Available Influence of gravity forces on the regulation of protein expression by healthy and malignant thyroid cells was studied with the aim to identify protein interactions. Western blot analyses of a limited number of proteins suggested a time-dependent regulation of protein expression by simulated microgravity. After applying free flow isoelectric focusing and mass spectrometry to search for differently expressed proteins by thyroid cells exposed to simulated microgravity for three days, a considerable number of candidates for gravi-sensitive proteins were detected. In order to show how proteins sensitive to microgravity could directly influence other proteins, we investigated all polypeptide chains identified with Mascot scores above 100, looking for groups of interacting proteins. Hence, UniProtKB entry numbers of all detected proteins were entered into the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING and processed. The program indicated that we had detected various groups of interacting proteins in each of the three cell lines studied. The major groups of interacting proteins play a role in pathways of carbohydrate and protein metabolism, regulation of cell growth and cell membrane structuring. Analyzing these groups, networks of interaction could be established which show how a punctual influence of simulated microgravity may propagate via various members of interaction chains.

Molecular characterization of cytoplasmic male sterility (CMS) in perennial ryegrass ( Lolium perenne L.)

DEFF Research Database (Denmark)

Islam, Md. Shofiqul; Møller, Ian Max; Studer, Bruno

2011-01-01

to increase biomass yield, improve nutritional value and tolerance towards abiotic and biotic stress. Cytoplasmic male sterility (CMS) is an efficient tool to control pollination for hybrid seed production. In order to identify the causative polymorphism of the CMS phenotype, a cytoplasmic male sterile plant...

SitesIdentify: a protein functional site prediction tool

Directory of Open Access Journals (Sweden)

Doig Andrew J

2009-11-01

Full Text Available Abstract Background The rate of protein structures being deposited in the Protein Data Bank surpasses the capacity to experimentally characterise them and therefore computational methods to analyse these structures have become increasingly important. Identifying the region of the protein most likely to be involved in function is useful in order to gain information about its potential role. There are many available approaches to predict functional site, but many are not made available via a publicly-accessible application. Results Here we present a functional site prediction tool (SitesIdentify, based on combining sequence conservation information with geometry-based cleft identification, that is freely available via a web-server. We have shown that SitesIdentify compares favourably to other functional site prediction tools in a comparison of seven methods on a non-redundant set of 237 enzymes with annotated active sites. Conclusion SitesIdentify is able to produce comparable accuracy in predicting functional sites to its closest available counterpart, but in addition achieves improved accuracy for proteins with few characterised homologues. SitesIdentify is available via a webserver at http://www.manchester.ac.uk/bioinformatics/sitesidentify/

Wild Nicotiana Species as a Source of Cytoplasmic Male Sterility in Nicotianatabacum

Directory of Open Access Journals (Sweden)

Nikova V

2014-12-01

Full Text Available The results of our experiments executed to obtain tobacco male sterile lines through interspecific hybridization are summarized. Ten wild species from the genus Nicotiana: N. excelsior (exc, N. amplexicaulis (amp, N. rustica (rus, Nicotianaglauca (gla, N. velutina (vel, N. benthamiana (ben, N. maritima (mar, N. paniculata (pan, N. longiflora (lon and N. africana (afr were used as cytoplasmic donors and N. tabacum, cv. HarmanliiskaBasma (HB as a donor of the nucleus. Genetic effects of cytoplasmic-nuclear interaction of the studied species are discussed. Our results suggested that cytoplasmic male sterility (CMS was expressed when the cytoplasms of the above mentioned wild Nicotiana species were combined with the nucleus of N. tabacum. The 10 sources of CMS obtained in tobacco were characterized by altered flower phenotypes. Flowers are classified into types according the stamen, pistil and corolla modification. All these CMS sources were backcrossed to Oriental tobaccos, cvs. Tekne, Nevrokop B-12, Kroumovgrad 90 and Djebel 576, to develop corresponding CMS lines. The investigated cytoplasms produced compete male sterility in all those cultivars. The CMS lines preserved flower types, specific for every “sterile” cytoplasm. The extent of male organ modifications varied from apparently normal (but pollenless stamens in CMS (pan, (afr, some plants of (vel (mar through different degrees of malformations (shriveled anther on shortened filaments (lon, pinnate-like anthers on filaments of normal length (amp, petal - (ben, pistil- or stigma-like structures (rus, (gla to lack of male reproductive organs in (exc and in some plants of (vel, (mar, (rus and (gla. Most of the above mentioned cytoplasms had normal female gametophyte and good seed productivity. Alterations of the pistils were observed in CMS (rus, (exc and (ben causing reduction of the seed set. Electrophoresis of seed proteins of the tobacco cultivars and their CMS lines also suggested that

Control of nuclear-cytoplasmic shuttling of Ankrd54 by PKCδ

Institute of Scientific and Technical Information of China (English)

Amy L Samuels; Alison Louw; Reza Zareie; Evan Ingley

2017-01-01

AIM To identify and characterize the effect of phosphorylation on the subcellular localization of Ankrd54.METHODS HEK293 T cells were treated with calyculin A, staurosporin or phorbol 12-myristate 13-acetate(PMA). Cells were transfected with eG FP-tagged Ankrd54 with or without Lyn tyrosine kinase(wild-type, Y397 F mutant, or Y508 F mutant). The subcellular localization was assessed by immunofluorescence imaging of cells, immunoblotting of subcellular fractionations. The phosphorylation of Ankrd54 was monitored using Phos-tagT M gel retardation. Phosphorylated peptides were analysed by multiplereaction-monitoring(MRM) proteomic analysis.RESULTS Activation of PKC kinases using PMA promoted nuclear export of Ankrd54 and correlated with increased Ankrd54 phosphorylation, assayed using Phos-tag TM gel retardation. Co-expression of an active form of the PKCδisoform specifically promoted both phosphorylation and cytoplasmic localization of Ankrd54, while PKCδ, Akt and PKA did not. Alanine mutation of several serine residues in the amino-terminal region of Ankrd54(Ser14, Ser17, Ser18, Ser19) reduced both PMA induced cytoplasmic localization and phosphorylation of Ankrd54. Using MRM proteomic analysis, phosphorylation of the Ser18 residue of Ankrd54 was readily detectable in response to PMA stimulation. PMA stimulation of cells co-expressing Ankrd54 and Lyn tyrosine kinase displayed increased coimmunoprecipitation and enhanced co-localization in the cytoplasm.CONCLUSION We identify phosphorylation by PKCδ as a major regulator of nuclear-cytoplasmic shuttling of Ankrd54, and its interaction with the tyrosine kinase Lyn.

Localization and function of KLF4 in cytoplasm of vascular smooth muscle cell

International Nuclear Information System (INIS)

Liu, Yan; Zheng, Bin; Zhang, Xin-hua; Nie, Chan-juan; Li, Yong-hui; Wen, Jin-kun

2013-01-01

Highlights: •PDGF-BB prompts the translocation of KLF4 to the cytoplasm. •PDGF-BB promotes interaction between KLF4 and actin in the cytoplasm. •Phosphorylation and SUMOylation of KLF4 participates in regulation of cytoskeletal organization. •KLF4 regulates cytoskeleton by promoting the expression of contraction-associated genes. -- Abstract: The Krüppel-like factor 4 is a DNA-binding transcriptional regulator that regulates a diverse array of cellular processes, including development, differentiation, proliferation, and apoptosis. The previous studies about KLF4 functions mainly focused on its role as a transcription factor, its functions in the cytoplasm are still unknown. In this study, we found that PDGF-BB could prompt the translocation of KLF4 to the cytoplasm through CRM1-mediated nuclear export pathway in vascular smooth muscle cells (VSMCs) and increased the interaction of KLF4 with actin in the cytoplasm. Further study showed that both KLF4 phosphorylation and SUMOylation induced by PDGF-BB participates in regulation of cytoskeletal organization by stabilizing the actin cytoskeleton in VSMCs. In conclusion, these results identify that KLF4 participates in the cytoskeletal organization by stabilizing cytoskeleton in the cytoplasm of VSMCs

Localization and function of KLF4 in cytoplasm of vascular smooth muscle cell

Energy Technology Data Exchange (ETDEWEB)

Liu, Yan [Department of Biochemistry and Molecular Biology, The Key Laboratory of Neurobiology and Vascular Biology (China); The Third Hospital of Hebei Medical University, Shijazhuang (China); Zheng, Bin; Zhang, Xin-hua; Nie, Chan-juan; Li, Yong-hui [Department of Biochemistry and Molecular Biology, The Key Laboratory of Neurobiology and Vascular Biology (China); Wen, Jin-kun, E-mail: wjk@hebmu.edu.cn [Department of Biochemistry and Molecular Biology, The Key Laboratory of Neurobiology and Vascular Biology (China)

2013-06-28

Highlights: •PDGF-BB prompts the translocation of KLF4 to the cytoplasm. •PDGF-BB promotes interaction between KLF4 and actin in the cytoplasm. •Phosphorylation and SUMOylation of KLF4 participates in regulation of cytoskeletal organization. •KLF4 regulates cytoskeleton by promoting the expression of contraction-associated genes. -- Abstract: The Krüppel-like factor 4 is a DNA-binding transcriptional regulator that regulates a diverse array of cellular processes, including development, differentiation, proliferation, and apoptosis. The previous studies about KLF4 functions mainly focused on its role as a transcription factor, its functions in the cytoplasm are still unknown. In this study, we found that PDGF-BB could prompt the translocation of KLF4 to the cytoplasm through CRM1-mediated nuclear export pathway in vascular smooth muscle cells (VSMCs) and increased the interaction of KLF4 with actin in the cytoplasm. Further study showed that both KLF4 phosphorylation and SUMOylation induced by PDGF-BB participates in regulation of cytoskeletal organization by stabilizing the actin cytoskeleton in VSMCs. In conclusion, these results identify that KLF4 participates in the cytoskeletal organization by stabilizing cytoskeleton in the cytoplasm of VSMCs.

A lanthipeptide library used to identify a protein-protein interaction inhibitor.

Science.gov (United States)

Yang, Xiao; Lennard, Katherine R; He, Chang; Walker, Mark C; Ball, Andrew T; Doigneaux, Cyrielle; Tavassoli, Ali; van der Donk, Wilfred A

2018-04-01

In this article we describe the production and screening of a genetically encoded library of 10 6 lanthipeptides in Escherichia coli using the substrate-tolerant lanthipeptide synthetase ProcM. This plasmid-encoded library was combined with a bacterial reverse two-hybrid system for the interaction of the HIV p6 protein with the UEV domain of the human TSG101 protein, which is a critical protein-protein interaction for HIV budding from infected cells. Using this approach, we identified an inhibitor of this interaction from the lanthipeptide library, whose activity was verified in vitro and in cell-based virus-like particle-budding assays. Given the variety of lanthipeptide backbone scaffolds that may be produced with ProcM, this method may be used for the generation of genetically encoded libraries of natural product-like lanthipeptides containing substantial structural diversity. Such libraries may be combined with any cell-based assay to identify lanthipeptides with new biological activities.

Identification of proteins from tuberculin purified protein derivative (PPD) by LC-MS/MS.

Science.gov (United States)

Borsuk, Sibele; Newcombe, Jane; Mendum, Tom A; Dellagostin, Odir A; McFadden, Johnjoe

2009-11-01

The tuberculin purified protein derivative (PPD) is a widely used diagnostic antigen for tuberculosis, however it is poorly defined. Most mycobacterial proteins are extensively denatured by the procedure employed in its preparation, which explains previous difficulties in identifying constituents from PPD to characterize their behaviour in B- and T-cell reactions. We here described a proteomics-based characterization of PPD from several different sources by LC-MS/MS, which combines the solute separation power of HPLC, with the detection power of a mass spectrometer. The technique is able to identify proteins from complex mixtures of peptide fragments. A total of 171 different proteins were identified among the four PPD samples (two bovine PPD and two avium PPD) from Brazil and UK. The majority of the proteins were cytoplasmic (77.9%) and involved in intermediary metabolism and respiration (24.25%) but there was a preponderance of proteins involved in lipid metabolism. We identified a group of 21 proteins that are present in both bovine PPD but were not detected in avium PPD preparation. In addition, four proteins found in bovine PPD are absent in Mycobacterium bovis BCG vaccine strain. This study provides a better understanding of the tuberculin PPD components leading to the identification of additional antigens useful as reagents for specific diagnosis of tuberculosis.

Disruption of reducing pathways is not essential for efficient disulfide bond formation in the cytoplasm of E. coli

Directory of Open Access Journals (Sweden)

Hatahet Feras

2010-09-01

Full Text Available Abstract Background The formation of native disulfide bonds is a complex and essential post-translational modification for many proteins. The large scale production of these proteins can be difficult and depends on targeting the protein to a compartment in which disulfide bond formation naturally occurs, usually the endoplasmic reticulum of eukaryotes or the periplasm of prokaryotes. It is currently thought to be impossible to produce large amounts of disulfide bond containing protein in the cytoplasm of wild-type bacteria such as E. coli due to the presence of multiple pathways for their reduction. Results Here we show that the introduction of Erv1p, a sulfhydryl oxidase and FAD-dependent catalyst of disulfide bond formation found in the inter membrane space of mitochondria, allows the efficient formation of native disulfide bonds in heterologously expressed proteins in the cytoplasm of E. coli even without the disruption of genes involved in disulfide bond reduction, for example trxB and/or gor. Indeed yields of active disulfide bonded proteins were higher in BL21 (DE3 pLysSRARE, an E. coli strain with the reducing pathways intact, than in the commercial Δgor ΔtrxB strain rosetta-gami upon co-expression of Erv1p. Conclusions Our results refute the current paradigm in the field that disruption of at least one of the reducing pathways is essential for the efficient production of disulfide bond containing proteins in the cytoplasm of E. coli and open up new possibilities for the use of E. coli as a microbial cell factory.

Cytoplasmic Domain of MscS Interacts with Cell Division Protein FtsZ: A Possible Non-Channel Function of the Mechanosensitive Channel in Escherichia Coli.

Directory of Open Access Journals (Sweden)

Piotr Koprowski

Full Text Available Bacterial mechano-sensitive (MS channels reside in the inner membrane and are considered to act as emergency valves whose role is to lower cell turgor when bacteria enter hypo-osmotic environments. However, there is emerging evidence that members of the Mechano-sensitive channel Small (MscS family play additional roles in bacterial and plant cell physiology. MscS has a large cytoplasmic C-terminal region that changes its shape upon activation and inactivation of the channel. Our pull-down and co-sedimentation assays show that this domain interacts with FtsZ, a bacterial tubulin-like protein. We identify point mutations in the MscS C-terminal domain that reduce binding to FtsZ and show that bacteria expressing these mutants are compromised in growth on sublethal concentrations of β-lactam antibiotics. Our results suggest that interaction between MscS and FtsZ could occur upon inactivation and/or opening of the channel and could be important for the bacterial cell response against sustained stress upon stationary phase and in the presence of β-lactam antibiotics.

Cytoplasmic Domain of MscS Interacts with Cell Division Protein FtsZ: A Possible Non-Channel Function of the Mechanosensitive Channel in Escherichia Coli.

Science.gov (United States)

Koprowski, Piotr; Grajkowski, Wojciech; Balcerzak, Marcin; Filipiuk, Iwona; Fabczak, Hanna; Kubalski, Andrzej

2015-01-01

Bacterial mechano-sensitive (MS) channels reside in the inner membrane and are considered to act as emergency valves whose role is to lower cell turgor when bacteria enter hypo-osmotic environments. However, there is emerging evidence that members of the Mechano-sensitive channel Small (MscS) family play additional roles in bacterial and plant cell physiology. MscS has a large cytoplasmic C-terminal region that changes its shape upon activation and inactivation of the channel. Our pull-down and co-sedimentation assays show that this domain interacts with FtsZ, a bacterial tubulin-like protein. We identify point mutations in the MscS C-terminal domain that reduce binding to FtsZ and show that bacteria expressing these mutants are compromised in growth on sublethal concentrations of β-lactam antibiotics. Our results suggest that interaction between MscS and FtsZ could occur upon inactivation and/or opening of the channel and could be important for the bacterial cell response against sustained stress upon stationary phase and in the presence of β-lactam antibiotics.

Expression of Anion Exchanger 1 Sequestrates p16 in the Cytoplasm in Gastric, Colonic Adenocarcinoma

Directory of Open Access Journals (Sweden)

Wei-Wei Shen

2007-10-01

Full Text Available p16INK4A (p16 binds to cyclin-dependent kinase 4/6, negatively regulates cell growth. Recent studies have led to an understanding of additional biologic functions for p16; however, the detailed mechanisms involved are still elusive. In this article, we show an unexpected expression of anion exchanger 1 (AEi in the cytoplasm in poorly, moderately differentiated gastric, colonic adenocarcinoma cells, in its interaction with p16, thereby sequestrating the protein in the cytoplasm. Genetic alterations of p16, AEi were not detectable. Forced expression of AEi in these cells sequestrated more p16 in the cytoplasm, whereas small interfering RNA-mediated silencing of AEi in the cells induced the release of p16 from the cytoplasm to the nucleus, leading to cell death, growth inhibition of tumor cells. By analyzing tissue samples obtained from patients with gastric, colonic cancers, we found that 83.33% of gastric cancers, 56.52% of colonic cancers coexpressed AEi, p16 in the cytoplasm. We conclude that AEi plays a crucial role in the pathogenesis of gastric, colonic adenocarcinoma, that p16 dysfunction is a novel pathway of carcinogenesis.

Diffusive Promotion by Velocity Gradient of Cytoplasmic Streaming (CPS in Nitella Internodal Cells.

Directory of Open Access Journals (Sweden)

Kenji Kikuchi

Full Text Available Cytoplasmic streaming (CPS is well known to assist the movement of nutrients, organelles and genetic material by transporting all of the cytoplasmic contents of a cell. CPS is generated by motility organelles that are driven by motor proteins near a membrane surface, where the CPS has been found to have a flat velocity profile in the flow field according to the sliding theory. There is a consistent mixing of contents inside the cell by CPS if the velocity gradient profile is flattened, which is not assisted by advection diffusion but is only supported by Brownian diffusion. Although the precise flow structure of the cytoplasm has an important role for cellular metabolism, the hydrodynamic mechanism of its convection has not been clarified. We conducted an experiment to visualise the flow of cytoplasm in Nitella cells by injecting tracer fluorescent nanoparticles and using a flow visualisation system in order to understand how the flow profile affects their metabolic system. We determined that the velocity field in the cytosol has an obvious velocity gradient, not a flattened gradient, which suggests that the gradient assists cytosolic mixing by Taylor-Aris dispersion more than by Brownian diffusion.

Cytoplasmic influence of nucleolar development

International Nuclear Information System (INIS)

Ghosh, Sibdas

1974-01-01

The role of cytoplasmic factors on the development of nucleolus in nucleus has been investigated in Ehrlich mouse ascites tumour cells using tritiated thymidine/uridine for autoradiography. It is inferred from the observations that the cytoplasmic factors has some but not absolute control over the development of nucleolus. (M.G.B.)

The novel 2Fe–2S outer mitochondrial protein mitoNEET displays conformational flexibility in its N-terminal cytoplasmic tethering domain

International Nuclear Information System (INIS)

Conlan, Andrea R.; Paddock, Mark L.; Axelrod, Herbert L.; Cohen, Aina E.; Abresch, Edward C.; Wiley, Sandra; Roy, Melinda; Nechushtai, Rachel; Jennings, Patricia A.

2009-01-01

The crystal structure of the anti-diabetic drug target mitoNEET obtained from a GFP fusion construct (1.4 Å resolution, R factor = 20.2%) shows that the CDGSH 2Fe–2S binding domains are superimposable with previously determined non-fused constructs. However, there is considerable flexibility in the position of the outer mitochondrial tethering arms resulting in two different conformations in the crystal structure. A primary role for mitochondrial dysfunction is indicated in the pathogenesis of insulin resistance. A widely used drug for the treatment of type 2 diabetes is pioglitazone, a member of the thiazolidinedione class of molecules. MitoNEET, a 2Fe–2S outer mitochondrial membrane protein, binds pioglitazone [Colca et al. (2004 ▶), Am. J. Physiol. Endocrinol. Metab.286, E252–E260]. The soluble domain of the human mitoNEET protein has been expressed C-terminal to the superfolder green fluorescent protein and the mitoNEET protein has been isolated. Comparison of the crystal structure of mitoNEET isolated from cleavage of the fusion protein (1.4 Å resolution, R factor = 20.2%) with other solved structures shows that the CDGSH domains are superimposable, indicating proper assembly of mitoNEET. Furthermore, there is considerable flexibility in the position of the cytoplasmic tethering arms, resulting in two different conformations in the crystal structure. This flexibility affords multiple orientations on the outer mitochondrial membrane

Secretion of bacterial lipoproteins: through the cytoplasmic membrane, the periplasm and beyond.

Science.gov (United States)

Zückert, Wolfram R

2014-08-01

Bacterial lipoproteins are peripherally anchored membrane proteins that play a variety of roles in bacterial physiology and virulence in monoderm (single membrane-enveloped, e.g., gram-positive) and diderm (double membrane-enveloped, e.g., gram-negative) bacteria. After export of prolipoproteins through the cytoplasmic membrane, which occurs predominantly but not exclusively via the general secretory or Sec pathway, the proteins are lipid-modified at the cytoplasmic membrane in a multistep process that involves sequential modification of a cysteine residue and cleavage of the signal peptide by the signal II peptidase Lsp. In both monoderms and diderms, signal peptide processing is preceded by acylation with a diacylglycerol through preprolipoprotein diacylglycerol transferase (Lgt). In diderms but also some monoderms, lipoproteins are further modified with a third acyl chain through lipoprotein N-acyl transferase (Lnt). Fully modified lipoproteins that are destined to be anchored in the inner leaflet of the outer membrane (OM) are selected, transported and inserted by the Lol (lipoprotein outer membrane localization) pathway machinery, which consists of the inner-membrane (IM) ABC transporter-like LolCDE complex, the periplasmic LolA chaperone and the OM LolB lipoprotein receptor. Retention of lipoproteins in the cytoplasmic membrane results from Lol avoidance signals that were originally described as the "+2 rule". Surface localization of lipoproteins in diderms is rare in most bacteria, with the exception of several spirochetal species. Type 2 (T2SS) and type 5 (T5SS) secretion systems are involved in secretion of specific surface lipoproteins of γ-proteobacteria. In the model spirochete Borrelia burgdorferi, surface lipoprotein secretion does not follow established sorting rules, but remains dependent on N-terminal peptide sequences. Secretion through the outer membrane requires maintenance of lipoproteins in a translocation-competent unfolded conformation

Identification of the Specific Interactors of the Human Lariat RNA Debranching Enzyme 1 Protein

Directory of Open Access Journals (Sweden)

So Masaki

2015-02-01

Full Text Available In eukaryotes, pre-mRNA splicing is an essential step for gene expression. We have been analyzing post-splicing intron turnover steps in higher eukaryotes. Here, we report protein interaction between human Debranching enzyme 1 (hDbr1 and several factors found in the Intron Large (IL complex, which is an intermediate complex of the intron degradation pathway. The hDbr1 protein specifically interacts with xeroderma pigmentosum, complementeation group A (XPA-binding protein 2 (Xab2. We also attempted to identify specific interactors of hDbr1. Co-immunoprecipitation experiments followed by mass spectrometry analysis identified a novel protein as one of the specific interactors of hDbr1. This protein is well conserved among many species and shows the highest similarity to yeast Drn1, so it is designated as human Dbr1 associated ribonuclease 1 (hDrn1. hDrn1 directly interacts with hDbr1 through protein–protein interaction. Furthermore, hDrn1 shuttles between the nucleus and the cytoplasm, as hDbr1 protein does. These findings suggest that hDrn1 has roles in both the nucleus and the cytoplasm, which are highly likely to involve hDbr1.

Secretion of Bacterial Lipoproteins: Through the Cytoplasmic Membrane, the Periplasm and Beyond

Science.gov (United States)

Zückert, Wolfram R.

2014-01-01

Bacterial lipoproteins are peripherally anchored membrane proteins that play a variety of roles in bacterial physiology and virulence in monoderm (single membrane-enveloped, e.g., grampositive) and diderm (double membrane-enveloped, e.g., gram-negative) bacteria. After export of prolipoproteins through the cytoplasmic membrane, which occurs predominantly but not exclusively via the general secretory or Sec pathway, the proteins are lipid-modified at the cytoplasmic membrane in a multistep process that involves sequential modification of a cysteine residue and cleavage of the signal peptide by the signal II peptidase Lsp. In both monoderms and diderms, signal peptide processing is preceded by acylation with a diacylglycerol through preprolipoprotein diacylglycerol transferase (Lgt). In diderms but also some monoderms, lipoproteins are further modified with a third acyl chain through lipoprotein N-acyl transferase (Lnt). Fully modified lipoproteins that are destined to be anchored in the inner leaflet of the outer membrane (OM) are selected, transported and inserted by the Lol (lipoprotein outer membrane localization) pathway machinery, which consists of the inner-membrane (IM) ABC transporterlike LolCDE complex, the periplasmic LolA chaperone and the OM LolB lipoprotein receptor. Retention of lipoproteins in the cytoplasmic membrane results from Lol avoidance signals that were originally described as the “+2 rule”. Surface localization of lipoproteins in diderms is rare in most bacteria, with the exception of several spirochetal species. Type 2 (T2SS) and type 5 (T5SS) secretion systems are involved in secretion of specific surface lipoproteins of γ-proteobacteria. In the model spirochete Borrelia burgdorferi, surface lipoprotein secretion does not follow established sorting rules, but remains dependent on N-terminal peptide sequences. Secretion through the outer membrane requires maintenance of lipoproteins in a translocation-competent unfolded conformation

Identification of FUSE-binding proteins as interacting partners of TIA proteins

International Nuclear Information System (INIS)

Rothe, Francoise; Gueydan, Cyril; Bellefroid, Eric; Huez, Georges; Kruys, Veronique

2006-01-01

TIA-1 and TIAR are closely related RNA-binding proteins involved in several mechanisms of RNA metabolism, including alternative hnRNA splicing and mRNA translation regulation. In particular, TIA-1 represses tumor necrosis factor (TNF) mRNA translation by binding to the AU-rich element (ARE) present in the mRNA 3' untranslated region. Here, we demonstrate that TIA proteins interact with FUSE-binding proteins (FBPs) and that fbp genes are co-expressed with tia genes during Xenopus embryogenesis. FBPs participate in various steps of RNA processing and degradation. In Cos cells, FBPs co-localize with TIA proteins in the nucleus and migrate into TIA-enriched cytoplasmic granules upon oxidative stress. Overexpression of FBP2-KH3 RNA-binding domain fused to EGFP induces the specific sequestration of TIA proteins in cytoplasmic foci, thereby precluding their nuclear accumulation. In cytosolic RAW 264.7 macrophage extracts, FBPs are found associated in EMSA to the TIA-1/TNF-ARE complex. Together, our results indicate that TIA and FBP proteins may thus be relevant biological involved in common events of RNA metabolism occurring both in the nucleus and the cytoplasm

Yeast ribosomal proteins

International Nuclear Information System (INIS)

Otaka, E.; Kobata, K.

1978-01-01

The cytoplasmic 80s ribosomal proteins from the cells of yeast Saccharomyces cerevisiae were analyzed by SDS two-dimensional polyacrylamide gel electrophoresis. Seventyfour proteins were identified and consecutively numbered from 1 to 74. Upon oxidation of the 80s proteins with performic acid, ten proteins (no. 15, 20, 35, 40, 44, 46, 49, 51, 54 and 55) were dislocated on the gel without change of the total number of protein spots. Five proteins (no. 8, 14, 16, 36 and 74) were phosphorylated in vivo as seen in 32 P-labelling experiments. The large and small subunits separated in low magnesium medium were analyzed by the above gel electrophoresis. At least forty-five and twenty-eight proteins were assumed to be in the large and small subunits, respectively. All proteins found in the 80s ribosomes, except for no. 3, were detected in either subunit without appearance of new spots. The acidic protein no. 3 seems to be lost during subunit dissociation. (orig.) [de

Cytoplasmic RAP1 mediates cisplatin resistance of non-small cell lung cancer.

Science.gov (United States)

Xiao, Lu; Lan, Xiaoying; Shi, Xianping; Zhao, Kai; Wang, Dongrui; Wang, Xuejun; Li, Faqian; Huang, Hongbiao; Liu, Jinbao

2017-05-18

Cytotoxic chemotherapy agents (e.g., cisplatin) are the first-line drugs to treat non-small cell lung cancer (NSCLC) but NSCLC develops resistance to the agent, limiting therapeutic efficacy. Despite many approaches to identifying the underlying mechanism for cisplatin resistance, there remains a lack of effective targets in the population that resist cisplatin treatment. In this study, we sought to investigate the role of cytoplasmic RAP1, a previously identified positive regulator of NF-κB signaling, in the development of cisplatin resistance in NSCLC cells. We found that the expression of cytoplasmic RAP1 was significantly higher in high-grade NSCLC tissues than in low-grade NSCLC; compared with a normal pulmonary epithelial cell line, the A549 NSCLC cells exhibited more cytoplasmic RAP1 expression as well as increased NF-κB activity; cisplatin treatment resulted in a further increase of cytoplasmic RAP1 in A549 cells; overexpression of RAP1 desensitized the A549 cells to cisplatin, and conversely, RAP1 depletion in the NSCLC cells reduced their proliferation and increased their sensitivity to cisplatin, indicating that RAP1 is required for cell growth and has a key mediating role in the development of cisplatin resistance in NSCLC cells. The RAP1-mediated cisplatin resistance was associated with the activation of NF-κB signaling and the upregulation of the antiapoptosis factor BCL-2. Intriguingly, in the small portion of RAP1-depleted cells that survived cisplatin treatment, no induction of NF-κB activity and BCL-2 expression was observed. Furthermore, in established cisplatin-resistant A549 cells, RAP1 depletion caused BCL2 depletion, caspase activation and dramatic lethality to the cells. Hence, our results demonstrate that the cytoplasmic RAP1-NF-κB-BCL2 axis represents a key pathway to cisplatin resistance in NSCLC cells, identifying RAP1 as a marker and a potential therapeutic target for cisplatin resistance of NSCLC.

The effects of 60Co γ-ray irradiation on cytoplasmic microtubules of mouse macrophages and lymphocytes

International Nuclear Information System (INIS)

Li Qianqian; Mao Zijun; Yin Zhiwei; Hu Yumin

1989-05-01

The effects of 60 Co γ-ray irradiation on cytoplasmic microtubules of mouse macrophages and lymphocytes were investigated by immunofluorescence microscopy and scanning electron microscope. The results indicated. (1) microtubule organization of the irradiated cells remarkably differed from that of the control since the doses over 4 Gy; (2) 144 hours after irradiation the alterations of microtubules have been shown to be basically r epaired ; (3) the cytoplasmic microtubules and centrioles disappeared under transmission electron microscope, the membranes irradiated and microvilli showed changes under scanning electron microscope too. From these observations and those of other workers who studied the radiation effect on extracted microtubule proteins in vitro, the authors support that 60 Co γ-ray irradiation can inhabits cytoplasmic microtubule assembling

Automatically identifying gene/protein terms in MEDLINE abstracts.

Science.gov (United States)

Yu, Hong; Hatzivassiloglou, Vasileios; Rzhetsky, Andrey; Wilbur, W John

2002-01-01

Natural language processing (NLP) techniques are used to extract information automatically from computer-readable literature. In biology, the identification of terms corresponding to biological substances (e.g., genes and proteins) is a necessary step that precedes the application of other NLP systems that extract biological information (e.g., protein-protein interactions, gene regulation events, and biochemical pathways). We have developed GPmarkup (for "gene/protein-full name mark up"), a software system that automatically identifies gene/protein terms (i.e., symbols or full names) in MEDLINE abstracts. As a part of marking up process, we also generated automatically a knowledge source of paired gene/protein symbols and full names (e.g., LARD for lymphocyte associated receptor of death) from MEDLINE. We found that many of the pairs in our knowledge source do not appear in the current GenBank database. Therefore our methods may also be used for automatic lexicon generation. GPmarkup has 73% recall and 93% precision in identifying and marking up gene/protein terms in MEDLINE abstracts. A random sample of gene/protein symbols and full names and a sample set of marked up abstracts can be viewed at http://www.cpmc.columbia.edu/homepages/yuh9001/GPmarkup/. Contact. hy52@columbia.edu. Voice: 212-939-7028; fax: 212-666-0140.

Identifying Hierarchical and Overlapping Protein Complexes Based on Essential Protein-Protein Interactions and “Seed-Expanding” Method

Directory of Open Access Journals (Sweden)

Jun Ren

2014-01-01

Full Text Available Many evidences have demonstrated that protein complexes are overlapping and hierarchically organized in PPI networks. Meanwhile, the large size of PPI network wants complex detection methods have low time complexity. Up to now, few methods can identify overlapping and hierarchical protein complexes in a PPI network quickly. In this paper, a novel method, called MCSE, is proposed based on λ-module and “seed-expanding.” First, it chooses seeds as essential PPIs or edges with high edge clustering values. Then, it identifies protein complexes by expanding each seed to a λ-module. MCSE is suitable for large PPI networks because of its low time complexity. MCSE can identify overlapping protein complexes naturally because a protein can be visited by different seeds. MCSE uses the parameter λ_th to control the range of seed expanding and can detect a hierarchical organization of protein complexes by tuning the value of λ_th. Experimental results of S. cerevisiae show that this hierarchical organization is similar to that of known complexes in MIPS database. The experimental results also show that MCSE outperforms other previous competing algorithms, such as CPM, CMC, Core-Attachment, Dpclus, HC-PIN, MCL, and NFC, in terms of the functional enrichment and matching with known protein complexes.

Versatile function of the circadian protein CIPC as a regulator of Erk activation

International Nuclear Information System (INIS)

Matsunaga, Ryota; Nishino, Tasuku; Yokoyama, Atsushi; Nakashima, Akio; Kikkawa, Ushio; Konishi, Hiroaki

2016-01-01

The CLOCK-interacting protein, Circadian (CIPC), has been identified as an additional negative-feedback regulator of the circadian clock. However, recent study on CIPC knockout mice has shown that CIPC is not critically required for basic circadian clock function, suggesting other unknown biological roles for CIPC. In this study, we focused on the cell cycle dependent nuclear-cytoplasmic shuttling function of CIPC and on identifying its binding proteins. Lys186 and 187 were identified as the essential amino acid residues within the nuclear localization signal (NLS) of CIPC. We identified CIPC-binding proteins such as the multifunctional enzyme CAD protein (carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotase), which is a key enzyme for de novo pyrimidine synthesis. Compared to control cells, HEK293 cells overexpressing wild-type CIPC showed suppressed cell proliferation and retardation of cell cycle. We also found that PMA-induced Erk activation was inhibited with expression of wild-type CIPC. In contrast, the NLS mutant of CIPC, which reduced the ability of CIPC to translocate into the nucleus, did not exhibit these biological effects. Since CAD and Erk have significant roles in cell proliferation and cell cycle, CIPC may work as a cell cycle regulator by interacting with these binding proteins. - Highlights: • CIPC is a cell cycle dependent nuclear-cytoplasmic shuttling protein. • K186 and 187are the essential amino acid residues within the NLS of CIPC. • CAD was identified as a novel CIPC-binding protein. • CIPC might regulate the activity and translocation of CAD in the cells.

Versatile function of the circadian protein CIPC as a regulator of Erk activation

Energy Technology Data Exchange (ETDEWEB)

Matsunaga, Ryota; Nishino, Tasuku [Faculty of Life and Environmental Sciences, Prefectural University of Hiroshima, Shobara, Hiroshima 727-0023 (Japan); Yokoyama, Atsushi [Department of Molecular Endocrinology, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575 (Japan); Nakashima, Akio; Kikkawa, Ushio [Biosignal Research Center, Kobe University, Kobe 657-8501 (Japan); Konishi, Hiroaki, E-mail: hkonishi@pu-hiroshima.ac.jp [Faculty of Life and Environmental Sciences, Prefectural University of Hiroshima, Shobara, Hiroshima 727-0023 (Japan)

2016-01-15

The CLOCK-interacting protein, Circadian (CIPC), has been identified as an additional negative-feedback regulator of the circadian clock. However, recent study on CIPC knockout mice has shown that CIPC is not critically required for basic circadian clock function, suggesting other unknown biological roles for CIPC. In this study, we focused on the cell cycle dependent nuclear-cytoplasmic shuttling function of CIPC and on identifying its binding proteins. Lys186 and 187 were identified as the essential amino acid residues within the nuclear localization signal (NLS) of CIPC. We identified CIPC-binding proteins such as the multifunctional enzyme CAD protein (carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotase), which is a key enzyme for de novo pyrimidine synthesis. Compared to control cells, HEK293 cells overexpressing wild-type CIPC showed suppressed cell proliferation and retardation of cell cycle. We also found that PMA-induced Erk activation was inhibited with expression of wild-type CIPC. In contrast, the NLS mutant of CIPC, which reduced the ability of CIPC to translocate into the nucleus, did not exhibit these biological effects. Since CAD and Erk have significant roles in cell proliferation and cell cycle, CIPC may work as a cell cycle regulator by interacting with these binding proteins. - Highlights: • CIPC is a cell cycle dependent nuclear-cytoplasmic shuttling protein. • K186 and 187are the essential amino acid residues within the NLS of CIPC. • CAD was identified as a novel CIPC-binding protein. • CIPC might regulate the activity and translocation of CAD in the cells.

The Capsicum annuum class IV chitinase ChitIV interacts with receptor-like cytoplasmic protein kinase PIK1 to accelerate PIK1-triggered cell death and defence responses

Science.gov (United States)

Kim, Dae Sung; Kim, Nak Hyun; Hwang, Byung Kook

2015-01-01

The pepper receptor-like cytoplasmic protein kinase, CaPIK1, which mediates signalling of plant cell death and defence responses was previously identified. Here, the identification of a class IV chitinase, CaChitIV, from pepper plants (Capsicum annuum), which interacts with CaPIK1 and promotes CaPIK1-triggered cell death and defence responses, is reported. CaChitIV contains a signal peptide, chitin-binding domain, and glycol hydrolase domain. CaChitIV expression was up-regulated by Xanthomonas campestris pv. vesicatoria (Xcv) infection. Notably, avirulent Xcv infection rapidly induced CaChitIV expression in pepper leaves. Bimolecular fluorescence complementation and co-immunoprecipitation revealed that CaPIK1 interacts with CaChitIV in planta, and that the CaPIK1–CaChitIV complex is localized mainly in the cytoplasm and plasma membrane. CaChitIV is also localized in the endoplasmic reticulum. Transient co-expression of CaChitIV with CaPIK1 enhanced CaPIK1-triggered cell death response and reactive oxygen species (ROS) and nitric oxide (NO) bursts. Co-silencing of both CaChitIV and CaPIK1 in pepper plants conferred enhanced susceptibility to Xcv infection, which was accompanied by a reduced induction of cell death response, ROS and NO bursts, and defence response genes. Ectopic expression of CaPIK1 in Arabidopsis enhanced basal resistance to Hyaloperonospora arabidopsidis infection. Together, the results suggest that CaChitIV positively regulates CaPIK1-triggered cell death and defence responses through its interaction with CaPIK1. PMID:25694549

Analysis of Pseudomonas aeruginosa Cell Envelope Proteome by Capture of Surface-Exposed Proteins on Activated Magnetic Nanoparticles

OpenAIRE

Vecchietti, Davide; Di Silvestre, Dario; Miriani, Matteo; Bonomi, Francesco; Marengo, Mauro; Bragonzi, Alessandra; Cova, Lara; Franceschi, Eleonora; Mauri, Pierluigi; Bertoni, Giovanni

2012-01-01

We report on specific magneto-capturing followed by Multidimensional Protein Identification Technology (MudPIT) for the analysis of surface-exposed proteins of intact cells of the bacterial opportunistic pathogen Pseudomonas aeruginosa. The magneto-separation of cell envelope fragments from the soluble cytoplasmic fraction allowed the MudPIT identification of the captured and neighboring proteins. Remarkably, we identified 63 proteins captured directly by nanoparticles and 67 proteins embedde...

Improvements in the Protein Identifier Cross-Reference service.

Science.gov (United States)

Wein, Samuel P; Côté, Richard G; Dumousseau, Marine; Reisinger, Florian; Hermjakob, Henning; Vizcaíno, Juan A

2012-07-01

The Protein Identifier Cross-Reference (PICR) service is a tool that allows users to map protein identifiers, protein sequences and gene identifiers across over 100 different source databases. PICR takes input through an interactive website as well as Representational State Transfer (REST) and Simple Object Access Protocol (SOAP) services. It returns the results as HTML pages, XLS and CSV files. It has been in production since 2007 and has been recently enhanced to add new functionality and increase the number of databases it covers. Protein subsequences can be Basic Local Alignment Search Tool (BLAST) against the UniProt Knowledgebase (UniProtKB) to provide an entry point to the standard PICR mapping algorithm. In addition, gene identifiers from UniProtKB and Ensembl can now be submitted as input or mapped to as output from PICR. We have also implemented a 'best-guess' mapping algorithm for UniProt. In this article, we describe the usefulness of PICR, how these changes have been implemented, and the corresponding additions to the web services. Finally, we explain that the number of source databases covered by PICR has increased from the initial 73 to the current 102. New resources include several new species-specific Ensembl databases as well as the Ensembl Genome ones. PICR can be accessed at http://www.ebi.ac.uk/Tools/picr/.

Production of ABA responses requires both the nuclear and cytoplasmic functional involvement of PYR1

International Nuclear Information System (INIS)

Park, EunJoo; Kim, Tae-Houn

2017-01-01

Abscisic acid (ABA) enhances stress tolerant responses in plants against unfavorable environmental conditions. In Arabidopsis, ABA promotes interactions between PYR/PYL/RCARs and PP2C, thereby allowing SnRK2s to phosphorylate downstream components required for the regulation of gene expression or for gating ion channels. Because PYR1 is known to localize to nucleus and cytoplasm it is a question whether nuclear or cytoplasmic PYR1 confer different functions to the ABA signaling pathway, as has been previously shown for regulatory proteins. In order to answer this question, transgenic lines expressing nuclear PYR1 were generated in an ABA insensitive mutant background. Enforced nuclear expression of PYR1 was examined by confocal microscopy and western blot analysis. Physiological analyses of the transgenic lines demonstrated that nuclear PYR1 is sufficient to generate ABA responses, such as, the inhibition of seed germination, root growth inhibition, the induction of gene expression, and stomatal closing movement. However, for the full recovery of ABA responses in the mutant background cytoplasmic PYR1 was required. The study suggests both nuclear and cytoplasmic PYR1 participate in the control of ABA signal transduction. - Highlights: • Nuclear and cytoplasmic functions of PYR1 were studied in the mutant which lacked majority of ABA responses. • Nuclear PYR1 reconstituted partially the ABA responses during seed germination, root growth, and guard cell movement. • Both the nuclear and cytoplasmic functions of PYR1 were required for the full generation of ABA responses.

Identification of a functional nuclear export signal in the green fluorescent protein asFP499

International Nuclear Information System (INIS)

Mustafa, Huseyin; Strasser, Bernd; Rauth, Sabine; Irving, Robert A.; Wark, Kim L.

2006-01-01

The green fluorescent protein (GFP) asFP499 from Anemonia sulcata is a distant homologue of the GFP from Aequorea victoria. We cloned the asFP499 gene into a mammalian expression vector and showed that this protein was expressed in the human lymphoblast cell line Ramos RA1 and in the embryonic kidney 293T cell line (HEK 293T). In HEK 293T cells, asFP499 was localized mainly in the cytoplasm, suggesting that the protein was excluded from the nucleus. We identified 194 LRMEKLNI 201 as a candidate nuclear export signal in asFP499 and mutated the isoleucine at position 201 to an alanine. Unlike the wildtype form, the mutant protein was distributed throughout the cytoplasm and nucleus. This is First report of a GFP that contains a functional NES

The Rules and Functions of Nucleocytoplasmic Shuttling Proteins.

Science.gov (United States)

Fu, Xuekun; Liang, Chao; Li, Fangfei; Wang, Luyao; Wu, Xiaoqiu; Lu, Aiping; Xiao, Guozhi; Zhang, Ge

2018-05-12

Biological macromolecules are the basis of life activities. There is a separation of spatial dimension between DNA replication and RNA biogenesis, and protein synthesis, which is an interesting phenomenon. The former occurs in the cell nucleus, while the latter in the cytoplasm. The separation requires protein to transport across the nuclear envelope to realize a variety of biological functions. Nucleocytoplasmic transport of protein including import to the nucleus and export to the cytoplasm is a complicated process that requires involvement and interaction of many proteins. In recent years, many studies have found that proteins constantly shuttle between the cytoplasm and the nucleus. These shuttling proteins play a crucial role as transport carriers and signal transduction regulators within cells. In this review, we describe the mechanism of nucleocytoplasmic transport of shuttling proteins and summarize some important diseases related shuttling proteins.

Cytoplasmic Streaming - Skylab Student Experiment ED-63

Science.gov (United States)

1973-01-01

This chart describes the Skylab student experiment (ED-63), Cytoplasmic Streaming, proposed by Cheryl A. Peitz of Arapahoe High School, Littleton, Colorado. Experiment ED-63 was to observe the effect of zero-gravity on cytoplasmic streaming in the aquatic plant named Elodea, commonly called water weed or water thyme. The phenomenon of cytoplasmic streaming is not well understood, but it is recognized as the circulation mechanism of the internal materials or cytoplasm of a cell. Cytoplasm is a gelatinous substance that has the ability to change its viscosity and flow, carrying various cell materials with it. The activity can be stimulated by sunlight or heat. In March 1972, NASA and the National Science Teachers Association selected 25 experiment proposals for flight on Skylab. Science advisors from the Marshall Space Flight Center aided and assisted the students in developing the proposals for flight on Skylab.

An early cytoplasmic step of peptidoglycan synthesis is associated to MreB in Bacillus subtilis.

Science.gov (United States)

Rueff, Anne-Stéphanie; Chastanet, Arnaud; Domínguez-Escobar, Julia; Yao, Zhizhong; Yates, James; Prejean, Maria-Victoria; Delumeau, Olivier; Noirot, Philippe; Wedlich-Söldner, Roland; Filipe, Sergio R; Carballido-López, Rut

2014-01-01

MreB proteins play a major role during morphogenesis of rod-shaped bacteria by organizing biosynthesis of the peptidoglycan cell wall. However, the mechanisms underlying this process are not well understood. In Bacillus subtilis, membrane-associated MreB polymers have been shown to be associated to elongation-specific complexes containing transmembrane morphogenetic factors and extracellular cell wall assembly proteins. We have now found that an early intracellular step of cell wall synthesis is also associated to MreB. We show that the previously uncharacterized protein YkuR (renamed DapI) is required for synthesis of meso-diaminopimelate (m-DAP), an essential constituent of the peptidoglycan precursor, and that it physically interacts with MreB. Highly inclined laminated optical sheet microscopy revealed that YkuR forms uniformly distributed foci that exhibit fast motion in the cytoplasm, and are not detected in cells lacking MreB. We propose a model in which soluble MreB organizes intracellular steps of peptidoglycan synthesis in the cytoplasm to feed the membrane-associated cell wall synthesizing machineries. © 2013 John Wiley & Sons Ltd.

The Arabidopsis splicing factors, AtU2AF65, AtU2AF35, and AtSF1 shuttle between nuclei and cytoplasms

KAUST Repository

Park, Hyo-Young

2017-04-21

The Arabidopsis splicing factors, AtU2AF65, AtU2AF35, and AtSF1 shuttle between nuclei and cytoplasms. These proteins also move rapidly and continuously in the nuclei, and their movements are affected by ATP depletion. The U2AF65 proteins are splicing factors that interact with SF1 and U2AF35 proteins to promote U2snRNP for the recognition of the pre-mRNA 3\\' splice site during early spliceosome assembly. We have determined the subcellular localization and movement of these proteins\\' Arabidopsis homologs. It was found that Arabidopsis U2AF65 homologs, AtU2AF65a, and AtU2AF65b proteins interact with AtU2AF35a and AtU2AF35b, which are Arabidopsis U2AF35 homologs. We have examined the mobility of these proteins including AtSF1 using fluorescence recovery after photobleaching and fluorescence loss in photobleaching analyses. These proteins displayed dynamic movements in nuclei and their movements were affected by ATP depletion. We have also demonstrated that these proteins shuttle between nuclei and cytoplasms, suggesting that they may also function in cytoplasm. These results indicate that such splicing factors show very similar characteristics to their human counterparts, suggesting evolutionary conservation.

The Arabidopsis splicing factors, AtU2AF65, AtU2AF35, and AtSF1 shuttle between nuclei and cytoplasms

KAUST Repository

Park, Hyo-Young; Lee, Keh Chien; Jang, Yun Hee; Kim, SoonKap; Thu, May Phyo; Lee, Jeong Hwan; Kim, Jeong-Kook

2017-01-01

The Arabidopsis splicing factors, AtU2AF65, AtU2AF35, and AtSF1 shuttle between nuclei and cytoplasms. These proteins also move rapidly and continuously in the nuclei, and their movements are affected by ATP depletion. The U2AF65 proteins are splicing factors that interact with SF1 and U2AF35 proteins to promote U2snRNP for the recognition of the pre-mRNA 3' splice site during early spliceosome assembly. We have determined the subcellular localization and movement of these proteins' Arabidopsis homologs. It was found that Arabidopsis U2AF65 homologs, AtU2AF65a, and AtU2AF65b proteins interact with AtU2AF35a and AtU2AF35b, which are Arabidopsis U2AF35 homologs. We have examined the mobility of these proteins including AtSF1 using fluorescence recovery after photobleaching and fluorescence loss in photobleaching analyses. These proteins displayed dynamic movements in nuclei and their movements were affected by ATP depletion. We have also demonstrated that these proteins shuttle between nuclei and cytoplasms, suggesting that they may also function in cytoplasm. These results indicate that such splicing factors show very similar characteristics to their human counterparts, suggesting evolutionary conservation.

Myosin-Powered Membrane Compartment Drives Cytoplasmic Streaming, Cell Expansion and Plant Development.

Science.gov (United States)

Peremyslov, Valera V; Cole, Rex A; Fowler, John E; Dolja, Valerian V

2015-01-01

Using genetic approaches, particle image velocimetry and an inert tracer of cytoplasmic streaming, we have made a mechanistic connection between the motor proteins (myosins XI), cargo transported by these motors (distinct endomembrane compartment defined by membrane-anchored MyoB receptors) and the process of cytoplasmic streaming in plant cells. It is shown that the MyoB compartment in Nicotiana benthamiana is highly dynamic moving with the mean velocity of ~3 μm/sec. In contrast, Golgi, mitochondria, peroxisomes, carrier vesicles and a cytosol flow tracer share distinct velocity profile with mean velocities of 0.6-1.5 μm/sec. Dominant negative inhibition of the myosins XI or MyoB receptors using overexpression of the N. benthamiana myosin cargo-binding domain or MyoB myosin-binding domain, respectively, resulted in velocity reduction for not only the MyoB compartment, but also each of the tested organelles, vesicles and cytoplasmic streaming. Furthermore, the extents of this reduction were similar for each of these compartments suggesting that MyoB compartment plays primary role in cytosol dynamics. Using gene knockout analysis in Arabidopsis thaliana, it is demonstrated that inactivation of MyoB1-4 results in reduced velocity of mitochondria implying slower cytoplasmic streaming. It is also shown that myosins XI and MyoB receptors genetically interact to contribute to cell expansion, plant growth, morphogenesis and proper onset of flowering. These results support a model according to which myosin-dependent, MyoB receptor-mediated transport of a specialized membrane compartment that is conserved in all land plants drives cytoplasmic streaming that carries organelles and vesicles and facilitates cell growth and plant development.

Cytoplasmic HIV-1 RNA is mainly transported by diffusion in the presence or absence of Gag protein

DEFF Research Database (Denmark)

Chen, Jianbo; Grunwald, David; Sardo, Luca

2014-01-01

. In this report, we visualized HIV-1 RNA and monitored its movement in the cytoplasm by using single-molecule tracking. We observed that most of the HIV-1 RNA molecules move in a nondirectional, random-walk manner, which does not require an intact cytoskeletal structure, and that the mean-squared distance...... traveled by the RNA increases linearly with time, indicative of diffusive movement. We also observed that a single HIV-1 RNA molecule can move at various speeds when traveling through the cytoplasm, indicating that its movement is strongly affected by the immediate environment. To examine the effect of Gag...

Involvement of hGLD-2 in cytoplasmic polyadenylation of human p53 mRNA

DEFF Research Database (Denmark)

Glahder, Jacob-Andreas Harald; Norrild, Bodil

2011-01-01

Cytoplasmic polyadenylation is a post-transcriptional mechanism regulating mRNA stability and translation. The human p53 3'-untranslated region (3'-UTR) contains two regions similar to cytoplasmic polyadenylation elements (CPEs) just upstream of the poly(A) hexanucleotide. Evaluation of the p53 CPE......-like elements was performed by luciferase reporter assays, qPCR, and poly(A) assays. Herein, we report the down regulation of a luciferase reporter fused to the p53 3'-UTR, when human CPE-binding protein 1 (hCPEB1) is overexpressed. This inhibition is partially rescued when hCPEB1fused to hGLD-2 [a human...... cytoplasmic poly(A) polymerase] is overexpressed instead. The stability of a luciferase mRNA containing the p53 3'-UTR downstream, is decreased when hCPEB1 is overexpressed as seen by qPCR. Expression of hGLD-2 restores the mRNA stability. This is due to elongation of the poly(A) tail as seen by a PCR...

Uniparental Inheritance Promotes Adaptive Evolution in Cytoplasmic Genomes

Science.gov (United States)

Christie, Joshua R.; Beekman, Madeleine

2017-01-01

Eukaryotes carry numerous asexual cytoplasmic genomes (mitochondria and plastids). Lacking recombination, asexual genomes should theoretically suffer from impaired adaptive evolution. Yet, empirical evidence indicates that cytoplasmic genomes experience higher levels of adaptive evolution than predicted by theory. In this study, we use a computational model to show that the unique biology of cytoplasmic genomes—specifically their organization into host cells and their uniparental (maternal) inheritance—enable them to undergo effective adaptive evolution. Uniparental inheritance of cytoplasmic genomes decreases competition between different beneficial substitutions (clonal interference), promoting the accumulation of beneficial substitutions. Uniparental inheritance also facilitates selection against deleterious cytoplasmic substitutions, slowing Muller’s ratchet. In addition, uniparental inheritance generally reduces genetic hitchhiking of deleterious substitutions during selective sweeps. Overall, uniparental inheritance promotes adaptive evolution by increasing the level of beneficial substitutions relative to deleterious substitutions. When we assume that cytoplasmic genome inheritance is biparental, decreasing the number of genomes transmitted during gametogenesis (bottleneck) aids adaptive evolution. Nevertheless, adaptive evolution is always more efficient when inheritance is uniparental. Our findings explain empirical observations that cytoplasmic genomes—despite their asexual mode of reproduction—can readily undergo adaptive evolution. PMID:28025277

Protein misfolding specifies recruitment to cytoplasmic inclusion bodies.

Science.gov (United States)

Bersuker, Kirill; Brandeis, Michael; Kopito, Ron R

2016-04-25

Inclusion bodies (IBs) containing aggregated disease-associated proteins and polyubiquitin (poly-Ub) conjugates are universal histopathological features of neurodegenerative diseases. Ub has been proposed to target proteins to IBs for degradation via autophagy, but the mechanisms that govern recruitment of ubiquitylated proteins to IBs are not well understood. In this paper, we use conditionally destabilized reporters that undergo misfolding and ubiquitylation upon removal of a stabilizing ligand to examine the role of Ub conjugation in targeting proteins to IBs that are composed of an N-terminal fragment of mutant huntingtin, the causative protein of Huntington's disease. We show that reporters are excluded from IBs in the presence of the stabilizing ligand but are recruited to IBs after ligand washout. However, we find that Ub conjugation is not necessary to target reporters to IBs. We also report that forced Ub conjugation by the Ub fusion degradation pathway is not sufficient for recruitment to IBs. Finally, we find that reporters and Ub conjugates are stable at IBs. These data indicate that compromised folding states, rather than conjugation to Ub, can specify recruitment to IBs. © 2016 Bersuker et al.

An Extremely Halophilic Proteobacterium Combines a Highly Acidic Proteome with a Low Cytoplasmic Potassium Content*

Science.gov (United States)

Deole, Ratnakar; Challacombe, Jean; Raiford, Douglas W.; Hoff, Wouter D.

2013-01-01

Halophilic archaea accumulate molar concentrations of KCl in their cytoplasm as an osmoprotectant and have evolved highly acidic proteomes that function only at high salinity. We examined osmoprotection in the photosynthetic Proteobacteria Halorhodospira halophila and Halorhodospira halochloris. Genome sequencing and isoelectric focusing gel electrophoresis showed that the proteome of H. halophila is acidic. In line with this finding, H. halophila accumulated molar concentrations of KCl when grown in high salt medium as detected by x-ray microanalysis and plasma emission spectrometry. This result extends the taxonomic range of organisms using KCl as a main osmoprotectant to the Proteobacteria. The closely related organism H. halochloris does not exhibit an acidic proteome, matching its inability to accumulate K+. This observation indicates recent evolutionary changes in the osmoprotection strategy of these organisms. Upon growth of H. halophila in low salt medium, its cytoplasmic K+ content matches that of Escherichia coli, revealing an acidic proteome that can function in the absence of high cytoplasmic salt concentrations. These findings necessitate a reassessment of two central aspects of theories for understanding extreme halophiles. First, we conclude that proteome acidity is not driven by stabilizing interactions between K+ ions and acidic side chains but by the need for maintaining sufficient solvation and hydration of the protein surface at high salinity through strongly hydrated carboxylates. Second, we propose that obligate protein halophilicity is a non-adaptive property resulting from genetic drift in which constructive neutral evolution progressively incorporates weakly stabilizing K+-binding sites on an increasingly acidic protein surface. PMID:23144460

Organelle Simple Sequence Repeat Markers Help to Distinguish Carpelloid Stamen and Normal Cytoplasmic Male Sterile Sources in Broccoli

Science.gov (United States)

Shu, Jinshuai; Liu, Yumei; Li, Zhansheng; Zhang, Lili; Fang, Zhiyuan; Yang, Limei; Zhuang, Mu; Zhang, Yangyong; Lv, Honghao

2015-01-01

We previously discovered carpelloid stamens when breeding cytoplasmic male sterile lines in broccoli (Brassica oleracea var. italica). In this study, hybrids and multiple backcrosses were produced from different cytoplasmic male sterile carpelloid stamen sources and maintainer lines. Carpelloid stamens caused dysplasia of the flower structure and led to hooked or coiled siliques with poor seed setting, which were inherited in a maternal fashion. Using four distinct carpelloid stamens and twelve distinct normal stamens from cytoplasmic male sterile sources and one maintainer, we used 21 mitochondrial simple sequence repeat (mtSSR) primers and 32 chloroplast SSR primers to identify a mitochondrial marker, mtSSR2, that can differentiate between the cytoplasm of carpelloid and normal stamens. Thereafter, mtSSR2 was used to identify another 34 broccoli accessions, with an accuracy rate of 100%. Analysis of the polymorphic sequences revealed that the mtSSR2 open reading frame of carpelloid stamen sterile sources had a deletion of 51 bases (encoding 18 amino acids) compared with normal stamen materials. The open reading frame is located in the coding region of orf125 and orf108 of the mitochondrial genomes in Brassica crops and had the highest similarity with Raphanus sativus and Brassica carinata. The current study has not only identified a useful molecular marker to detect the cytoplasm of carpelloid stamens during broccoli breeding, but it also provides evidence that the mitochondrial genome is maternally inherited and provides a basis for studying the effect of the cytoplasm on flower organ development in plants. PMID:26407159

An MHC-I cytoplasmic domain/HIV-1 Nef fusion protein binds directly to the mu subunit of the AP-1 endosomal coat complex.

Directory of Open Access Journals (Sweden)

Rajendra Kumar Singh

2009-12-01

Full Text Available The down-regulation of the major histocompatibility complex class I (MHC-I from the surface of infected cells by the Nef proteins of primate immunodeficiency viruses likely contributes to pathogenesis by providing evasion of cell-mediated immunity. HIV-1 Nef-induced down-regulation involves endosomal trafficking and a cooperative interaction between the cytoplasmic domain (CD of MHC-I, Nef, and the clathrin adaptor protein complex-1 (AP-1. The CD of MHC-I contains a key tyrosine within the sequence YSQA that is required for down-regulation by Nef, but this sequence does not conform to the canonical AP-binding tyrosine-based motif Yxxphi, which mediates binding to the medium (micro subunits of AP complexes. We previously proposed that Nef allows the MHC-I CD to bind the mu subunit of AP-1 (micro1 as if it contained a Yxxphimotif.Here, we show that a direct interaction between the MHC-I CD/Nef and micro1 plays a primary role in the down-regulation of MHC-I: GST pulldown assays using recombinant proteins indicated that most of the MHC-I CD and Nef residues that are required for the down-regulation in human cells contribute to direct interactions with a truncated version of micro1. Specifically, the tyrosine residue of the YSQA sequence in the MHC-I CD as well as Nef residues E62-65 and P78 each contributed to the interaction between MHC-I CD/Nef and micro1 in vitro, whereas Nef M20 had little to no role. Conversely, residues F172/D174 and V392/L395 of the binding pocket on micro1 for Yxxphi motifs were required for a robust interaction.These data indicate that the MHC-I cytoplasmic domain, Nef, and the C-terminal two thirds of the mu subunit of AP-1 are sufficient to constitute a biologically relevant interaction. The data also reveal an unexpected role for a hydrophobic pocket in micro1 for interaction with MHC-I CD/Nef.

Herpesvirus Genome Recognition Induced Acetylation of Nuclear IFI16 Is Essential for Its Cytoplasmic Translocation, Inflammasome and IFN-β Responses.

Directory of Open Access Journals (Sweden)

Mairaj Ahmed Ansari

2015-07-01

Full Text Available The IL-1β and type I interferon-β (IFN-β molecules are important inflammatory cytokines elicited by the eukaryotic host as innate immune responses against invading pathogens and danger signals. Recently, a predominantly nuclear gamma-interferon-inducible protein 16 (IFI16 involved in transcriptional regulation has emerged as an innate DNA sensor which induced IL-1β and IFN-β production through inflammasome and STING activation, respectively. Herpesvirus (KSHV, EBV, and HSV-1 episomal dsDNA genome recognition by IFI16 leads to IFI16-ASC-procaspase-1 inflammasome association, cytoplasmic translocation and IL-1β production. Independent of ASC, HSV-1 genome recognition results in IFI16 interaction with STING in the cytoplasm to induce interferon-β production. However, the mechanisms of IFI16-inflammasome formation, cytoplasmic redistribution and STING activation are not known. Our studies here demonstrate that recognition of herpesvirus genomes in the nucleus by IFI16 leads into its interaction with histone acetyltransferase p300 and IFI16 acetylation resulting in IFI16-ASC interaction, inflammasome assembly, increased interaction with Ran-GTPase, cytoplasmic redistribution, caspase-1 activation, IL-1β production, and interaction with STING which results in IRF-3 phosphorylation, nuclear pIRF-3 localization and interferon-β production. ASC and STING knockdowns did not affect IFI16 acetylation indicating that this modification is upstream of inflammasome-assembly and STING-activation. Vaccinia virus replicating in the cytoplasm did not induce nuclear IFI16 acetylation and cytoplasmic translocation. IFI16 physically associates with KSHV and HSV-1 genomes as revealed by proximity ligation microscopy and chromatin-immunoprecipitation studies which is not hampered by the inhibition of acetylation, thus suggesting that acetylation of IFI16 is not required for its innate sensing of nuclear viral genomes. Collectively, these studies identify the

Cytoplasmic TRADD Confers a Worse Prognosis in Glioblastoma

Directory of Open Access Journals (Sweden)

Sharmistha Chakraborty

2013-08-01

Full Text Available Tumor necrosis factor receptor 1 (TNFR1-associated death domain protein (TRADD is an important adaptor in TNFR1 signaling and has an essential role in nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB activation and survival signaling. Increased expression of TRADD is sufficient to activate NF-κB. Recent studies have highlighted the importance of NF-κB activation as a key pathogenic mechanism in glioblastoma multiforme (GBM, the most common primary malignant brain tumor in adults.We examined the expression of TRADD by immunohistochemistry (IHC and find that TRADD is commonly expressed at high levels in GBM and is detected in both cytoplasmic and nuclear distribution. Cytoplasmic IHC TRADD scoring is significantly associated with worse progression-free survival (PFS both in univariate and multivariate analysis but is not associated with overall survival (n = 43 GBMs. PFS is a marker for responsiveness to treatment. We propose that TRADD-mediated NF-κB activation confers chemoresistance and thus a worse PFS in GBM. Consistent with the effect on PFS, silencing TRADD in glioma cells results in decreased NF-κB activity, decreased proliferation of cells, and increased sensitivity to temozolomide. TRADD expression is common in glioma-initiating cells. Importantly, silencing TRADD in GBM-initiating stem cell cultures results in decreased viability of stem cells, suggesting that TRADD may be required for maintenance of GBM stem cell populations. Thus, our study suggests that increased expression of cytoplasmic TRADD is both an important biomarker and a key driver of NF-κB activation in GBM and supports an oncogenic role for TRADD in GBM.

Cytoplasmic Estrogen Receptor in breast cancer

Science.gov (United States)

Welsh, Allison W.; Lannin, Donald R.; Young, Gregory S.; Sherman, Mark E.; Figueroa, Jonine D.; Henry, N. Lynn; Ryden, Lisa; Kim, Chungyeul; Love, Richard R.; Schiff, Rachel; Rimm, David L.

2011-01-01

Purpose In addition to genomic signaling, it is accepted that ERα has non-nuclear signaling functions, which correlate with tamoxifen resistance in preclinical models. However, evidence for cytoplasmic ER localization in human breast tumors is less established. We sought to determine the presence and implications of non-nuclear ER in clinical specimens. Experimental Design A panel of ERα-specific antibodies (SP1, MC20, F10, 60c, 1D5) were validated by western blot and quantitative immunofluorescent (QIF) analysis of cell lines and patient controls. Then eight retrospective cohorts collected on tissue microarrays were assessed for cytoplasmic ER. Four cohorts were from Yale (YTMA 49, 107, 130, 128) and four others (NCI YTMA 99, South Swedish Breast Cancer Group SBII, NSABP B14, and a Vietnamese Cohort) from other sites around the world. Results Four of the antibodies specifically recognized ER by western and QIF, showed linear increases in amounts of ER in cell line series with progressively increasing ER, and the antibodies were reproducible on YTMA 49 with pearson’s correlations (r2 values)ranging from 0.87-0.94. One antibody with striking cytoplasmic staining (MC20) failed validation. We found evidence for specific cytoplasmic staining with the other 4 antibodies across eight cohorts. The average incidence was 1.5%, ranging from 0 to 3.2%. Conclusions Our data shows ERα present in the cytoplasm in a number of cases using multiple antibodies, while reinforcing the importance of antibody validation. In nearly 3,200 cases, cytoplasmic ER is present at very low incidence, suggesting its measurement is unlikely to be of routine clinical value. PMID:21980134

High Prevalence of Autoantibodies to hLAMP-2 in Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis

NARCIS (Netherlands)

Kain, Renate; Tadema, Henko; McKinney, Eoin F.; Benharkou, Alexandra; Brandes, Ricarda; Peschel, Andrea; Hubert, Virginie; Feenstra, Tjerk; Sengoelge, Guerkan; Stegeman, Coen; Heeringa, Peter; Lyons, Paul A.; Smith, Kenneth G. C.; Kallenberg, Cees; Rees, Andrew J.

The involvement of autoantibodies to human lysosome-associated membrane protein-2 (hLAMP-2) in anti neutrophil cytoplasmic antibody (ANCA) associated vasculitis is controversial because of the absence of confirmatory data subsequent to the initial reports of their high prevalence in this disease. We

Molecular characterization of genome segments 1 and 3 encoding two capsid proteins of Antheraea mylitta cytoplasmic polyhedrosis virus

Directory of Open Access Journals (Sweden)

Chakrabarti Mrinmay

2010-08-01

Full Text Available Abstract Background Antheraea mylitta cytoplasmic polyhedrosis virus (AmCPV, a cypovirus of Reoviridae family, infects Indian non-mulberry silkworm, Antheraea mylitta, and contains 11 segmented double stranded RNA (S1-S11 in its genome. Some of its genome segments (S2 and S6-S11 have been previously characterized but genome segments encoding viral capsid have not been characterized. Results In this study genome segments 1 (S1 and 3 (S3 of AmCPV were converted to cDNA, cloned and sequenced. S1 consisted of 3852 nucleotides, with one long ORF of 3735 nucleotides and could encode a protein of 1245 amino acids with molecular mass of ~141 kDa. Similarly, S3 consisted of 3784 nucleotides having a long ORF of 3630 nucleotides and could encode a protein of 1210 amino acids with molecular mass of ~137 kDa. BLAST analysis showed 20-22% homology of S1 and S3 sequence with spike and capsid proteins, respectively, of other closely related cypoviruses like Bombyx mori CPV (BmCPV, Lymantria dispar CPV (LdCPV, and Dendrolimus punctatus CPV (DpCPV. The ORFs of S1 and S3 were expressed as 141 kDa and 137 kDa insoluble His-tagged fusion proteins, respectively, in Escherichia coli M15 cells via pQE-30 vector, purified through Ni-NTA chromatography and polyclonal antibodies were raised. Immunoblot analysis of purified polyhedra, virion particles and virus infected mid-gut cells with the raised anti-p137 and anti-p141 antibodies showed specific immunoreactive bands and suggest that S1 and S3 may code for viral structural proteins. Expression of S1 and S3 ORFs in insect cells via baculovirus recombinants showed to produce viral like particles (VLPs by transmission electron microscopy. Immunogold staining showed that S3 encoded proteins self assembled to form viral outer capsid and VLPs maintained their stability at different pH in presence of S1 encoded protein. Conclusion Our results of cloning, sequencing and functional analysis of AmCPV S1 and S3 indicate that S3

Generation of micronuclei during interphase by coupling between cytoplasmic membrane blebbing and nuclear budding.

Directory of Open Access Journals (Sweden)

Koh-ichi Utani

Full Text Available Micronucleation, mediated by interphase nuclear budding, has been repeatedly suggested, but the process is still enigmatic. In the present study, we confirmed the previous observation that there are lamin B1-negative micronuclei in addition to the positive ones. A large cytoplasmic bleb was found to frequently entrap lamin B1-negative micronuclei, which were connected to the nucleus by a thin chromatin stalk. At the bottom of the stalk, the nuclear lamin B1 structure appeared broken. Chromatin extrusion through lamina breaks has been referred to as herniation or a blister of the nucleus, and has been observed after the expression of viral proteins. A cell line in which extrachromosomal double minutes and lamin B1 protein were simultaneously visualized in different colors in live cells was established. By using these cells, time-lapse microscopy revealed that cytoplasmic membrane blebbing occurred simultaneously with the extrusion of nuclear content, which generated lamin B1-negative micronuclei during interphase. Furthermore, activation of cytoplasmic membrane blebbing by the addition of fresh serum or camptothecin induced nuclear budding within 1 to 10 minutes, which suggested that blebbing might be the cause of the budding. After the induction of blebbing, the frequency of lamin-negative micronuclei increased. The budding was most frequent during S phase and more efficiently entrapped small extrachromosomal chromatin than the large chromosome arm. Based on these results, we suggest a novel mechanism in which cytoplasmic membrane dynamics pulls the chromatin out of the nucleus through the lamina break. Evidence for such a mechanism was obtained in certain cancer cell lines including human COLO 320 and HeLa. The mechanism could significantly perturb the genome and influence cancer cell phenotypes.

Identifying Bacterial Immune Evasion Proteins Using Phage Display.

Science.gov (United States)

Fevre, Cindy; Scheepmaker, Lisette; Haas, Pieter-Jan

2017-01-01

Methods aimed at identification of immune evasion proteins are mainly rely on in silico prediction of sequence, structural homology to known evasion proteins or use a proteomics driven approach. Although proven successful these methods are limited by a low efficiency and or lack of functional identification. Here we describe a high-throughput genomic strategy to functionally identify bacterial immune evasion proteins using phage display technology. Genomic bacterial DNA is randomly fragmented and ligated into a phage display vector that is used to create a phage display library expressing bacterial secreted and membrane bound proteins. This library is used to select displayed bacterial secretome proteins that interact with host immune components.

Evidence for a cytoplasmic pathway of oxalate biosynthesis in Aspergillus niger

International Nuclear Information System (INIS)

Kubicek, C.P.; Schreferl-Kunar, G.; Woehrer, W.; Roehr, M.

1988-01-01

Oxalate accumulation of up to 8 g/liter was induced in Aspergillus niger by shifting the pH from 6 to 8. This required the presence of P/sub i/ and a nitrogen source and was inhibited by the protein synthesis inhibitor cycloheximide. Exogenously added 14 CO 2 was not incorporated into oxalate, but was incorporated into acetate and malate, thus indicating the biosynthesis of oxalate by hydrolytic cleavage of oxaloacetate. Inhibition of mitochondrial citrate metabolism by fluorocitrate did not significantly decrease the oxalate yield. The putative enzyme that was responsible for this oxaloacetate hydrolase (EC 3.7.1.1), which was induced de novo during the pH shift. Subcellular fractionation of oxalic acid-forming mycelia of A. niger showed that this enzyme is located in the cytoplasm of A. niger. The results are consistent with a cytoplasmic pathway of oxalate formation which does not involve the tricarboxylic acid cycle

Arrest of cytoplasmic streaming induces algal proliferation in green paramecia.

Directory of Open Access Journals (Sweden)

Toshiyuki Takahashi

Full Text Available A green ciliate Paramecium bursaria, bearing several hundreds of endosymbiotic algae, demonstrates rotational microtubule-based cytoplasmic streaming, in which cytoplasmic granules and endosymbiotic algae flow in a constant direction. However, its physiological significance is still unknown. We investigated physiological roles of cytoplasmic streaming in P. bursaria through host cell cycle using video-microscopy. Here, we found that cytoplasmic streaming was arrested in dividing green paramecia and the endosymbiotic algae proliferated only during the arrest of cytoplasmic streaming. Interestingly, arrest of cytoplasmic streaming with pressure or a microtubule drug also induced proliferation of endosymbiotic algae independently of host cell cycle. Thus, cytoplasmic streaming may control the algal proliferation in P. bursaria. Furthermore, confocal microscopic observation revealed that a division septum was formed in the constricted area of a dividing paramecium, producing arrest of cytoplasmic streaming. This is a first report to suggest that cytoplasmic streaming controls proliferation of eukaryotic cells.

Uniparental Inheritance Promotes Adaptive Evolution in Cytoplasmic Genomes.

Science.gov (United States)

Christie, Joshua R; Beekman, Madeleine

2017-03-01

Eukaryotes carry numerous asexual cytoplasmic genomes (mitochondria and plastids). Lacking recombination, asexual genomes should theoretically suffer from impaired adaptive evolution. Yet, empirical evidence indicates that cytoplasmic genomes experience higher levels of adaptive evolution than predicted by theory. In this study, we use a computational model to show that the unique biology of cytoplasmic genomes-specifically their organization into host cells and their uniparental (maternal) inheritance-enable them to undergo effective adaptive evolution. Uniparental inheritance of cytoplasmic genomes decreases competition between different beneficial substitutions (clonal interference), promoting the accumulation of beneficial substitutions. Uniparental inheritance also facilitates selection against deleterious cytoplasmic substitutions, slowing Muller's ratchet. In addition, uniparental inheritance generally reduces genetic hitchhiking of deleterious substitutions during selective sweeps. Overall, uniparental inheritance promotes adaptive evolution by increasing the level of beneficial substitutions relative to deleterious substitutions. When we assume that cytoplasmic genome inheritance is biparental, decreasing the number of genomes transmitted during gametogenesis (bottleneck) aids adaptive evolution. Nevertheless, adaptive evolution is always more efficient when inheritance is uniparental. Our findings explain empirical observations that cytoplasmic genomes-despite their asexual mode of reproduction-can readily undergo adaptive evolution. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Cytoplasmic Kaiso is associated with poor prognosis in non-small cell lung cancer

International Nuclear Information System (INIS)

Dai, Shun-Dong; Wang, Yan; Miao, Yuan; Zhao, Yue; Zhang, Yong; Jiang, Gui-Yang; Zhang, Peng-Xin; Yang, Zhi-Qiang; Wang, En-Hua

2009-01-01

Kaiso has been identified as a new member of the POZ-zinc finger family of transcription factors that are implicated in development and cancer. Although controversy still exists, Kaiso is supposed to be involved in human cancer. However, there is limited information regarding the clinical significance of cytoplasmic/nuclear Kaiso in human lung cancer. In this study, immunohistochemical studies were performed on 20 cases of normal lung tissues and 294 cases of non-small cell lung cancer (NSCLC), including 50 cases of paired lymph node metastases and 88 cases with complete follow-up records. Three lung cancer cell lines showing primarily nuclear localization of Kaiso were selected to examine whether roles of Kaiso in cytoplasm and in nucleus are identical. Nuclear Kaiso was down-regulated by shRNA technology or addition a specific Kaiso antibody in these cell lines. The proliferative and invasive abilities were evaluated by MTT and Matrigel invasive assay, transcription of Kaiso's target gene matrilysin was detected by RT-PCR. Kaiso was primarily expressed in the cytoplasm of lung cancer tissues. Overall positive cytoplasmic expression rate was 63.61% (187/294). The positive cytoplasmic expression of Kaiso was higher in advanced TNM stages (III+IV) of NSCLC, compared to lower stages (I+II) (p = 0.019). A correlation between cytoplasmic Kaiso expression and lymph node metastasis was found (p = 0.003). In 50 paired cases, cytoplasmic expression of Kaiso was 78.0% (41/50) in primary sites and 90.0% (45/50) in lymph node metastases (p = 0.001). The lung cancer-related 5-year survival rate was significantly lower in patients who were cytoplasmic Kaiso-positive (22.22%), compared to those with cytoplasmic Kaiso-negative tumors (64.00%) (p = 0.005). Nuclear Kaiso staining was seen in occasional cases with only a 5.10% (15/294) positive rate and was not associated with any clinicopathological features of NSCLC. Furthermore, after the down-regulation of the nuclear

Tubulinlike protein from Spirochaeta bajacaliforniensis

Science.gov (United States)

Bermudes, D.; Fracek, S. P. Jr; Laursen, R. A.; Margulis, L.; Obar, R.; Tzertzinis, G.

1987-01-01

Tubulin proteins are the fundamental subunits of all polymeric microtubule-based eukaryotic structures. Long, hollow structures each composed of 13 protofilaments as revealed by electron microscopy, microtubules (240 angstroms in diameter) are nearly ubiquitous in eukaryotes. These proteins have been the subject of intense biochemical and biophyiscal interest since the early 1970s and are of evolutionary interest as well. If tubulin-based structures (i.e., neurotubules, mitotic spindle tubules, centrioles, kinetosomes, axonemes, etc.) evolved from spirochetes by way of motility symbioses, tubulin homologies with spirochete proteins should be detectable. Tubulin proteins are widely thought to be limited to eukaryotes. Yet both azotobacters and spirochetes have shown immunological cross-reactivity with antitubulin antibodies. In neither of these studies was tubulin isolated nor any specific antigen identified as responsible for the immunoreactivity. Furthermore, although far less uniform in structure than eukaryotic microtubules, various cytoplasmic fibers and tubules (as seen by electron microscopy) have been reported in several types of prokaryotes (e.g., Spirochaeta; large termite spirochetes; treponemes; cyanobacteria; and Azotobacter. This work forms a part of our long-range study of the possible prokaryotic origin of tubulin and microtubules. Spirochetes are helically shaped gram-negative motile prokaryotes. They differ from all other bacterial in that the position of their flagella is periplasmic: their flagella lie between the inner and outer membranes of the gram-negative cell wall. Some of the largest spirochetes have longitudinally aligned 240 angstrom microtubules. Unfortunately, in spite of many attempts, all of the larger spirochetes (family Pillotaceae) with well-defined cytoplasmic tubules and antitubulin immunoreactivity are not cultivable. However, a newly described spirochete species (Spirochaeta bajacaliforniensis) possessing cytoplasmic fibers

Validation of cytoplasmic-to-nuclear ratio of survivin as an indicator of improved prognosis in breast cancer

International Nuclear Information System (INIS)

Rexhepaj, Elton; Jirstrom, Karin; O'Connor, Darran P; O'Brien, Sallyann L; Landberg, Goran; Duffy, Michael J; Brennan, Donal J; Gallagher, William M

2010-01-01

Conflicting data exist regarding the prognostic and predictive impact of survivin (BIRC5) in breast cancer. We previously reported survivin cytoplasmic-to-nuclear ratio (CNR) as an independent prognostic indicator in breast cancer. Here, we validate survivin CNR in a separate and extended cohort. Furthermore, we present new data suggesting that a low CNR may predict outcome in tamoxifen-treated patients. Survin expression was assessed using immunhistochemistry on a breast cancer tissue microarray (TMA) containing 512 tumours. Whole slide digital images were captured using an Aperio XT scanner. Automated image analysis was used to identify tumour from stroma and then to quantify tumour-specific nuclear and cytoplasmic survivin. A decision tree model selected using a 10-fold cross-validation approach was used to identify prognostic subgroups based on nuclear and cytoplasmic survivin expression. Following optimisation of the staining procedure, it was possible to evaluate survivin protein expression in 70.1% (n = 359) of the 512 tumours represented on the TMA. Decision tree analysis predicted that nuclear, as opposed to cytoplasmic, survivin was the most important determinant of overall survival (OS) and breast cancer-specific survival (BCSS). The decision tree model confirmed CNR of 5 as the optimum threshold for survival analysis. Univariate analysis demonstrated an association between a high CNR (>5) and a prolonged BCSS (HR 0.49, 95% CI 0.29-0.81, p = 0.006). Multivariate analysis revealed a high CNR (>5) was an independent predictor of BCSS (HR 0.47, 95% CI 0.27-0.82, p = 0.008). An increased CNR was associated with ER positive (p = 0.045), low grade (p = 0.007), Ki-67 (p = 0.001) and Her2 (p = 0.026) negative tumours. Finally, a high CNR was an independent predictor of OS in tamoxifen-treated ER-positive patients (HR 0.44, 95% CI 0.23-0.87, p = 0.018). Using the same threshold as our previous study, we have validated survivin CNR as a marker of good prognosis in

Validation of cytoplasmic-to-nuclear ratio of survivin as an indicator of improved prognosis in breast cancer

LENUS (Irish Health Repository)

Rexhepaj, Elton

2010-11-23

Abstract Background Conflicting data exist regarding the prognostic and predictive impact of survivin (BIRC5) in breast cancer. We previously reported survivin cytoplasmic-to-nuclear ratio (CNR) as an independent prognostic indicator in breast cancer. Here, we validate survivin CNR in a separate and extended cohort. Furthermore, we present new data suggesting that a low CNR may predict outcome in tamoxifen-treated patients. Methods Survin expression was assessed using immunhistochemistry on a breast cancer tissue microarray (TMA) containing 512 tumours. Whole slide digital images were captured using an Aperio XT scanner. Automated image analysis was used to identify tumour from stroma and then to quantify tumour-specific nuclear and cytoplasmic survivin. A decision tree model selected using a 10-fold cross-validation approach was used to identify prognostic subgroups based on nuclear and cytoplasmic survivin expression. Results Following optimisation of the staining procedure, it was possible to evaluate survivin protein expression in 70.1% (n = 359) of the 512 tumours represented on the TMA. Decision tree analysis predicted that nuclear, as opposed to cytoplasmic, survivin was the most important determinant of overall survival (OS) and breast cancer-specific survival (BCSS). The decision tree model confirmed CNR of 5 as the optimum threshold for survival analysis. Univariate analysis demonstrated an association between a high CNR (>5) and a prolonged BCSS (HR 0.49, 95% CI 0.29-0.81, p = 0.006). Multivariate analysis revealed a high CNR (>5) was an independent predictor of BCSS (HR 0.47, 95% CI 0.27-0.82, p = 0.008). An increased CNR was associated with ER positive (p = 0.045), low grade (p = 0.007), Ki-67 (p = 0.001) and Her2 (p = 0.026) negative tumours. Finally, a high CNR was an independent predictor of OS in tamoxifen-treated ER-positive patients (HR 0.44, 95% CI 0.23-0.87, p = 0.018). Conclusion Using the same threshold as our previous study, we have

Identifying New Small Proteins in Escherichia coli.

Science.gov (United States)

VanOrsdel, Caitlin E; Kelly, John P; Burke, Brittany N; Lein, Christina D; Oufiero, Christopher E; Sanchez, Joseph F; Wimmers, Larry E; Hearn, David J; Abuikhdair, Fatimeh J; Barnhart, Kathryn R; Duley, Michelle L; Ernst, Sarah E G; Kenerson, Briana A; Serafin, Aubrey J; Hemm, Matthew R

2018-04-12

The number of small proteins (SPs) encoded in the Escherichia coli genome is unknown, as current bioinformatics and biochemical techniques make short gene and small protein identification challenging. One method of small protein identification involves adding an epitope tag to the 3' end of a short open reading frame (sORF) on the chromosome, with synthesis confirmed by immunoblot assays. In this study, this strategy was used to identify new E. coli small proteins, tagging 80 sORFs in the E. coli genome, and assayed for protein synthesis. The selected sORFs represent diverse sequence characteristics, including degrees of sORF conservation, predicted transmembrane domains, sORF direction with respect to flanking genes, ribosome binding site (RBS) prediction, and ribosome profiling results. Of 80 sORFs, 36 resulted in encoded synthesized proteins-a 45% success rate. Modeling of detected versus non-detected small proteins analysis showed predictions based on RBS prediction, transcription data, and ribosome profiling had statistically-significant correlation with protein synthesis; however, there was no correlation between current sORF annotation and protein synthesis. These results suggest substantial numbers of small proteins remain undiscovered in E. coli, and existing bioinformatics techniques must continue to improve to facilitate identification. © 2018 The Authors. Proteomics Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim, Towson University.

Quantifying Multistate Cytoplasmic Molecular Diffusion in Bacterial Cells via Inverse Transform of Confined Displacement Distribution.

Science.gov (United States)

Chen, Tai-Yen; Jung, Won; Santiago, Ace George; Yang, Feng; Krzemiński, Łukasz; Chen, Peng

2015-11-12

Single-molecule tracking (SMT) of fluorescently tagged cytoplasmic proteins can provide valuable information on the underlying biological processes in living cells via subsequent analysis of the displacement distributions; however, the confinement effect originated from the small size of a bacterial cell skews the protein's displacement distribution and complicates the quantification of the intrinsic diffusive behaviors. Using the inverse transformation method, we convert the skewed displacement distribution (for both 2D and 3D imaging conditions) back to that in free space for systems containing one or multiple (non)interconverting Brownian diffusion states, from which we can reliably extract the number of diffusion states as well as their intrinsic diffusion coefficients and respective fractional populations. We further demonstrate a successful application to experimental SMT data of a transcription factor in living E. coli cells. This work allows a direct quantitative connection between cytoplasmic SMT data with diffusion theory for analyzing molecular diffusive behavior in live bacteria.

Algorithms for Cytoplasm Segmentation of Fluorescence Labelled Cells

OpenAIRE

Carolina Wählby; Joakim Lindblad; Mikael Vondrus; Ewert Bengtsson; Lennart Björkesten

2002-01-01

Automatic cell segmentation has various applications in cytometry, and while the nucleus is often very distinct and easy to identify, the cytoplasm provides a lot more challenge. A new combination of image analysis algorithms for segmentation of cells imaged by fluorescence microscopy is presented. The algorithm consists of an image pre?processing step, a general segmentation and merging step followed by a segmentation quality measurement. The quality measurement consists of a statistical ana...

Antineutrophil cytoplasmic autoantibody-associated small-vessel vasculitis

NARCIS (Netherlands)

Kallenberg, Cees G. M.

Purpose of reviews This review focuses on recent advance in the diagnosis pathogenesis and treatment of antineutrophil cytoplasmic autoantibody-associated small-vessel vasculitis. Recent findings Antineutrophil cytoplasmic autoantibodies are closely associated with Wegener's granulomatosis and

Identifying Floppy and Rigid Regions in Proteins

Science.gov (United States)

Jacobs, D. J.; Thorpe, M. F.; Kuhn, L. A.

1998-03-01

In proteins it is possible to separate hard covalent forces involving bond lengths and bond angles from other weak forces. We model the microstructure of the protein as a generic bar-joint truss framework, where the hard covalent forces and strong hydrogen bonds are regarded as rigid bar constraints. We study the mechanical stability of proteins using FIRST (Floppy Inclusions and Rigid Substructure Topography) based on a recently developed combinatorial constraint counting algorithm (the 3D Pebble Game), which is a generalization of the 2D pebble game (D. J. Jacobs and M. F. Thorpe, ``Generic Rigidity: The Pebble Game'', Phys. Rev. Lett.) 75, 4051-4054 (1995) for the special class of bond-bending networks (D. J. Jacobs, "Generic Rigidity in Three Dimensional Bond-bending Networks", Preprint Aug (1997)). This approach is useful in identifying rigid motifs and flexible linkages in proteins, and thereby determines the essential degrees of freedom. We will show some preliminary results from the FIRST analysis on the myohemerythrin and lyozyme proteins.

Endogenous RGS14 is a cytoplasmic-nuclear shuttling protein that localizes to juxtanuclear membranes and chromatin-rich regions of the nucleus

Science.gov (United States)

Hepler, John R.

2017-01-01

Regulator of G protein signaling 14 (RGS14) is a multifunctional scaffolding protein that integrates G protein and H-Ras/MAPkinase signaling pathways to regulate synaptic plasticity important for hippocampal learning and memory. However, to date, little is known about the subcellular distribution and roles of endogenous RGS14 in a neuronal cell line. Most of what is known about RGS14 cellular behavior is based on studies of tagged, recombinant RGS14 ectopically overexpressed in unnatural host cells. Here, we report for the first time a comprehensive assessment of the subcellular distribution and dynamic localization of endogenous RGS14 in rat B35 neuroblastoma cells. Using confocal imaging and 3D-structured illumination microscopy, we find that endogenous RGS14 localizes to subcellular compartments not previously recognized in studies of recombinant RGS14. RGS14 localization was observed most notably at juxtanuclear membranes encircling the nucleus, at nuclear pore complexes (NPC) on both sides of the nuclear envelope and within intranuclear membrane channels, and within both chromatin-poor and chromatin-rich regions of the nucleus in a cell cycle-dependent manner. In addition, a subset of nuclear RGS14 localized adjacent to active RNA polymerase II. Endogenous RGS14 was absent from the plasma membrane in resting cells; however, the protein could be trafficked to the plasma membrane from juxtanuclear membranes in endosomes derived from ER/Golgi, following constitutive activation of endogenous RGS14 G protein binding partners using AlF4¯. Finally, our findings show that endogenous RGS14 behaves as a cytoplasmic-nuclear shuttling protein confirming what has been shown previously for recombinant RGS14. Taken together, the findings highlight possible cellular roles for RGS14 not previously recognized that are distinct from the regulation of conventional GPCR-G protein signaling, in particular undefined roles for RGS14 in the nucleus. PMID:28934222

Protein-protein interactions within late pre-40S ribosomes.

Directory of Open Access Journals (Sweden)

Melody G Campbell

2011-01-01

Full Text Available Ribosome assembly in eukaryotic organisms requires more than 200 assembly factors to facilitate and coordinate rRNA transcription, processing, and folding with the binding of the ribosomal proteins. Many of these assembly factors bind and dissociate at defined times giving rise to discrete assembly intermediates, some of which have been partially characterized with regards to their protein and RNA composition. Here, we have analyzed the protein-protein interactions between the seven assembly factors bound to late cytoplasmic pre-40S ribosomes using recombinant proteins in binding assays. Our data show that these factors form two modules: one comprising Enp1 and the export adaptor Ltv1 near the beak structure, and the second comprising the kinase Rio2, the nuclease Nob1, and a regulatory RNA binding protein Dim2/Pno1 on the front of the head. The GTPase-like Tsr1 and the universally conserved methylase Dim1 are also peripherally connected to this second module. Additionally, in an effort to further define the locations for these essential proteins, we have analyzed the interactions between these assembly factors and six ribosomal proteins: Rps0, Rps3, Rps5, Rps14, Rps15 and Rps29. Together, these results and previous RNA-protein crosslinking data allow us to propose a model for the binding sites of these seven assembly factors. Furthermore, our data show that the essential kinase Rio2 is located at the center of the pre-ribosomal particle and interacts, directly or indirectly, with every other assembly factor, as well as three ribosomal proteins required for cytoplasmic 40S maturation. These data suggest that Rio2 could play a central role in regulating cytoplasmic maturation steps.

Urinary Biomarkers in Relapsing Antineutrophil Cytoplasmic Antibody-associated Vasculitis

Science.gov (United States)

Lieberthal, Jason G.; Cuthbertson, David; Carette, Simon; Hoffman, Gary S.; Khalidi, Nader A.; Koening, Curry L.; Langford, Carol A.; Maksimowicz-McKinnon, Kathleen; Seo, Philip; Specks, Ulrich; Ytterberg, Steven R.; Merkel, Peter A.; Monach, Paul A.

2015-01-01

Objective Glomerulonephritis (GN) is common in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), but tools for early detection of renal involvement are imperfect. We investigated 4 urinary proteins as markers of active renal AAV: alpha-1 acid glycoprotein (AGP), kidney injury molecule-1 (KIM-1), monocyte chemoattractant protein-1 (MCP-1), and neutrophil gelatinase-associated lipocalin (NGAL). Methods Patients with active renal AAV (n = 20), active nonrenal AAV (n = 16), and AAV in longterm remission (n = 14) were identified within a longitudinal cohort. Urinary biomarker concentrations (by ELISA) were normalized for urine creatinine. Marker levels during active AAV were compared to baseline remission levels (from 1–4 visits) for each patient. Areas under receiver-operating characteristic curves (AUC), sensitivities, specificities, and likelihood ratios (LR) comparing disease states were calculated. Results Baseline biomarker levels varied among patients. All 4 markers increased during renal flares (p < 0.05). MCP-1 discriminated best between active renal disease and remission: a 1.3-fold increase in MCP-1 had 94% sensitivity and 89% specificity for active renal disease (AUC = 0.93, positive LR 8.5, negative LR 0.07). Increased MCP-1 also characterized 50% of apparently nonrenal flares. Change in AGP, KIM-1, or NGAL showed more modest ability to distinguish active renal disease from remission (AUC 0.71–0.75). Hematuria was noted in 83% of active renal episodes, but also 43% of nonrenal flares and 25% of remission samples. Conclusion Either urinary MCP-1 is not specific for GN in AAV, or it identifies early GN not detected by standard assessment and thus has potential to improve care. A followup study with kidney biopsy as the gold standard is needed. PMID:23547217

Heart-type fatty-acid-binding protein (FABP3 is a lysophosphatidic acid-binding protein in human coronary artery endothelial cells

Directory of Open Access Journals (Sweden)

Ryoko Tsukahara

2014-01-01

Full Text Available Fatty-acid-binding protein 3, muscle and heart (FABP3, also known as heart-type FABP, is a member of the family of intracellular lipid-binding proteins. It is a small cytoplasmic protein with a molecular mass of about 15 kDa. FABPs are known to be carrier proteins for transporting fatty acids and other lipophilic substances from the cytoplasm to the nucleus, where these lipids are released to a group of nuclear receptors such as peroxisome proliferator-activated receptors (PPARs. In this study, using lysophosphatidic acid (LPA-coated agarose beads, we have identified FABP3 as an LPA carrier protein in human coronary artery endothelial cells (HCAECs. Administration of LPA to HCAECs resulted in a dose-dependent increase in PPARγ activation. Furthermore, the LPA-induced PPARγ activation was abolished when the FABP3 expression was reduced using small interfering RNA (siRNA. We further show that the nuclear fraction of control HCAECs contained a significant amount of exogenously added LPA, whereas FABP3 siRNA-transfected HCAECs had a decreased level of LPA in the nucleus. Taken together, these results suggest that FABP3 governs the transcriptional activities of LPA by targeting them to cognate PPARγ in the nucleus.

Solution structure of a syndecan-4 cytoplasmic domain and its interaction with phosphatidylinositol 4,5-bisphosphate

DEFF Research Database (Denmark)

Lee, D; Oh, E S; Woods, A

1998-01-01

Syndecan-4, a transmembrane heparan sulfate proteoglycan, is a coreceptor with integrins in cell adhesion. It has been suggested to form a ternary signaling complex with protein kinase Calpha and phosphatidylinositol 4,5-bisphosphate (PIP2). Syndecans each have a unique, central, and variable (V......) region in their cytoplasmic domains, and that of syndecan-4 is critical to its interaction with protein kinase C and PIP2. Two oligopeptides corresponding to the variable region (4V) and whole domain (4L) of syndecan-4 cytoplasmic domain were synthesized for nuclear magnetic resonance (NMR) studies. Data...... and dynamical simulated annealing calculations. The 4V peptide in the presence of PIP2 formed a compact dimer with two twisted strands packed parallel to each other and the exposed surface of the dimer consisted of highly charged and polar residues. The overall three-dimensional structure in solution exhibits...

Nuclear and Cytoplasmic Delivery of Lactoferrin in Glioma using Chitosan Nanoparticles: Cellular Location Dependent-Action of Lactoferrin.

Science.gov (United States)

Tammam, Salma N; Azzazy, Hassan M E; Lamprecht, Alf

2018-05-23

Lactoferrin (Lf) exerts anti-cancer effects on glioma, however, the exact mechanism remains unclear. Despite possessing a nuclear localization sequence (NLS), Lf was found to allocate only in the cytoplasm of glioma 261. Lf was therefore loaded into nuclear and cytoplasmic targeted nanoparticles (NPs) to determine whether nuclear delivery of Lf would enhance its anti-cancer effect. Upon treatment with 300 and 800 µg/mL Lf loaded chitosan NPs, nuclear targeted Lf-NPs showed 1.3 and 2.7 folds increase in cell viability, whereas cytoplasmic targeted Lf-NPs at 300 µg/mL decreased cell viability by 0.8 folds in comparison to free Lf and controls. Results suggest that the cytotoxicity of Lf on glioma is attributable to its cytoplasmic allocation. Nuclear delivery of Lf induced cell proliferation rather than cytotoxicity, indicating that the mode of action of Lf in glioma is cell location dependent. This calls for caution about the general use of Lf as an anti-cancer protein. Copyright © 2018. Published by Elsevier B.V.

Behavioral and other phenotypes in a cytoplasmic Dynein light intermediate chain 1 mutant mouse.

Science.gov (United States)

Banks, Gareth T; Haas, Matilda A; Line, Samantha; Shepherd, Hazel L; Alqatari, Mona; Stewart, Sammy; Rishal, Ida; Philpott, Amelia; Kalmar, Bernadett; Kuta, Anna; Groves, Michael; Parkinson, Nicholas; Acevedo-Arozena, Abraham; Brandner, Sebastian; Bannerman, David; Greensmith, Linda; Hafezparast, Majid; Koltzenburg, Martin; Deacon, Robert; Fainzilber, Mike; Fisher, Elizabeth M C

2011-04-06

The cytoplasmic dynein complex is fundamentally important to all eukaryotic cells for transporting a variety of essential cargoes along microtubules within the cell. This complex also plays more specialized roles in neurons. The complex consists of 11 types of protein that interact with each other and with external adaptors, regulators and cargoes. Despite the importance of the cytoplasmic dynein complex, we know comparatively little of the roles of each component protein, and in mammals few mutants exist that allow us to explore the effects of defects in dynein-controlled processes in the context of the whole organism. Here we have taken a genotype-driven approach in mouse (Mus musculus) to analyze the role of one subunit, the dynein light intermediate chain 1 (Dync1li1). We find that, surprisingly, an N235Y point mutation in this protein results in altered neuronal development, as shown from in vivo studies in the developing cortex, and analyses of electrophysiological function. Moreover, mutant mice display increased anxiety, thus linking dynein functions to a behavioral phenotype in mammals for the first time. These results demonstrate the important role that dynein-controlled processes play in the correct development and function of the mammalian nervous system.

MreB and MurG as scaffolds for the cytoplasmic steps of peptidoglycan biosynthesis.

Science.gov (United States)

Favini-Stabile, Sandy; Contreras-Martel, Carlos; Thielens, Nicole; Dessen, Andréa

2013-12-01

Peptidoglycan is a major determinant of cell shape in bacteria, and its biosynthesis involves the concerted action of cytoplasmic, membrane-associated and periplasmic enzymes. Within the cytoplasm, Mur enzymes catalyse the first steps leading to peptidoglycan precursor biosynthesis, and have been suggested as being part of a multicomponent complex that could also involve the transglycosylase MurG and the cytoskeletal protein MreB. In order to initialize the characterization of a potential Mur interaction network, we purified MurD, MurE, MurF, MurG and MreB from Thermotoga maritima and characterized their interactions using membrane blotting and surface plasmon resonance. MurD, MurE and MurF all recognize MurG and MreB, but not each other, while the two latter proteins interact. In addition, we solved the crystal structures of MurD, MurE and MurF, which indicate that their C-termini display high conformational flexibilities. The differences in Mur conformations could be important parameters for the stability of an intracytoplasmic murein biosynthesis complex. © 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.

Cytoplasmic chromatin triggers inflammation in senescence and cancer.

Science.gov (United States)

Dou, Zhixun; Ghosh, Kanad; Vizioli, Maria Grazia; Zhu, Jiajun; Sen, Payel; Wangensteen, Kirk J; Simithy, Johayra; Lan, Yemin; Lin, Yanping; Zhou, Zhuo; Capell, Brian C; Xu, Caiyue; Xu, Mingang; Kieckhaefer, Julia E; Jiang, Tianying; Shoshkes-Carmel, Michal; Tanim, K M Ahasan Al; Barber, Glen N; Seykora, John T; Millar, Sarah E; Kaestner, Klaus H; Garcia, Benjamin A; Adams, Peter D; Berger, Shelley L

2017-10-19

Chromatin is traditionally viewed as a nuclear entity that regulates gene expression and silencing. However, we recently discovered the presence of cytoplasmic chromatin fragments that pinch off from intact nuclei of primary cells during senescence, a form of terminal cell-cycle arrest associated with pro-inflammatory responses. The functional significance of chromatin in the cytoplasm is unclear. Here we show that cytoplasmic chromatin activates the innate immunity cytosolic DNA-sensing cGAS-STING (cyclic GMP-AMP synthase linked to stimulator of interferon genes) pathway, leading both to short-term inflammation to restrain activated oncogenes and to chronic inflammation that associates with tissue destruction and cancer. The cytoplasmic chromatin-cGAS-STING pathway promotes the senescence-associated secretory phenotype in primary human cells and in mice. Mice deficient in STING show impaired immuno-surveillance of oncogenic RAS and reduced tissue inflammation upon ionizing radiation. Furthermore, this pathway is activated in cancer cells, and correlates with pro-inflammatory gene expression in human cancers. Overall, our findings indicate that genomic DNA serves as a reservoir to initiate a pro-inflammatory pathway in the cytoplasm in senescence and cancer. Targeting the cytoplasmic chromatin-mediated pathway may hold promise in treating inflammation-related disorders.

Evidence for a cytoplasmic pathway of oxalate biosynthesis in Aspergillus niger

Energy Technology Data Exchange (ETDEWEB)

Kubicek, C.P.; Schreferl-Kunar, G.; Woehrer, W.; Roehr, M.

1988-03-01

Oxalate accumulation of up to 8 g/liter was induced in Aspergillus niger by shifting the pH from 6 to 8. This required the presence of P/sub i/ and a nitrogen source and was inhibited by the protein synthesis inhibitor cycloheximide. Exogenously added /sup 14/CO/sub 2/ was not incorporated into oxalate, but was incorporated into acetate and malate, thus indicating the biosynthesis of oxalate by hydrolytic cleavage of oxaloacetate. Inhibition of mitochondrial citrate metabolism by fluorocitrate did not significantly decrease the oxalate yield. The putative enzyme that was responsible for this oxaloacetate hydrolase (EC 3.7.1.1), which was induced de novo during the pH shift. Subcellular fractionation of oxalic acid-forming mycelia of A. niger showed that this enzyme is located in the cytoplasm of A. niger. The results are consistent with a cytoplasmic pathway of oxalate formation which does not involve the tricarboxylic acid cycle.

Surfing the wave, cycle, life history, and genes/proteins expressed by testicular germ cells. Part 3: developmental changes in spermatid flagellum and cytoplasmic droplet and interaction of sperm with the zona pellucida and egg plasma membrane.

Science.gov (United States)

Hermo, Louis; Pelletier, R-Marc; Cyr, Daniel G; Smith, Charles E

2010-04-01

Spermiogenesis constitutes the steps involved in the metamorphosis of spermatids into spermatozoa. It involves modification of several organelles in addition to the formation of several structures including the flagellum and cytoplasmic droplet. The flagellum is composed of a neck region and middle, principal, and end pieces. The axoneme composed of nine outer microtubular doublets circularly arranged to form a cylinder around a central pair of microtubules is present throughout the flagellum. The middle and principal pieces each contain specific components such as the mitochondrial sheath and fibrous sheath, respectively, while outer dense fibers are common to both. A plethora of proteins are constituents of each of these structures, with each playing key roles in functions related to the fertility of spermatozoa. At the end of spermiogenesis, a portion of spermatid cytoplasm remains associated with the released spermatozoa, referred to as the cytoplasmic droplet. The latter has as its main feature Golgi saccules, which appear to modify the plasma membrane of spermatozoa as they move down the epididymal duct and hence may be partly involved in male gamete maturation. The end product of spermatogenesis is highly streamlined and motile spermatozoa having a condensed nucleus equipped with an acrosome. Spermatozoa move through the female reproductive tract and eventually penetrate the zona pellucida and bind to the egg plasma membrane. Many proteins have been implicated in the process of fertilization as well as a plethora of proteins involved in the development of spermatids and sperm, and these are high lighted in this review. Copyright 2009 Wiley-Liss, Inc.

Newly identified protein Imi1 affects mitochondrial integrity and glutathione homeostasis in Saccharomyces cerevisiae.

Science.gov (United States)

Kowalec, Piotr; Grynberg, Marcin; Pająk, Beata; Socha, Anna; Winiarska, Katarzyna; Fronk, Jan; Kurlandzka, Anna

2015-09-01

Glutathione homeostasis is crucial for cell functioning. We describe a novel Imi1 protein of Saccharomyces cerevisiae affecting mitochondrial integrity and involved in controlling glutathione level. Imi1 is cytoplasmic and, except for its N-terminal Flo11 domain, has a distinct solenoid structure. A lack of Imi1 leads to mitochondrial lesions comprising aberrant morphology of cristae and multifarious mtDNA rearrangements and impaired respiration. The mitochondrial malfunctioning is coupled to significantly decrease the level of intracellular reduced glutathione without affecting oxidized glutathione, which decreases the reduced/oxidized glutathione ratio. These defects are accompanied by decreased cadmium sensitivity and increased phytochelatin-2 level. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Nucleocytoplasmic transport of nucleocapsid proteins of enveloped RNA viruses

Directory of Open Access Journals (Sweden)

Wahyu eWulan

2015-06-01

Full Text Available Most viruses with non-segmented single stranded RNA genomes complete their life cycle in the cytoplasm of infected cells. However, despite undergoing replication in the cytoplasm, the structural proteins of some of these RNA viruses localize to the nucleus at specific times in the virus life cycle, primarily early in infection. Limited evidence suggests that this enhances successful viral replication by interfering with or inhibiting the host antiviral response. Nucleocapsid proteins of RNA viruses have a well-established, essential cytoplasmic role in virus replication and assembly. Intriguingly, nucleocapsid proteins of some RNA viruses also localize to the nucleus/nucleolus of infected cells. Their nuclear function is less well understood although significant advances have been made in recent years. This review will focus on the nucleocapsid protein of cytoplasmic enveloped RNA viruses, including their localization to the nucleus/nucleolus and function therein. A greater understanding of the nuclear localization of nucleocapsid proteins has the potential to enhance therapeutic strategies as it can be a target for the development of live-attenuated vaccines or antiviral drugs.

Exploiting genomic data to identify proteins involved in abalone reproduction.

Science.gov (United States)

Mendoza-Porras, Omar; Botwright, Natasha A; McWilliam, Sean M; Cook, Mathew T; Harris, James O; Wijffels, Gene; Colgrave, Michelle L

2014-08-28

Aside from their critical role in reproduction, abalone gonads serve as an indicator of sexual maturity and energy balance, two key considerations for effective abalone culture. Temperate abalone farmers face issues with tank restocking with highly marketable abalone owing to inefficient spawning induction methods. The identification of key proteins in sexually mature abalone will serve as the foundation for a greater understanding of reproductive biology. Addressing this knowledge gap is the first step towards improving abalone aquaculture methods. Proteomic profiling of female and male gonads of greenlip abalone, Haliotis laevigata, was undertaken using liquid chromatography-mass spectrometry. Owing to the incomplete nature of abalone protein databases, in addition to searching against two publicly available databases, a custom database comprising genomic data was used. Overall, 162 and 110 proteins were identified in females and males respectively with 40 proteins common to both sexes. For proteins involved in sexual maturation, sperm and egg structure, motility, acrosomal reaction and fertilization, 23 were identified only in females, 18 only in males and 6 were common. Gene ontology analysis revealed clear differences between the female and male protein profiles reflecting a higher rate of protein synthesis in the ovary and higher metabolic activity in the testis. A comprehensive mass spectrometry-based analysis was performed to profile the abalone gonad proteome providing the foundation for future studies of reproduction in abalone. Key proteins involved in both reproduction and energy balance were identified. Genomic resources were utilised to build a database of molluscan proteins yielding >60% more protein identifications than in a standard workflow employing public protein databases. Copyright © 2014 Elsevier B.V. All rights reserved.

Cytoplasmic streaming velocity as a plant size determinant.

Science.gov (United States)

Tominaga, Motoki; Kimura, Atsushi; Yokota, Etsuo; Haraguchi, Takeshi; Shimmen, Teruo; Yamamoto, Keiichi; Nakano, Akihiko; Ito, Kohji

2013-11-11

Cytoplasmic streaming is active transport widely occurring in plant cells ranging from algae to angiosperms. Although it has been revealed that cytoplasmic streaming is generated by organelle-associated myosin XI moving along actin bundles, the fundamental function in plants remains unclear. We generated high- and low-speed chimeric myosin XI by replacing the motor domains of Arabidopsis thaliana myosin XI-2 with those of Chara corallina myosin XI and Homo sapiens myosin Vb, respectively. Surprisingly, the plant sizes of the transgenic Arabidopsis expressing high- and low-speed chimeric myosin XI-2 were larger and smaller, respectively, than that of the wild-type plant. This size change correlated with acceleration and deceleration, respectively, of cytoplasmic streaming. Our results strongly suggest that cytoplasmic streaming is a key determinant of plant size. Furthermore, because cytoplasmic streaming is a common system for intracellular transport in plants, our system could have applications in artificial size control in plants. Copyright © 2013 Elsevier Inc. All rights reserved.

SPRED: A machine learning approach for the identification of classical and non-classical secretory proteins in mammalian genomes

Energy Technology Data Exchange (ETDEWEB)

Kandaswamy, Krishna Kumar [Institute for Neuro- and Bioinformatics, University of Luebeck, 23538 Luebeck (Germany); Graduate School for Computing in Medicine and Life Sciences, University of Luebeck, 23538 Luebeck (Germany); Pugalenthi, Ganesan [School of Electrical and Electronic Engineering, Nanyang Technological University, Singapore 639798 (Singapore); Hartmann, Enno; Kalies, Kai-Uwe [Centre for Structural and Cell Biology in Medicine, Institute of Biology, University of Luebeck, 23538 Luebeck (Germany); Moeller, Steffen [Institute for Neuro- and Bioinformatics, University of Luebeck, 23538 Luebeck (Germany); Suganthan, P.N. [School of Electrical and Electronic Engineering, Nanyang Technological University, Singapore 639798 (Singapore); Martinetz, Thomas, E-mail: martinetz@inb.uni-luebeck.de [Institute for Neuro- and Bioinformatics, University of Luebeck, 23538 Luebeck (Germany)

2010-01-15

Eukaryotic protein secretion generally occurs via the classical secretory pathway that traverses the ER and Golgi apparatus. Secreted proteins usually contain a signal sequence with all the essential information required to target them for secretion. However, some proteins like fibroblast growth factors (FGF-1, FGF-2), interleukins (IL-1 alpha, IL-1 beta), galectins and thioredoxin are exported by an alternative pathway. This is known as leaderless or non-classical secretion and works without a signal sequence. Most computational methods for the identification of secretory proteins use the signal peptide as indicator and are therefore not able to identify substrates of non-classical secretion. In this work, we report a random forest method, SPRED, to identify secretory proteins from protein sequences irrespective of N-terminal signal peptides, thus allowing also correct classification of non-classical secretory proteins. Training was performed on a dataset containing 600 extracellular proteins and 600 cytoplasmic and/or nuclear proteins. The algorithm was tested on 180 extracellular proteins and 1380 cytoplasmic and/or nuclear proteins. We obtained 85.92% accuracy from training and 82.18% accuracy from testing. Since SPRED does not use N-terminal signals, it can detect non-classical secreted proteins by filtering those secreted proteins with an N-terminal signal by using SignalP. SPRED predicted 15 out of 19 experimentally verified non-classical secretory proteins. By scanning the entire human proteome we identified 566 protein sequences potentially undergoing non-classical secretion. The dataset and standalone version of the SPRED software is available at (http://www.inb.uni-luebeck.de/tools-demos/spred/spred).

SPRED: A machine learning approach for the identification of classical and non-classical secretory proteins in mammalian genomes

International Nuclear Information System (INIS)

Kandaswamy, Krishna Kumar; Pugalenthi, Ganesan; Hartmann, Enno; Kalies, Kai-Uwe; Moeller, Steffen; Suganthan, P.N.; Martinetz, Thomas

2010-01-01

Eukaryotic protein secretion generally occurs via the classical secretory pathway that traverses the ER and Golgi apparatus. Secreted proteins usually contain a signal sequence with all the essential information required to target them for secretion. However, some proteins like fibroblast growth factors (FGF-1, FGF-2), interleukins (IL-1 alpha, IL-1 beta), galectins and thioredoxin are exported by an alternative pathway. This is known as leaderless or non-classical secretion and works without a signal sequence. Most computational methods for the identification of secretory proteins use the signal peptide as indicator and are therefore not able to identify substrates of non-classical secretion. In this work, we report a random forest method, SPRED, to identify secretory proteins from protein sequences irrespective of N-terminal signal peptides, thus allowing also correct classification of non-classical secretory proteins. Training was performed on a dataset containing 600 extracellular proteins and 600 cytoplasmic and/or nuclear proteins. The algorithm was tested on 180 extracellular proteins and 1380 cytoplasmic and/or nuclear proteins. We obtained 85.92% accuracy from training and 82.18% accuracy from testing. Since SPRED does not use N-terminal signals, it can detect non-classical secreted proteins by filtering those secreted proteins with an N-terminal signal by using SignalP. SPRED predicted 15 out of 19 experimentally verified non-classical secretory proteins. By scanning the entire human proteome we identified 566 protein sequences potentially undergoing non-classical secretion. The dataset and standalone version of the SPRED software is available at (http://www.inb.uni-luebeck.de/tools-demos/spred/spred).

Quantification of cytosolic interactions identifies Ede1 oligomers as key organizers of endocytosis.

Science.gov (United States)

Boeke, Dominik; Trautmann, Susanne; Meurer, Matthias; Wachsmuth, Malte; Godlee, Camilla; Knop, Michael; Kaksonen, Marko

2014-11-03

Clathrin-mediated endocytosis is a highly conserved intracellular trafficking pathway that depends on dynamic protein-protein interactions between up to 60 different proteins. However, little is known about the spatio-temporal regulation of these interactions. Using fluorescence (cross)-correlation spectroscopy in yeast, we tested 41 previously reported interactions in vivo and found 16 to exist in the cytoplasm. These detected cytoplasmic interactions included the self-interaction of Ede1, homolog of mammalian Eps15. Ede1 is the crucial scaffold for the organization of the early stages of endocytosis. We show that oligomerization of Ede1 through its central coiled coil domain is necessary for its localization to the endocytic site and we link the oligomerization of Ede1 to its function in locally concentrating endocytic adaptors and organizing the endocytic machinery. Our study sheds light on the importance of the regulation of protein-protein interactions in the cytoplasm for the assembly of the endocytic machinery in vivo. © 2014 The Authors. Published under the terms of the CC BY 4.0 license.

Human hnRNP Q re-localizes to cytoplasmic granules upon PMA, thapsigargin, arsenite and heat-shock treatments

International Nuclear Information System (INIS)

Quaresma, Alexandre J.C.; Bressan, G.C.; Gava, L.M.; Lanza, D.C.F.; Ramos, C.H.I; Kobarg, Joerg

2009-01-01

Eukaryotic gene expression is regulated on different levels ranging from pre-mRNA processing to translation. One of the most characterized families of RNA-binding proteins is the group of hnRNPs: heterogenous nuclear ribonucleoproteins. Members of this protein family play important roles in gene expression control and mRNAs metabolism. In the cytoplasm, several hnRNPs proteins are involved in RNA-related processes and they can be frequently found in two specialized structures, known as GW-bodies (GWbs), previously known as processing bodies: PBs, and stress granules, which may be formed in response to specific stimuli. GWbs have been early reported to be involved in the mRNA decay process, acting as a site of mRNA degradation. In a similar way, stress granules (SGs) have been described as cytoplasmic aggregates, which contain accumulated mRNAs in cells under stress conditions and present reduced or inhibited translation. Here, we characterized the hnRNP Q localization after different stress conditions. hnRNP Q is a predominantly nuclear protein that exhibits a modular organization and several RNA-related functions. Our data suggest that the nuclear localization of hnRNP Q might be modified after different treatments, such as: PMA, thapsigargin, arsenite and heat shock. Under different stress conditions, hnRNP Q can fully co-localize with the endoplasmatic reticulum specific chaperone, BiP. However, under stress, this protein only co-localizes partially with the proteins: GW182 - GWbs marker protein and TIA-1 stress granule component

Inhibition of chloroplast protein synthesis following light chilling of tomato

International Nuclear Information System (INIS)

Kent, J.; Ort, D.

1989-01-01

In the present study we looked at the effects of a high light chill on the pulsed incorporation of 35 S methionine into total, stromal, and thylakoid proteins of lightly abraded leaflets of 18-21 day old tomato (Lycopersicon esculentum Mill ca. Floramerica) seedlings. Based on gel fluorographic patterns of marker proteins that are indicative of the net rates of chloroplast and cytoplasmic protein synthesis, there appears to be a nearly complete cessation of chloroplastic protein synthesis. No labeling is observed for either the stromal large subunit of Rubisco or the thylakoid-bound alpha and beta subunits of the coupling factor. One notable exception, however, appears to be the 32 kd, D1 protein. Its net synthetic rate remains high despite the inhibition of other chloroplastically synthesized proteins. The small subunit of Rubicso, LHCP-II, as well as several other proteins of known cytoplasmic origin, were still synthesized, albeit, at lower than control rates. Light chilling of chill-insensitive spinach produced a similar, but less dramatic differential behavior between chloroplastic and cytoplasmic protein synthesis. It appears, in chilling-sensitive plants, that chloroplast protein synthesis exhibits a greater sensitivity to low temperature inhibition than does cytoplasmic protein synthesis and that recovery of chloroplast protein synthesis may play an important role in recovery of photosynthetic activity following chilling

Studies on Antifungal Potential, Primary Characterization and Mode of Action of a De Novo Cytoplasmic Protein (EAF) from Human Commensal Escherichia coli Against Aspergillus spp.

Science.gov (United States)

Balhara, Meenakshi; Ruhil, Sonam; Dhankhar, Sandeep; Chhillar, Anil K

2015-01-01

A de novo protein named as EAF (Escherichia antifungal protein) from the cytoplasmic pool of an Escherichia coli strain (MTCC 1652), has been purified to homogeneity using anion exchange (Q-XL Sepharose) and cation exchange (SP-Sepharose) chromatography. The MIC (minimum inhibitory concentration) values of purified protein against A. fumigatus (the major pathogenic species) were found to be comparable with standard drugs i.e. 3.90 µg/ml, 3.90 µg/ml and 1.25 µg/disc via microbroth dilution assay (MDA), percentage spore germination inhibition (PSGI) and disc diffusion assay (DDA) respectively. Toxicity results confirmed that it causes no haemolysis against human RBCs upto a concentration of 1000.0 µg/ml as compared to Amphotericin B (conventional antifungal drug) that causes hundred percent haemolysis at a concentration of 37.50 µg/ml only.The purified protein demonstrated a molecular mass of 28 kDa on SDS-PAGE which was further authenticated by MALDI-TOF. Proteomic and bioinformatics studies deciphered its significant homology (72 %) with chain A-D-ribose binding protein (cluster 2 sugar binding periplasmic proteins; sequence homologues of transcription regulatory proteins) from E. coli. Single dimensional page analysis of A. fumigatusproteins with due effect of EAF (at MIC50) revealed the inhibition of two major proteins; a heat shock protein 70-Hsp70 (68 kDa); having role in protein folding and functioning andphenylanalyl-t RNA synthetase PodG subunit protein (74 kDa); involved in growth polarity in fungi. Scanning electron microscopic studies depicted homologous results. We suggest that EAF most likely belongs to a new group of proteins with potent antifungal characteristics, negligible toxicity and targeting vital proteins of fungal metabolism.

Imaging Nuclear-Cytoplasmic Dynamics in Primary and Metastatic Colon Cancer in Nude Mice.

Science.gov (United States)

Hasegawa, Kosuke; Suetsugu, Atsushi; Nakamura, Miki; Matsumoto, Takuro; Aoki, Hitomi; Kunisada, Takahiro; Bouvet, Michael; Shimizu, Masahito; Hoffman, Robert M

2016-05-01

Colon cancer frequently results in metastasis to the liver, where it becomes the main cause of death. However, the cell cycle in primary tumors and metastases is poorly understood. We developed a mouse model of liver metastasis using the human colon cancer cell line HCT-116, which expresses green fluorescent protein (GFP) in the nucleus and red fluorescent protein (RFP) in the cytoplasm (HCT-116-GFP-RFP). HCT-116 GFP-RFP cells were injected into the spleen of nu/nu nude mice. HCT-116-GFP-RFP cells subsequently formed primary tumors in the spleen, as well as metastatic colonies in the liver and retroperitoneum by 28 days after cell transplantation. Using an Olympus FV1000 confocal microscope, it was possible to clearly image mitosis of the dual-colored colon cancer cells in the primary tumor as well as liver and other metastases. Multi-nucleate cancer cells, in addition to mono-nucleate cancer cells and their mitosis, were observed in the primary tumor and metastasis. Multi-nucleate HCT-116-GFP-RFP cells were also observed after culture of the primary and metastatic tumors. A similar ratio of mono-nucleate, multi-nucleate, and mitotic cells grew from the primary and metastatic tumors in culture, suggesting similarity of the nuclear-cytoplasmic dynamics of primary and metastatic cancer cells, further emphasizing the stochastic nature of metastasis. Our results demonstrate a similar heterogeneity of nuclear-cytoplasmic dynamics within primary tumors and metastases, which may be an important factor in the stochastic nature of metastasis. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Algorithms for Cytoplasm Segmentation of Fluorescence Labelled Cells

Directory of Open Access Journals (Sweden)

Carolina Wählby

2002-01-01

Full Text Available Automatic cell segmentation has various applications in cytometry, and while the nucleus is often very distinct and easy to identify, the cytoplasm provides a lot more challenge. A new combination of image analysis algorithms for segmentation of cells imaged by fluorescence microscopy is presented. The algorithm consists of an image pre‐processing step, a general segmentation and merging step followed by a segmentation quality measurement. The quality measurement consists of a statistical analysis of a number of shape descriptive features. Objects that have features that differ to that of correctly segmented single cells can be further processed by a splitting step. By statistical analysis we therefore get a feedback system for separation of clustered cells. After the segmentation is completed, the quality of the final segmentation is evaluated. By training the algorithm on a representative set of training images, the algorithm is made fully automatic for subsequent images created under similar conditions. Automatic cytoplasm segmentation was tested on CHO‐cells stained with calcein. The fully automatic method showed between 89% and 97% correct segmentation as compared to manual segmentation.

Mitochondrial Protein Synthesis, Import, and Assembly

Science.gov (United States)

Fox, Thomas D.

2012-01-01

The mitochondrion is arguably the most complex organelle in the budding yeast cell cytoplasm. It is essential for viability as well as respiratory growth. Its innermost aqueous compartment, the matrix, is bounded by the highly structured inner membrane, which in turn is bounded by the intermembrane space and the outer membrane. Approximately 1000 proteins are present in these organelles, of which eight major constituents are coded and synthesized in the matrix. The import of mitochondrial proteins synthesized in the cytoplasm, and their direction to the correct soluble compartments, correct membranes, and correct membrane surfaces/topologies, involves multiple pathways and macromolecular machines. The targeting of some, but not all, cytoplasmically synthesized mitochondrial proteins begins with translation of messenger RNAs localized to the organelle. Most proteins then pass through the translocase of the outer membrane to the intermembrane space, where divergent pathways sort them to the outer membrane, inner membrane, and matrix or trap them in the intermembrane space. Roughly 25% of mitochondrial proteins participate in maintenance or expression of the organellar genome at the inner surface of the inner membrane, providing 7 membrane proteins whose synthesis nucleates the assembly of three respiratory complexes. PMID:23212899

Use of a Granulocyte Immunofluorescence Assay Designed for Humans for Detection of Antineutrophil Cytoplasmic Antibodies in Dogs with Chronic Enteropathies.

Science.gov (United States)

Florey, J; Viall, A; Streu, S; DiMuro, V; Riddle, A; Kirk, J; Perazzotti, L; Affeldt, K; Wagner, R; Vaden, S; Harris, T; Allenspach, K

2017-07-01

Perinuclear antineutrophil cytoplasmic antibodies (pANCA) previously have been shown to be serum markers in dogs with chronic enteropathies, with dogs that have food-responsive disease (FRD) having higher frequencies of seropositivity than dogs with steroid-responsive disease (SRD). The indirect immunofluorescence (IIF) assay used in previous publications is time-consuming to perform, with low interobserver agreement. We hypothesized that a commercially available granulocyte IIF assay designed for humans could be used to detect perinuclear antineutrophil cytoplasmic antibodies in dogs. Forty-four dogs with FRD, 20 dogs with SRD, 20 control dogs, and 38 soft-coated wheaten terrier (SCWT) or SCWT-cross dogs. A granulocyte assay designed for humans was used to detect pANCA, cANCA, and antinuclear antibodies (ANA), as well as antibodies against proteinase-3 protein (PR-3) and myeloperoxidase protein (MPO) in archived serum samples. Sensitivity of the granulocyte assay to predict FRD in dogs was 0.61 (95% confidence interval (CI), 0.45, 0.75), and specificity was 1.00 (95% CI, 0.91, 1.00). A significant association was identified between positive pANCA or cANCA result and diagnosis of FRD (P < 0.0001). Agreement between the two assays to detect ANCA in the same serum samples from SCWT with protein-losing enteropathy/protein-losing nephropathy (PLE/PLN) was substantial (kappa, 0.77; 95% CI, 0.53, 1.00). Eight ANCA-positive cases were positive for MPO or PR-3 antibodies. The granulocyte immunofluorescence assay used in our pilot study was easy and quick to perform. Agreement with the previously published method was good. Copyright © 2017 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.

The coat protein complex II, COPII, protein Sec13 directly interacts with presenilin-1

International Nuclear Information System (INIS)

Nielsen, Anders Lade

2009-01-01

Mutations in the human gene encoding presenilin-1, PS1, account for most cases of early-onset familial Alzheimer's disease. PS1 has nine transmembrane domains and a large loop orientated towards the cytoplasm. PS1 locates to cellular compartments as endoplasmic reticulum (ER), Golgi apparatus, vesicular structures, and plasma membrane, and is an integral member of γ-secretase, a protein protease complex with specificity for intra-membranous cleavage of substrates such as β-amyloid precursor protein. Here, an interaction between PS1 and the Sec13 protein is described. Sec13 takes part in coat protein complex II, COPII, vesicular trafficking, nuclear pore function, and ER directed protein sequestering and degradation control. The interaction maps to the N-terminal part of the large hydrophilic PS1 loop and the first of the six WD40-repeats present in Sec13. The identified Sec13 interaction to PS1 is a new candidate interaction for linking PS1 to secretory and protein degrading vesicular circuits.

The coat protein complex II, COPII, protein Sec13 directly interacts with presenilin-1

Energy Technology Data Exchange (ETDEWEB)

Nielsen, Anders Lade, E-mail: aln@humgen.au.dk [Department of Human Genetics, The Bartholin Building, University of Aarhus, DK-8000 Aarhus C (Denmark)

2009-10-23

Mutations in the human gene encoding presenilin-1, PS1, account for most cases of early-onset familial Alzheimer's disease. PS1 has nine transmembrane domains and a large loop orientated towards the cytoplasm. PS1 locates to cellular compartments as endoplasmic reticulum (ER), Golgi apparatus, vesicular structures, and plasma membrane, and is an integral member of {gamma}-secretase, a protein protease complex with specificity for intra-membranous cleavage of substrates such as {beta}-amyloid precursor protein. Here, an interaction between PS1 and the Sec13 protein is described. Sec13 takes part in coat protein complex II, COPII, vesicular trafficking, nuclear pore function, and ER directed protein sequestering and degradation control. The interaction maps to the N-terminal part of the large hydrophilic PS1 loop and the first of the six WD40-repeats present in Sec13. The identified Sec13 interaction to PS1 is a new candidate interaction for linking PS1 to secretory and protein degrading vesicular circuits.

HuR cytoplasmic expression is associated with increased cyclin A expression and poor outcome with upper urinary tract urothelial carcinoma

International Nuclear Information System (INIS)

Liang, Peir-In; Shiue, Yow-Ling; Huang, Hsuan-Ying; Hsu, Han-Ping; Chen, Li-Tzon; Lin, Ching-Yih; Tai, Chein; Lin, Chun-Mao; Li, Chien-Feng; Li, Wei-Ming; Wang, Yu-Hui; Wu, Ting-Feng; Wu, Wen-Ren; Liao, Alex C; Shen, Kun-Hung; Wei, Yu-Ching; Hsing, Chung-Hsi

2012-01-01

HuR is an RNA-binding protein that post-transcriptionally modulates the expressions of various target genes implicated in carcinogenesis, such as CCNA2 encoding cyclin A. No prior study attempted to evaluate the significance of HuR expression in a large cohort with upper urinary tract urothelial carcinomas (UTUCs). In total, 340 cases of primary localized UTUC without previous or concordant bladder carcinoma were selected. All of these patients received ureterectomy or radical nephroureterectomy with curative intents. Pathological slides were reviewed, and clinical findings were collected. Immunostaining for HuR and cyclin A was performed and evaluated by using H-score. The results of cytoplasmic HuR and nuclear cyclin A expressions were correlated with disease-specific survival (DSS), metastasis-free survival (MeFS), urinary bladder recurrence-free survival (UBRFS), and various clinicopathological factors. HuR cytoplasmic expression was significantly related to the pT status, lymph node metastasis, a higher histological grade, the pattern of invasion, vascular and perineurial invasion, and cyclin A expression (p = 0.005). Importantly, HuR cytoplasmic expression was strongly associated with a worse DSS (p < 0.0001), MeFS (p < 0.0001), and UBRFS (p = 0.0370) in the univariate analysis, and the first two results remained independently predictive of adverse outcomes (p = 0.038, relative risk [RR] = 1.996 for DSS; p = 0.027, RR = 1.880 for MeFS). Cyclin A nuclear expression was associated with a poor DSS (p = 0.0035) and MeFS (p = 0.0015) in the univariate analysis but was not prognosticatory in the multivariate analyses. High-risk patients (pT3 or pT4 with/without nodal metastasis) with high HuR cytoplasmic expression had better DSS if adjuvant chemotherapy was performed (p = 0.015). HuR cytoplasmic expression was correlated with adverse phenotypes and cyclin A overexpression and also independently predictive of worse DSS and MeFS, suggesting its roles in

Tolerance induction to cytoplasmic beta-galactosidase by hepatic AAV gene transfer: implications for antigen presentation and immunotoxicity.

Directory of Open Access Journals (Sweden)

Ashley T Martino

2009-08-01

Full Text Available Hepatic gene transfer, in particular using adeno-associated viral (AAV vectors, has been shown to induce immune tolerance to several protein antigens. This approach has been exploited in animal models of inherited protein deficiency for systemic delivery of therapeutic proteins. Adequate levels of transgene expression in hepatocytes induce a suppressive T cell response, thereby promoting immune tolerance. This study addresses the question of whether AAV gene transfer can induce tolerance to a cytoplasmic protein.AAV-2 vector-mediated hepatic gene transfer for expression of cytoplasmic beta-galactosidase (beta-gal was performed in immune competent mice, followed by a secondary beta-gal gene transfer with E1/E3-deleted adenoviral Ad-LacZ vector to provoke a severe immunotoxic response. Transgene expression from the AAV-2 vector in approximately 2% of hepatocytes almost completely protected from inflammatory T cell responses against beta-gal, eliminated antibody formation, and significantly reduced adenovirus-induced hepatotoxicity. Consequently, approximately 10% of hepatocytes continued to express beta-gal 45 days after secondary Ad-LacZ gene transfer, a time point when control mice had lost all Ad-LacZ derived expression. Suppression of inflammatory T cell infiltration in the liver and liver damage was linked to specific transgene expression and was not seen for secondary gene transfer with Ad-GFP. A combination of adoptive transfer studies and flow cytometric analyses demonstrated induction of Treg that actively suppressed CD8(+ T cell responses to beta-gal and that was amplified in liver and spleen upon secondary Ad-LacZ gene transfer.These data demonstrate that tolerance induction by hepatic AAV gene transfer does not require systemic delivery of the transgene product and that expression of a cytoplasmic neo-antigen in few hepatocytes can induce Treg and provide long-term suppression of inflammatory responses and immunotoxicity.

Gene Unprediction with Spurio: A tool to identify spurious protein sequences.

Science.gov (United States)

Höps, Wolfram; Jeffryes, Matt; Bateman, Alex

2018-01-01

We now have access to the sequences of tens of millions of proteins. These protein sequences are essential for modern molecular biology and computational biology. The vast majority of protein sequences are derived from gene prediction tools and have no experimental supporting evidence for their translation.  Despite the increasing accuracy of gene prediction tools there likely exists a large number of spurious protein predictions in the sequence databases.  We have developed the Spurio tool to help identify spurious protein predictions in prokaryotes.  Spurio searches the query protein sequence against a prokaryotic nucleotide database using tblastn and identifies homologous sequences. The tblastn matches are used to score the query sequence's likelihood of being a spurious protein prediction using a Gaussian process model. The most informative feature is the appearance of stop codons within the presumed translation of homologous DNA sequences. Benchmarking shows that the Spurio tool is able to distinguish spurious from true proteins. However, transposon proteins are prone to be predicted as spurious because of the frequency of degraded homologs found in the DNA sequence databases. Our initial experiments suggest that less than 1% of the proteins in the UniProtKB sequence database are likely to be spurious and that Spurio is able to identify over 60 times more spurious proteins than the AntiFam resource. The Spurio software and source code is available under an MIT license at the following URL: https://bitbucket.org/bateman-group/spurio.

Cytoplasmic expression of survivin is an independent predictor of poor prognosis in patients with salivary gland cancer.

Science.gov (United States)

Stenner, Markus; Weinell, Antje; Ponert, Tobias; Hardt, Aline; Hahn, Moritz; Preuss, Simon F; Guntinas-Lichius, Orlando; Klussmann, Jens Peter

2010-11-01

The expression of the inhibitor of apoptosis protein survivin has been shown to be a significant prognostic indicator in various human cancers. The aim was to assess its expression and prognostic value in salivary gland adenocarcinoma and muco-epidermoid carcinoma. Survivin expression was analysed in 48 patients with parotid gland cancer (21 muco-epidermoid, 27 adenocarcinomas) by means of immunohistochemistry. The experimental findings were correlated with clinicopathological and survival parameters. A high cytoplasmic expression of survivin was found in 30% of the examined tumours without any significant correlation with the patients' clinicopathological characteristics (P > 0.05). Within all patients, the estimated overall survival rate of muco-epidermoid carcinomas was significantly better than that of adenocarcinomas (P = 0.013). A high cytoplasmic survivin expression significantly indicated a poor 5-year disease-free survival rate compared to patients with a low cytoplasmic survivin expression in the whole group (P = 0.001) and in adenocarcinomas (P = 0.004). In a multivariate analysis, a high cytoplasmic survivin expression was the only independent prognostic indicator for a significantly poorer 5-year disease-free survival rate (P = 0.001). The correlation between cytoplasmic survivin expression and survival in salivary gland malignancies might make this an effective tool in patient follow-up, prognosis and targeted therapy in future. © 2010 Blackwell Publishing Limited.

Cytoplasmic Streaming in the Drosophila Oocyte.

Science.gov (United States)

Quinlan, Margot E

2016-10-06

Objects are commonly moved within the cell by either passive diffusion or active directed transport. A third possibility is advection, in which objects within the cytoplasm are moved with the flow of the cytoplasm. Bulk movement of the cytoplasm, or streaming, as required for advection, is more common in large cells than in small cells. For example, streaming is observed in elongated plant cells and the oocytes of several species. In the Drosophila oocyte, two stages of streaming are observed: relatively slow streaming during mid-oogenesis and streaming that is approximately ten times faster during late oogenesis. These flows are implicated in two processes: polarity establishment and mixing. In this review, I discuss the underlying mechanism of streaming, how slow and fast streaming are differentiated, and what we know about the physiological roles of the two types of streaming.

Coordinated movement of cytoplasmic and transmembrane domains of RyR1 upon gating.

Directory of Open Access Journals (Sweden)

Montserrat Samsó

2009-04-01

Full Text Available Ryanodine receptor type 1 (RyR1 produces spatially and temporally defined Ca2+ signals in several cell types. How signals received in the cytoplasmic domain are transmitted to the ion gate and how the channel gates are unknown. We used EGTA or neuroactive PCB 95 to stabilize the full closed or open states of RyR1. Single-channel measurements in the presence of FKBP12 indicate that PCB 95 inverts the thermodynamic stability of RyR1 and locks it in a long-lived open state whose unitary current is indistinguishable from the native open state. We analyzed two datasets of 15,625 and 18,527 frozen-hydrated RyR1-FKBP12 particles in the closed and open conformations, respectively, by cryo-electron microscopy. Their corresponding three-dimensional structures at 10.2 A resolution refine the structure surrounding the ion pathway previously identified in the closed conformation: two right-handed bundles emerging from the putative ion gate (the cytoplasmic "inner branches" and the transmembrane "inner helices". Furthermore, six of the identifiable transmembrane segments of RyR1 have similar organization to those of the mammalian Kv1.2 potassium channel. Upon gating, the distal cytoplasmic domains move towards the transmembrane domain while the central cytoplasmic domains move away from it, and also away from the 4-fold axis. Along the ion pathway, precise relocation of the inner helices and inner branches results in an approximately 4 A diameter increase of the ion gate. Whereas the inner helices of the K+ channels and of the RyR1 channel cross-correlate best with their corresponding open/closed states, the cytoplasmic inner branches, which are not observed in the K+ channels, appear to have at least as important a role as the inner helices for RyR1 gating. We propose a theoretical model whereby the inner helices, the inner branches, and the h1 densities together create an efficient novel gating mechanism for channel opening by relaxing two right

Semantic integration to identify overlapping functional modules in protein interaction networks

Directory of Open Access Journals (Sweden)

Ramanathan Murali

2007-07-01

Full Text Available Abstract Background The systematic analysis of protein-protein interactions can enable a better understanding of cellular organization, processes and functions. Functional modules can be identified from the protein interaction networks derived from experimental data sets. However, these analyses are challenging because of the presence of unreliable interactions and the complex connectivity of the network. The integration of protein-protein interactions with the data from other sources can be leveraged for improving the effectiveness of functional module detection algorithms. Results We have developed novel metrics, called semantic similarity and semantic interactivity, which use Gene Ontology (GO annotations to measure the reliability of protein-protein interactions. The protein interaction networks can be converted into a weighted graph representation by assigning the reliability values to each interaction as a weight. We presented a flow-based modularization algorithm to efficiently identify overlapping modules in the weighted interaction networks. The experimental results show that the semantic similarity and semantic interactivity of interacting pairs were positively correlated with functional co-occurrence. The effectiveness of the algorithm for identifying modules was evaluated using functional categories from the MIPS database. We demonstrated that our algorithm had higher accuracy compared to other competing approaches. Conclusion The integration of protein interaction networks with GO annotation data and the capability of detecting overlapping modules substantially improve the accuracy of module identification.

The antimicrobial peptides lactoferricin B and magainin 2 cross over the bacterial cytoplasmic membrane and reside in the cytoplasm.

Science.gov (United States)

Haukland, H H; Ulvatne, H; Sandvik, K; Vorland, L H

2001-11-23

The localization of immunolabelled antimicrobial peptides was studied using transmission electron microscopy. Staphylococcus aureus and Escherichia coli were exposed to lactoferricin B (17-41), lactoferricin B (17-31) and D-lactoferricin B (17-31). E. coli was also exposed to cecropin P1 and magainin 2. The lactoferricins were found in the cytoplasm of both bacteria. In S. aureus the amount of cytoplasmic lactoferricin B (17-41) was time- and concentration-dependent, reaching a maximum within 30 min. Cecropin P1 was confined to the cell wall, while magainin 2 was found in the cytoplasm of E. coli. The finding of intracellularly localized magainin is not reported previously.

A coevolution analysis for identifying protein-protein interactions by Fourier transform

Science.gov (United States)

Yin, Changchuan; Yau, Stephen S. -T.

2017-01-01

Protein-protein interactions (PPIs) play key roles in life processes, such as signal transduction, transcription regulations, and immune response, etc. Identification of PPIs enables better understanding of the functional networks within a cell. Common experimental methods for identifying PPIs are time consuming and expensive. However, recent developments in computational approaches for inferring PPIs from protein sequences based on coevolution theory avoid these problems. In the coevolution theory model, interacted proteins may show coevolutionary mutations and have similar phylogenetic trees. The existing coevolution methods depend on multiple sequence alignments (MSA); however, the MSA-based coevolution methods often produce high false positive interactions. In this paper, we present a computational method using an alignment-free approach to accurately detect PPIs and reduce false positives. In the method, protein sequences are numerically represented by biochemical properties of amino acids, which reflect the structural and functional differences of proteins. Fourier transform is applied to the numerical representation of protein sequences to capture the dissimilarities of protein sequences in biophysical context. The method is assessed for predicting PPIs in Ebola virus. The results indicate strong coevolution between the protein pairs (NP-VP24, NP-VP30, NP-VP40, VP24-VP30, VP24-VP40, and VP30-VP40). The method is also validated for PPIs in influenza and E.coli genomes. Since our method can reduce false positive and increase the specificity of PPI prediction, it offers an effective tool to understand mechanisms of disease pathogens and find potential targets for drug design. The Python programs in this study are available to public at URL (https://github.com/cyinbox/PPI). PMID:28430779

A coevolution analysis for identifying protein-protein interactions by Fourier transform.

Directory of Open Access Journals (Sweden)

Changchuan Yin

Full Text Available Protein-protein interactions (PPIs play key roles in life processes, such as signal transduction, transcription regulations, and immune response, etc. Identification of PPIs enables better understanding of the functional networks within a cell. Common experimental methods for identifying PPIs are time consuming and expensive. However, recent developments in computational approaches for inferring PPIs from protein sequences based on coevolution theory avoid these problems. In the coevolution theory model, interacted proteins may show coevolutionary mutations and have similar phylogenetic trees. The existing coevolution methods depend on multiple sequence alignments (MSA; however, the MSA-based coevolution methods often produce high false positive interactions. In this paper, we present a computational method using an alignment-free approach to accurately detect PPIs and reduce false positives. In the method, protein sequences are numerically represented by biochemical properties of amino acids, which reflect the structural and functional differences of proteins. Fourier transform is applied to the numerical representation of protein sequences to capture the dissimilarities of protein sequences in biophysical context. The method is assessed for predicting PPIs in Ebola virus. The results indicate strong coevolution between the protein pairs (NP-VP24, NP-VP30, NP-VP40, VP24-VP30, VP24-VP40, and VP30-VP40. The method is also validated for PPIs in influenza and E.coli genomes. Since our method can reduce false positive and increase the specificity of PPI prediction, it offers an effective tool to understand mechanisms of disease pathogens and find potential targets for drug design. The Python programs in this study are available to public at URL (https://github.com/cyinbox/PPI.

Extracellular signal-regulated kinase 2 (ERK-2) mediated phosphorylation regulates nucleo-cytoplasmic shuttling and cell growth control of Ras-associated tumor suppressor protein, RASSF2

International Nuclear Information System (INIS)

Kumari, Gita; Mahalingam, S.

2009-01-01

Ras GTPase controls the normal cell growth through binding with an array of effector molecules, such as Raf and PI3-kinase in a GTP-dependent manner. RASSF2, a member of the Ras association domain family, is known to be involved in the suppression of cell growth and is frequently down-regulated in various tumor tissues by promoter hypermethylation. In the present study, we demonstrate that RASSF2 shuttles between nucleus and cytoplasm by a signal-mediated process and its export from the nucleus is sensitive to leptomycin B. Amino acids between 240 to 260 in the C-terminus of RASSF2 harbor a functional nuclear export signal (NES), which is necessary and sufficient for efficient export of RASSF2 from the nucleus. Substitution of conserved Ile254, Val257 and Leu259 within the minimal NES impaired RASSF2 export from the nucleus. In addition, wild type but not the nuclear export defective RASSF2 mutant interacts with export receptor, CRM-1 and exported from the nucleus. Surprisingly, we observed nucleolar localization for the nuclear export defective mutant suggesting the possibility that RASSF2 may localize in different cellular compartments transiently in a cell cycle dependent manner and the observed nuclear localization for wild type protein may be due to faster export kinetics from the nucleolus. Furthermore, our data suggest that RASSF2 is specifically phosphorylated by MAPK/ERK-2 and the inhibitors of MAPK pathway impair the phosphorylation and subsequently block the export of RASSF2 from the nucleus. These data clearly suggest that ERK-2 mediated phosphorylation plays an important role in regulating the nucleo-cytoplasmic shuttling of RASSF2. Interestingly, nuclear import defective mutant of RASSF2 failed to induce cell cycle arrest at G1/S phase and apoptosis suggesting that RASSF2 regulates cell growth in a nuclear localization dependent manner. Collectively, these data provided evidence for the first time that MAPK/ERK-2 mediated phosphorylation regulates

Extracellular signal-regulated kinase 2 (ERK-2) mediated phosphorylation regulates nucleo-cytoplasmic shuttling and cell growth control of Ras-associated tumor suppressor protein, RASSF2

Energy Technology Data Exchange (ETDEWEB)

Kumari, Gita [Laboratory of Molecular Virology, Centre for DNA Fingerprinting and Diagnostics, Hyderabad 500076 (India); Mahalingam, S., E-mail: mahalingam@iitm.ac.in [Laboratory of Molecular Virology, Centre for DNA Fingerprinting and Diagnostics, Hyderabad 500076 (India); Department of Biotechnology, Laboratory of Molecular Virology and Cell Biology, Indian Institute of Technology-Madras, Chennai 600 036 (India)

2009-10-01

Ras GTPase controls the normal cell growth through binding with an array of effector molecules, such as Raf and PI3-kinase in a GTP-dependent manner. RASSF2, a member of the Ras association domain family, is known to be involved in the suppression of cell growth and is frequently down-regulated in various tumor tissues by promoter hypermethylation. In the present study, we demonstrate that RASSF2 shuttles between nucleus and cytoplasm by a signal-mediated process and its export from the nucleus is sensitive to leptomycin B. Amino acids between 240 to 260 in the C-terminus of RASSF2 harbor a functional nuclear export signal (NES), which is necessary and sufficient for efficient export of RASSF2 from the nucleus. Substitution of conserved Ile254, Val257 and Leu259 within the minimal NES impaired RASSF2 export from the nucleus. In addition, wild type but not the nuclear export defective RASSF2 mutant interacts with export receptor, CRM-1 and exported from the nucleus. Surprisingly, we observed nucleolar localization for the nuclear export defective mutant suggesting the possibility that RASSF2 may localize in different cellular compartments transiently in a cell cycle dependent manner and the observed nuclear localization for wild type protein may be due to faster export kinetics from the nucleolus. Furthermore, our data suggest that RASSF2 is specifically phosphorylated by MAPK/ERK-2 and the inhibitors of MAPK pathway impair the phosphorylation and subsequently block the export of RASSF2 from the nucleus. These data clearly suggest that ERK-2 mediated phosphorylation plays an important role in regulating the nucleo-cytoplasmic shuttling of RASSF2. Interestingly, nuclear import defective mutant of RASSF2 failed to induce cell cycle arrest at G1/S phase and apoptosis suggesting that RASSF2 regulates cell growth in a nuclear localization dependent manner. Collectively, these data provided evidence for the first time that MAPK/ERK-2 mediated phosphorylation regulates

Recognition of secretory proteins in Escherichia coli requires signals in addition to the signal sequence and slow folding

Directory of Open Access Journals (Sweden)

Flower Ann M

2002-11-01

Full Text Available Abstract Background The Sec-dependent protein export apparatus of Escherichia coli is very efficient at correctly identifying proteins to be exported from the cytoplasm. Even bacterial strains that carry prl mutations, which allow export of signal sequence-defective precursors, accurately differentiate between cytoplasmic and mutant secretory proteins. It was proposed previously that the basis for this precise discrimination is the slow folding rate of secretory proteins, resulting in binding by the secretory chaperone, SecB, and subsequent targeting to translocase. Based on this proposal, we hypothesized that a cytoplasmic protein containing a mutation that slows its rate of folding would be recognized by SecB and therefore targeted to the Sec pathway. In a Prl suppressor strain the mutant protein would be exported to the periplasm due to loss of ability to reject non-secretory proteins from the pathway. Results In the current work, we tested this hypothesis using a mutant form of λ repressor that folds slowly. No export of the mutant protein was observed, even in a prl strain. We then examined binding of the mutant λ repressor to SecB. We did not observe interaction by either of two assays, indicating that slow folding is not sufficient for SecB binding and targeting to translocase. Conclusions These results strongly suggest that to be targeted to the export pathway, secretory proteins contain signals in addition to the canonical signal sequence and the rate of folding.

Functional display of proteins, mutant proteins, fragments of proteins and peptides on the surface of filamentous (bacterio) phages: A review

NARCIS (Netherlands)

Pannekoek, H.; van Meijer, M.; Gaardsvoll, H.; van Zonneveld, A. J.

1995-01-01

Cytoplasmic expression of complex eukaryotic proteins inEscherichia coli usually yields inactive protein preparations. In some cases, (part) of the biological activity can be recovered by rather inefficient denaturation-renaturation procedures. Recently, novel concepts have been developed for the

Microaspiration of esophageal gland cells and cDNA library construction for identifying parasitism genes of plant-parasitic nematodes.

Science.gov (United States)

Hussey, Richard S; Huang, Guozhong; Allen, Rex

2011-01-01

Identifying parasitism genes encoding proteins secreted from a plant-parasitic nematode's esophageal gland cells and injected through its stylet into plant tissue is the key to understanding the molecular basis of nematode parasitism of plants. Parasitism genes have been cloned by directly microaspirating the cytoplasm from the esophageal gland cells of different parasitic stages of cyst or root-knot nematodes to provide mRNA to create a gland cell-specific cDNA library by long-distance reverse-transcriptase polymerase chain reaction. cDNA clones are sequenced and deduced protein sequences with a signal peptide for secretion are identified for high-throughput in situ hybridization to confirm gland-specific expression.

Protein-protein interaction networks identify targets which rescue the MPP+ cellular model of Parkinson’s disease

Science.gov (United States)

Keane, Harriet; Ryan, Brent J.; Jackson, Brendan; Whitmore, Alan; Wade-Martins, Richard

2015-11-01

Neurodegenerative diseases are complex multifactorial disorders characterised by the interplay of many dysregulated physiological processes. As an exemplar, Parkinson’s disease (PD) involves multiple perturbed cellular functions, including mitochondrial dysfunction and autophagic dysregulation in preferentially-sensitive dopamine neurons, a selective pathophysiology recapitulated in vitro using the neurotoxin MPP+. Here we explore a network science approach for the selection of therapeutic protein targets in the cellular MPP+ model. We hypothesised that analysis of protein-protein interaction networks modelling MPP+ toxicity could identify proteins critical for mediating MPP+ toxicity. Analysis of protein-protein interaction networks constructed to model the interplay of mitochondrial dysfunction and autophagic dysregulation (key aspects of MPP+ toxicity) enabled us to identify four proteins predicted to be key for MPP+ toxicity (P62, GABARAP, GBRL1 and GBRL2). Combined, but not individual, knockdown of these proteins increased cellular susceptibility to MPP+ toxicity. Conversely, combined, but not individual, over-expression of the network targets provided rescue of MPP+ toxicity associated with the formation of autophagosome-like structures. We also found that modulation of two distinct proteins in the protein-protein interaction network was necessary and sufficient to mitigate neurotoxicity. Together, these findings validate our network science approach to multi-target identification in complex neurological diseases.

Hydroxychloroquine binding to cytoplasmic domain of Band 3 in human erythrocytes: Novel mechanistic insights into drug structure, efficacy and toxicity.

Science.gov (United States)

Nakagawa, Mizuki; Sugawara, Kotomi; Goto, Tatsufumi; Wakui, Hideki; Nunomura, Wataru

2016-05-13

Hydroxychloroquine (HCQ) is a widely used drug in the treatment of autoimmune diseases, such as arthritis and systemic lupus erythematosus. It has also been prescribed for the treatment of malaria owing to its lower toxicity compared to its closely related compound chloroquine (CQ). However, the mechanisms of action of HCQ in erythrocytes (which bind preferentially this drug) have not been documented and the reasons underlying the lower side effects of HCQ compared to CQ remain unclear. Here we show that, although the activity of erythrocyte lactate dehydrogenase (LDH), but not GAPDH, was inhibited by both HCQ and CQ in vitro, LDH activity in erythrocytes incubated with 20 mM HCQ was not significantly reduced within 5 h in contrast to CQ did. Using HCQ coupled Sepharose chromatography (HCQ-Sepharose), we identified Band 3, spectrin, ankyrin, protein 4.1R and protein 4.2 as HCQ binding proteins in human erythrocyte plasma membrane. Recombinant cytoplasmic N-terminal 43 kDa domain of Band 3 bound to HCQ-Sepharose and was eluted with 40 mM (but not 20 mM) HCQ. Band 3 transport activity was reduced by only 23% in the presence of 20 mM HCQ. Taken together, these data demonstrate that HCQ binds to the cytoplasmic N-terminal domain of Band 3 in human erythrocytes but does not inhibit dramatically its transport activity. We hypothesize that the trapping of HCQ on Band 3 contributes to the lower side effects of the drug on energy production in erythrocytes. Copyright © 2016 Elsevier Inc. All rights reserved.

Rice black-streaked dwarf virus P6 self-interacts to form punctate, viroplasm-like structures in the cytoplasm and recruits viroplasm-associated protein P9-1

Directory of Open Access Journals (Sweden)

Yu Jialin

2011-01-01

Full Text Available Abstract Background Rice black-streaked dwarf virus (RBSDV, a member of the genus Fijivirus within the family Reoviridae, can infect several graminaceous plant species including rice, maize and wheat, and is transmitted by planthoppers. Although several RBSDV proteins have been studied in detail, functions of the nonstructural protein P6 are still largely unknown. Results In the current study, we employed yeast two-hybrid assays, bimolecular fluorescence complementation and subcellular localization experiments to show that P6 can self-interact to form punctate, cytoplasmic viroplasm-like structures (VLS when expressed alone in plant cells. The region from residues 395 to 659 is necessary for P6 self-interaction, whereas two polypeptides (residues 580-620 and 615-655 are involved in the subcellular localization of P6. Furthermore, P6 strongly interacts with the viroplasm-associated protein P9-1 and recruits P9-1 to localize in VLS. The P6 395-659 region is also important for the P6-P9-1 interaction, and deleting any region of P9-1 abolishes this heterologous interaction. Conclusions RBSDV P6 protein has an intrinsic ability to self-interact and forms VLS without other RBSDV proteins or RNAs. P6 recruits P9-1 to VLS by direct protein-protein interaction. This is the first report on the functionality of RBSDV P6 protein. P6 may be involved in the process of viroplasm nucleation and virus morphogenesis.

Profound regulation of Na/K pump activity by transient elevations of cytoplasmic calcium in murine cardiac myocytes.

Science.gov (United States)

Lu, Fang-Min; Deisl, Christine; Hilgemann, Donald W

2016-09-14

Small changes of Na/K pump activity regulate internal Ca release in cardiac myocytes via Na/Ca exchange. We now show conversely that transient elevations of cytoplasmic Ca strongly regulate cardiac Na/K pumps. When cytoplasmic Na is submaximal, Na/K pump currents decay rapidly during extracellular K application and multiple results suggest that an inactivation mechanism is involved. Brief activation of Ca influx by reverse Na/Ca exchange enhances pump currents and attenuates current decay, while repeated Ca elevations suppress pump currents. Pump current enhancement reverses over 3 min, and results are similar in myocytes lacking the regulatory protein, phospholemman. Classical signaling mechanisms, including Ca-activated protein kinases and reactive oxygen, are evidently not involved. Electrogenic signals mediated by intramembrane movement of hydrophobic ions, such as hexyltriphenylphosphonium (C6TPP), increase and decrease in parallel with pump currents. Thus, transient Ca elevation and Na/K pump inactivation cause opposing sarcolemma changes that may affect diverse membrane processes.

Reduced RAN expression and disrupted transport between cytoplasm and nucleus; a key event in Alzheimer's disease pathophysiology.

Directory of Open Access Journals (Sweden)

Diego Mastroeni

Full Text Available Transcription of DNA is essential for cell maintenance and survival; inappropriate localization of proteins that are involved in transcription would be catastrophic. In Alzheimer's disease brains, and in vitro studies, we have found qualitative and quantitative deficits in transport into the nucleus of DNA methyltransferase 1 (DNMT1 and RNA polymerase II (RNA pol II, accompanied by their abnormal sequestration in the cytoplasm. RAN (RAs-related Nuclear protein knockdown, by siRNA and oligomeric Aβ42 treatment in neurons, replicate human data which indicate that transport disruption in AD may be mechanistically linked to reduced expression of RAN, a pivotal molecule in nucleocytoplasmic transport. In vitro studies also indicate a significant role for oligomeric Aβ42 in the observed phenomena. We propose a model in which reduced transcription regulators in the nucleus and their increased presence in the cytoplasm may lead to many of the cellular manifestations of Alzheimer's disease.

Lead-Binding Proteins: A Review

Directory of Open Access Journals (Sweden)

Harvey C. Gonick

2011-01-01

Full Text Available Lead-binding proteins are a series of low molecular weight proteins, analogous to metallothionein, which segregate lead in a nontoxic form in several organs (kidney, brain, lung, liver, erythrocyte. Whether the lead-binding proteins in every organ are identical or different remains to be determined. In the erythrocyte, delta-aminolevulinic acid dehydratase (ALAD isoforms have commanded the greatest attention as proteins and enzymes that are both inhibitable and inducible by lead. ALAD-2, although it binds lead to a greater degree than ALAD-1, appears to bind lead in a less toxic form. What may be of greater significance is that a low molecular weight lead-binding protein, approximately 10 kDa, appears in the erythrocyte once blood lead exceeds 39 μg/dL and eventually surpasses the lead-binding capacity of ALAD. In brain and kidney of environmentally exposed humans and animals, a cytoplasmic lead-binding protein has been identified as thymosin β4, a 5 kDa protein. In kidney, but not brain, another lead-binding protein has been identified as acyl-CoA binding protein, a 9 kDa protein. Each of these proteins, when coincubated with liver ALAD and titrated with lead, diminishes the inhibition of ALAD by lead, verifying their ability to segregate lead in a nontoxic form.

Preliminary X-ray diffraction analysis of YqjH from Escherichia coli: a putative cytoplasmic ferri-siderophore reductase.

Science.gov (United States)

Bamford, Vicki A; Armour, Maria; Mitchell, Sue A; Cartron, Michaël; Andrews, Simon C; Watson, Kimberly A

2008-09-01

YqjH is a cytoplasmic FAD-containing protein from Escherichia coli; based on homology to ViuB of Vibrio cholerae, it potentially acts as a ferri-siderophore reductase. This work describes its overexpression, purification, crystallization and structure solution at 3.0 A resolution. YqjH shares high sequence similarity with a number of known siderophore-interacting proteins and its structure was solved by molecular replacement using the siderophore-interacting protein from Shewanella putrefaciens as the search model. The YqjH structure resembles those of other members of the NAD(P)H:flavin oxidoreductase superfamily.

Zebrafish P54 RNA helicases are cytoplasmic granule residents that are required for development and stress resilience

Directory of Open Access Journals (Sweden)

Cecilia Zampedri

2016-10-01

Full Text Available Stress granules are cytoplasmic foci that directly respond to the protein synthesis status of the cell. Various environmental insults, such as oxidative stress or extreme heat, block protein synthesis; consequently, mRNA will stall in translation, and stress granules will immediately form and become enriched with mRNAs. P54 DEAD box RNA helicases are components of RNA granules such as P-bodies and stress granules. We studied the expression, in cytoplasmic foci, of both zebrafish P54 RNA helicases (P54a and P54b during development and found that they are expressed in cytoplasmic granules under both normal conditions and stress conditions. In zebrafish embryos exposed to heat shock, some proportion of P54a and P54b helicases move to larger granules that exhibit the properties of genuine stress granules. Knockdown of P54a and/or P54b in zebrafish embryos produces developmental abnormalities restricted to the posterior trunk; further, these embryos do not form stress granules, and their survival upon exposure to heat-shock conditions is compromised. Our observations fit the model that cells lacking stress granules have no resilience or ability to recover once the stress has ended, indicating that stress granules play an essential role in the way organisms adapt to a changing environment.

Conserved CPEs in the p53 3' untranslated region influence mRNA stability and protein synthesis

DEFF Research Database (Denmark)

Rosenstierne, Maiken W; Vinther, Jeppe; Mittler, Gerhard

2008-01-01

CaT skin and MCF-7 breast cancer cell lines were established. Quantitative PCR and an enzymatic assay were used to quantify the reporter mRNA and protein levels, respectively. Proteins binding to the CPEs were identified by RNA-immunoprecipitation (IP) and quantitative mass spectroscopy. RESULTS: The wild...... irradiation. Several proteins (including GAPDH, heterogeneous nuclear ribonucleoprotein (hnRNP) D and A/B) were identified from the MCF-7 cytoplasmic extracts that bound specifically to the CPEs. CONCLUSION: Two conserved CPEs in the p53 3'UTR regulate stability and translation of a reporter mRNA in non...

The RanGTP pathway: from nucleo-cytoplasmic transport to spindle assembly and beyond

Directory of Open Access Journals (Sweden)

Tommaso eCavazza

2016-01-01

Full Text Available The small GTPase Ran regulates the interaction of transport receptors with a number of cellular cargo proteins. The high affinity binding of the GTP-bound form of Ran to import receptors promotes cargo release, whereas its binding to export receptors stabilizes their interaction with the cargo. This basic mechanism linked to the asymmetric distribution of the two nucleotide-bound forms of Ran between the nucleus and the cytoplasm generates a switch like mechanism controlling nucleo-cytoplasmic transport. Since 1999, we have known that after nuclear envelope breakdown (NEBD Ran and the above transport receptors also provide a local control over the activity of factors driving spindle assembly and regulating other aspects of cell division. The identification and functional characterization of RanGTP mitotic targets is providing novel insights into mechanisms essential for cell division. Here we review our current knowledge on the RanGTP system and its regulation and we focus on the recent advances made through the characterization of its mitotic targets. We then briefly review the novel functions of the pathway that were recently described. Altogether, the RanGTP system has moonlighting functions exerting a spatial control over protein interactions that drive specific functions depending on the cellular context.

Interspecific variation of intracellular localization and postirradiation movement of Ku70-protein in fibroblastic cells

International Nuclear Information System (INIS)

Endoh, Daiji; Hayashi, Masanobu; Okui, Toyo; Kawase, Shiro; Kon, Yasushiro

2003-01-01

Ku (Ku70 and Ku80) Proteins are known as components of DNA-dependent protein kinase (DNA-PK) and play an important role for DNA repair. We previously reported that more than 70% of Ku proteins were located in cytoplasm of rat cells, the Ku proteins moved into nuclei of normal rat cells after X-irradiation, Ku proteins also moved into nuclei after X-irradiation but were not retained in nucleus of radiosensitive LEC rat cells. While reports have been shown about mechanisms on nuclear localization of Ku proteins, how Ku proteins export from nucleus is poorly understood. Here we show that C-terminal region of Ku70 protein is important for its cytoplasmic localization. When transfected into LEC rat cells, exogenous intact Ku70 (1-609) tagged with enhanced green fluorescent protein (EGFP-Ku70) localized mainly in the cytoplasm, whereas C-terminal-deletion mutant of Ku70 (1-593) tagged with EGFP (EGFP-Ku70D) was mainly localized in the nucleus. After X-irradiation, the endogenous intact EGFP-Ku70 once moved into nucleus, but returned into the cytoplasm. On the other hand, EGFP-Ku70D was retained in nucleus for two hours after X-irradiation. These results suggest that C-terminal region of Ku70 is included in the postirradiation nuclear export. Next, we investigated the intracellular localization of Ku70 proteins and the movement after X-irradiation of fibroblastic cells prepared from some mammalian species. Ku70 proteins were localized in nucleus and the postirradiation-extranuclear transport was not observed in human and African green monkey cells. On the other hand, Ku70 proteins were mainly localized in cytoplasm and moved into nucleus in mouse, Chinese hamster, Golden hamster, cotton rat, squirrel, cat and dog cells. These results may show that alternatively Ku70 protein is localized in the cytoplasm or nucleus depends on species and translocation of cytoplasmic Ku70 into nucleus is a response against low dose irradiation in fibroblasts of rodents, cats and dogs

Affinity of hemoglobin for the cytoplasmic fragment of human erythrocyte membrane band 3. Equilibrium measurements at physiological pH using matrix-bound proteins: the effects of ionic strength, deoxygenation and of 2,3-diphosphoglycerate.

Science.gov (United States)

Chétrite, G; Cassoly, R

1985-10-05

The cytoplasmic fragment of band 3 protein isolated from the human erythrocyte membrane was linked to a CNBr-activated Sepharose matrix in an attempt to measure, in batch experiments, its equilibrium binding constant with oxy- and deoxyhemoglobin at physiological pH and ionic strength values and in the presence or the absence of 2,3-diphosphoglycerate. All the experiments were done at pH 7.2, and equilibrium constants were computed on the basis of one hemoglobin tetramer bound per monomer of fragment. In 10 mM-phosphate buffer, a dissociation constant KD = 2 X 10(-4)M was measured for oxyhemoglobin and was shown to increase to 8 X 10(-4)M in the presence of 50 mM-NaCl. Association could not be demonstrated at higher salt concentrations. Diphosphoglycerate-stripped deoxyhemoglobin was shown to associate more strongly with the cytoplasmic fragment of band 3. In 10 mM-bis-Tris (pH 7.2) and in the presence of 120 mM-NaCl, a dissociation constant KD = 4 X 10(-4)M was measured. Upon addition of increasing amounts of 2,3-diphosphoglycerate, the complex formed between deoxyhemoglobin and the cytoplasmic fragment of band 3 was dissociated. On the reasonable assumption that the hemoglobin binding site present on band 3 fragment was not modified upon linking the protein to the Sepharose matrix, the results indicated that diphosphoglycerate-stripped deoxyhemoglobin or partially liganded hemoglobin tetramers in the T state could bind band 3 inside the intact human red blood cell.

Poliovirus 2A protease triggers a selective nucleo-cytoplasmic redistribution of splicing factors to regulate alternative pre-mRNA splicing.

Directory of Open Access Journals (Sweden)

Enrique Álvarez

Full Text Available Poliovirus protease 2A (2A(pro obstructs host gene expression by reprogramming transcriptional and post-transcriptional regulatory events during infection. Here we demonstrate that expression of 2A(pro induces a selective nucleo-cytoplasm translocation of several important RNA binding proteins and splicing factors. Subcellular fractionation studies, together with immunofluorescence microscopy revealed an asymmetric distribution of HuR and TIA1/TIAR in 2A(pro expressing cells, which modulates splicing of the human Fas exon 6. Consistent with this result, knockdown of HuR or overexpression of TIA1/TIAR, leads to Fas exon 6 inclusion in 2A(pro-expressing cells. Therefore, poliovirus 2A(pro can target alternative pre-mRNA splicing by regulating protein shuttling between the nucleus and the cytoplasm.

In Vitro Analysis of Metabolite Transport Proteins.

Science.gov (United States)

Roell, Marc-Sven; Kuhnert, Franziska; Zamani-Nour, Shirin; Weber, Andreas P M

2017-01-01

The photorespiratory cycle is distributed over four cellular compartments, the chloroplast, peroxisomes, cytoplasm, and mitochondria. Shuttling of photorespiratory intermediates between these compartments is essential to maintain the function of photorespiration. Specific transport proteins mediate the transport across biological membranes and represent important components of the cellular metabolism. Although significant progress was made in the last years on identifying and characterizing new transport proteins, the overall picture of intracellular metabolite transporters is still rather incomplete. The photorespiratory cycle requires at least 25 transmembrane transport steps; however to date only plastidic glycolate/glycerate transporter and the accessory 2-oxoglutarate/malate and glutamate/malate transporters as well as the mitochondrial transporter BOU1 have been identified. The characterization of transport proteins and defining their substrates and kinetics are still major challenges.Here we present a detailed set of protocols for the in vitro characterization of transport proteins. We provide protocols for the isolation of recombinant transport protein expressed in E. coli or Saccharomyces cerevisiae and the extraction of total leaf membrane protein for in vitro analysis of transporter proteins. Further we explain the process of reconstituting transport proteins in artificial lipid vesicles and elucidate the details of transport assays.

Subcellular sites for bacterial protein export

NARCIS (Netherlands)

Campo, Nathalie; Tjalsma, Harold; Buist, Girbe; Stepniak, Dariusz; Meijer, Michel; Veenhuis, Marten; Westermann, Martin; Müller, Jörg P.; Bron, Sierd; Kok, Jan; Kuipers, Oscar P.; Jongbloed, Jan D.H.

2004-01-01

Most bacterial proteins destined to leave the cytoplasm are exported to extracellular compartments or imported into the cytoplasmic membrane via the highly conserved SecA-YEG pathway. In the present studies, the subcellular distributions of core components of this pathway, SecA and SecY, and of the

Subcellular sites for bacterial protein export.

NARCIS (Netherlands)

Campo, N.; Tjalsma, H.; Buist, G.; Stepniak, D.; Meijer, M.; Veenhuis, M.; Westermann, M.; Muller, J.P.; Bron, S.; Kok, J.; Kuipers, O.P.; Jongbloed, J.D.

2004-01-01

Most bacterial proteins destined to leave the cytoplasm are exported to extracellular compartments or imported into the cytoplasmic membrane via the highly conserved SecA-YEG pathway. In the present studies, the subcellular distributions of core components of this pathway, SecA and SecY, and of the

The Composition and Organization of Cytoplasm in Prebiotic Cells

Directory of Open Access Journals (Sweden)

Jack T. Trevors

2011-03-01

Full Text Available This article discusses the hypothesized composition and organization of cytoplasm in prebiotic cells from a theoretical perspective and also based upon what is currently known about bacterial cytoplasm. It is unknown if the first prebiotic, microscopic scale, cytoplasm was initially contained within a primitive, continuous, semipermeable membrane, or was an uncontained gel substance, that later became enclosed by a continuous membrane. Another possibility is that the first cytoplasm in prebiotic cells and a primitive membrane organized at the same time, permitting a rapid transition to the first cell(s capable of growth and division, thus assisting with the emergence of life on Earth less than a billion years after the formation of the Earth. It is hypothesized that the organization and composition of cytoplasm progressed initially from an unstructured, microscopic hydrogel to a more complex cytoplasm, that may have been in the volume magnitude of about 0.1–0.2 µm3 (possibly less if a nanocell prior to the first cell division.

Xenopus egg cytoplasm with intact actin.

Science.gov (United States)

Field, Christine M; Nguyen, Phuong A; Ishihara, Keisuke; Groen, Aaron C; Mitchison, Timothy J

2014-01-01

We report optimized methods for preparing Xenopus egg extracts without cytochalasin D, that we term "actin-intact egg extract." These are undiluted egg cytoplasm that contains abundant organelles, and glycogen which supplies energy, and represents the least perturbed cell-free cytoplasm preparation we know of. We used this system to probe cell cycle regulation of actin and myosin-II dynamics (Field et al., 2011), and to reconstitute the large, interphase asters that organize early Xenopus embryos (Mitchison et al., 2012; Wühr, Tan, Parker, Detrich, & Mitchison, 2010). Actin-intact Xenopus egg extracts are useful for analysis of actin dynamics, and interaction of actin with other cytoplasmic systems, in a cell-free system that closely mimics egg physiology, and more generally for probing the biochemistry and biophysics of the egg, zygote, and early embryo. Detailed protocols are provided along with assays used to check cell cycle state and tips for handling and storing undiluted egg extracts. © 2014 Elsevier Inc. All rights reserved.

Cellular Protein WDR11 Interacts with Specific Herpes Simplex Virus Proteins at the trans-Golgi Network To Promote Virus Replication

Science.gov (United States)

Taylor, Kathryne E.

2015-01-01

ABSTRACT It has recently been proposed that the herpes simplex virus (HSV) protein ICP0 has cytoplasmic roles in blocking antiviral signaling and in promoting viral replication in addition to its well-known proteasome-dependent functions in the nucleus. However, the mechanisms through which it produces these effects remain unclear. While investigating this further, we identified a novel cytoplasmic interaction between ICP0 and the poorly characterized cellular protein WDR11. During an HSV infection, WDR11 undergoes a dramatic change in localization at late times in the viral replication cycle, moving from defined perinuclear structures to a dispersed cytoplasmic distribution. While this relocation was not observed during infection with viruses other than HSV-1 and correlated with efficient HSV-1 replication, the redistribution was found to occur independently of ICP0 expression, instead requiring viral late gene expression. We demonstrate for the first time that WDR11 is localized to the trans-Golgi network (TGN), where it interacts specifically with some, but not all, HSV virion components, in addition to ICP0. Knockdown of WDR11 in cultured human cells resulted in a modest but consistent decrease in yields of both wild-type and ICP0-null viruses, in the supernatant and cell-associated fractions, without affecting viral gene expression. Although further study is required, we propose that WDR11 participates in viral assembly and/or secondary envelopment. IMPORTANCE While the TGN has been proposed to be the major site of HSV-1 secondary envelopment, this process is incompletely understood, and in particular, the role of cellular TGN components in this pathway is unknown. Additionally, little is known about the cellular functions of WDR11, although the disruption of this protein has been implicated in multiple human diseases. Therefore, our finding that WDR11 is a TGN-resident protein that interacts with specific viral proteins to enhance viral yields improves both

HIV-1 tat protein recruits CIS to the cytoplasmic tail of CD127 to induce receptor ubiquitination and proteasomal degradation

Energy Technology Data Exchange (ETDEWEB)

Sugden, Scott, E-mail: scott.sugden@ircm.qc.ca [The Ottawa Hospital Research Institute, 501 Smyth Road, Ottawa, Ontario, Canada K1H 8L6 (Canada); Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, 451 Smyth Road, Ottawa, Ontario, Canada K1H 8M5 (Canada); Ghazawi, Feras [The Ottawa Hospital Research Institute, 501 Smyth Road, Ottawa, Ontario, Canada K1H 8L6 (Canada); Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, 451 Smyth Road, Ottawa, Ontario, Canada K1H 8M5 (Canada); MacPherson, Paul, E-mail: pmacpherson@toh.on.ca [The Ottawa Hospital Research Institute, 501 Smyth Road, Ottawa, Ontario, Canada K1H 8L6 (Canada); Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, 451 Smyth Road, Ottawa, Ontario, Canada K1H 8M5 (Canada); Division of Infectious Diseases, The Ottawa Hospital General Campus, 501 Smyth Road, Ottawa, Ontario, Canada K1H 8L6 (Canada)

2016-11-15

HIV-1 Tat protein down regulates expression of the IL-7 receptor alpha-chain (CD127) from the surface of CD8 T cells resulting in impaired T cell proliferation and cytolytic capacity. We have previously shown that soluble Tat protein is taken up by CD8 T cells and interacts with the cytoplasmic tail of CD127 to induce receptor degradation. The N-terminal domain of Tat interacts with CD127 while the basic domain directs CD127 to the proteasome. We have also shown that upon IL-7 binding to its receptor, CD127 is phosphorylated resulting in CIS-mediated proteasomal degradation. Here, we show that Tat mimics this process by recruiting CIS to CD127 in the absence of IL-7 and receptor phosphorylation, leading to CD127 ubiquitination and degradation. Tat therefore acts as an adapter to induce cellular responses under conditions where they may not otherwise occur. Thusly, Tat reduces IL-7 signaling and impairs CD8 T cell survival and function. -- Highlights: •Soluble HIV-1 Tat decreases CD127 expression on CD8 T cells, causing dysfunction. •Tat induces CD127 ubiquitination without activating IL-7 signaling. •Tat binds CD127 and recruits the E3 ubiquitin ligase CIS via its basic domain. •Tat hijacks a normal cellular mechanism to degrade CD127 without IL-7 signaling.

HIV-1 tat protein recruits CIS to the cytoplasmic tail of CD127 to induce receptor ubiquitination and proteasomal degradation

International Nuclear Information System (INIS)

Sugden, Scott; Ghazawi, Feras; MacPherson, Paul

2016-01-01

HIV-1 Tat protein down regulates expression of the IL-7 receptor alpha-chain (CD127) from the surface of CD8 T cells resulting in impaired T cell proliferation and cytolytic capacity. We have previously shown that soluble Tat protein is taken up by CD8 T cells and interacts with the cytoplasmic tail of CD127 to induce receptor degradation. The N-terminal domain of Tat interacts with CD127 while the basic domain directs CD127 to the proteasome. We have also shown that upon IL-7 binding to its receptor, CD127 is phosphorylated resulting in CIS-mediated proteasomal degradation. Here, we show that Tat mimics this process by recruiting CIS to CD127 in the absence of IL-7 and receptor phosphorylation, leading to CD127 ubiquitination and degradation. Tat therefore acts as an adapter to induce cellular responses under conditions where they may not otherwise occur. Thusly, Tat reduces IL-7 signaling and impairs CD8 T cell survival and function. -- Highlights: •Soluble HIV-1 Tat decreases CD127 expression on CD8 T cells, causing dysfunction. •Tat induces CD127 ubiquitination without activating IL-7 signaling. •Tat binds CD127 and recruits the E3 ubiquitin ligase CIS via its basic domain. •Tat hijacks a normal cellular mechanism to degrade CD127 without IL-7 signaling.

Peptide domains involved in the localization of the porcine reproductive and respiratory syndrome virus nucleocapsid protein to the nucleolus

International Nuclear Information System (INIS)

Rowland, Raymond R.R.; Schneider, Paula; Fang Ying; Wootton, Sarah; Yoo, Dongwan; Benfield, David A.

2003-01-01

The nucleocapsid (N) protein of porcine reproductive and respiratory syndrome virus (PRRSV) is the principal component of the viral nucleocapsid and localizes to the nucleolus. Peptide sequence analysis of the N protein of several North American isolates identified two potential nuclear localization signal (NLS) sequences located at amino acids 10-13 and 41-42, which were labeled NLS-1 and NLS-2, respectively. Peptides containing NLS-1 or NLS-2 were sufficient to accumulate enhanced green fluorescent protein (EGFP) in the nucleus. The inactivation of NLS-1 by site-directed mutagenesis or the deletion of the first 14 amino acids did not affect N protein localization to the nucleolus. The substitution of key lysine residues with uncharged amino acids in NLS-2 blocked nuclear/nucleolar localization. Site-directed mutagenesis within NLS-2 identified the sequence, KKNKK, as forming the core localization domain within NLS-2. Using an in vitro pull-down assay, the N protein was able to bind importin-α, importin-β nuclear transport proteins. The localization pattern of N-EGFP fusion peptides represented by a series of deletions from the C- and N-terminal ends of the N protein identified a region covering amino acids 41-72, which contained a nucleolar localization signal (NoLS) sequence. The 41-72 N peptide when fused to EGFP mimicked the nucleolar-cytoplasmic distribution of native N. These results identify a single NLS involved in the transport of N from the cytoplasm and into nucleus. An additional peptide sequence, overlapping NLS-2, is involved in the further targeting of N to the nucleolus

Plant transducers of the endoplasmic reticulum unfolded protein response

KAUST Repository

Iwata, Yuji; Koizumi, Nozomu

2012-01-01

The unfolded protein response (UPR) activates a set of genes to overcome accumulation of unfolded proteins in the endoplasmic reticulum (ER), a condition termed ER stress, and constitutes an essential part of ER protein quality control that ensures efficient maturation of secretory and membrane proteins in eukaryotes. Recent studies on Arabidopsis and rice identified the signaling pathway in which the ER membrane-localized ribonuclease IRE1 (inositol-requiring enzyme 1) catalyzes unconventional cytoplasmic splicing of mRNA, thereby producing the active transcription factor Arabidopsis bZIP60 (basic leucine zipper 60) and its ortholog in rice. Here we review recent findings identifying the molecular components of the plant UPR, including IRE1/bZIP60 and the membrane-bound transcription factors bZIP17 and bZIP28, and implicating its importance in several physiological phenomena such as pathogen response. © 2012 Elsevier Ltd.

Plant transducers of the endoplasmic reticulum unfolded protein response

KAUST Repository

Iwata, Yuji

2012-12-01

The unfolded protein response (UPR) activates a set of genes to overcome accumulation of unfolded proteins in the endoplasmic reticulum (ER), a condition termed ER stress, and constitutes an essential part of ER protein quality control that ensures efficient maturation of secretory and membrane proteins in eukaryotes. Recent studies on Arabidopsis and rice identified the signaling pathway in which the ER membrane-localized ribonuclease IRE1 (inositol-requiring enzyme 1) catalyzes unconventional cytoplasmic splicing of mRNA, thereby producing the active transcription factor Arabidopsis bZIP60 (basic leucine zipper 60) and its ortholog in rice. Here we review recent findings identifying the molecular components of the plant UPR, including IRE1/bZIP60 and the membrane-bound transcription factors bZIP17 and bZIP28, and implicating its importance in several physiological phenomena such as pathogen response. © 2012 Elsevier Ltd.

Protein functional links in Trypanosoma brucei, identified by gene fusion analysis

Directory of Open Access Journals (Sweden)

Trimpalis Philip

2011-07-01

Full Text Available Abstract Background Domain or gene fusion analysis is a bioinformatics method for detecting gene fusions in one organism by comparing its genome to that of other organisms. The occurrence of gene fusions suggests that the two original genes that participated in the fusion are functionally linked, i.e. their gene products interact either as part of a multi-subunit protein complex, or in a metabolic pathway. Gene fusion analysis has been used to identify protein functional links in prokaryotes as well as in eukaryotic model organisms, such as yeast and Drosophila. Results In this study we have extended this approach to include a number of recently sequenced protists, four of which are pathogenic, to identify fusion linked proteins in Trypanosoma brucei, the causative agent of African sleeping sickness. We have also examined the evolution of the gene fusion events identified, to determine whether they can be attributed to fusion or fission, by looking at the conservation of the fused genes and of the individual component genes across the major eukaryotic and prokaryotic lineages. We find relatively limited occurrence of gene fusions/fissions within the protist lineages examined. Our results point to two trypanosome-specific gene fissions, which have recently been experimentally confirmed, one fusion involving proteins involved in the same metabolic pathway, as well as two novel putative functional links between fusion-linked protein pairs. Conclusions This is the first study of protein functional links in T. brucei identified by gene fusion analysis. We have used strict thresholds and only discuss results which are highly likely to be genuine and which either have already been or can be experimentally verified. We discuss the possible impact of the identification of these novel putative protein-protein interactions, to the development of new trypanosome therapeutic drugs.

Identification of a nuclear export signal in the KSHV latent protein LANA2 mediating its export from the nucleus

International Nuclear Information System (INIS)

Munoz-Fontela, C.; Collado, M.; Rodriguez, E.; Garcia, M.A.; Alvarez-Barrientos, A.; Arroyo, J.; Nombela, C.; Rivas, C.

2005-01-01

LANA2 is a latent protein detected in Kaposi's sarcoma-associated herpesvirus (KSHV)-infected B cells that inhibits p53-dependent transcriptional transactivation and apoptosis and PKR-dependent apoptosis, suggesting an important role in the transforming activity of the virus. It has been reported that LANA2 localizes into the nucleus of both KSHV-infected B cells and transiently transfected HeLa cells. In this study, we show that LANA2 is a nucleocytoplasmic shuttling protein that requires a Rev-type nuclear export signal located in the C-terminus to direct the protein to the cytoplasm, through an association with the export receptor CRM1. In addition, a functional protein kinase B (PKB)/Akt phosphorylation motif partially overlapping with the nuclear export signal was identified. Nuclear exclusion of LANA2 was negatively regulated by the phosphorylation of threonine 564 by Akt. The ability of LANA2 to shuttle between nucleus and cytoplasm has implications for the function of this viral protein

Production of soluble mammalian proteins in Escherichia coli: identification of protein features that correlate with successful expression

Directory of Open Access Journals (Sweden)

Perera Rajika L

2004-12-01

Full Text Available Abstract Background In the search for generic expression strategies for mammalian protein families several bacterial expression vectors were examined for their ability to promote high yields of soluble protein. Proteins studied included cell surface receptors (Ephrins and Eph receptors, CD44, kinases (EGFR-cytoplasmic domain, CDK2 and 4, proteases (MMP1, CASP2, signal transduction proteins (GRB2, RAF1, HRAS and transcription factors (GATA2, Fli1, Trp53, Mdm2, JUN, FOS, MAD, MAX. Over 400 experiments were performed where expression of 30 full-length proteins and protein domains were evaluated with 6 different N-terminal and 8 C-terminal fusion partners. Expression of an additional set of 95 mammalian proteins was also performed to test the conclusions of this study. Results Several protein features correlated with soluble protein expression yield including molecular weight and the number of contiguous hydrophobic residues and low complexity regions. There was no relationship between successful expression and protein pI, grand average of hydropathicity (GRAVY, or sub-cellular location. Only small globular cytoplasmic proteins with an average molecular weight of 23 kDa did not require a solubility enhancing tag for high level soluble expression. Thioredoxin (Trx and maltose binding protein (MBP were the best N-terminal protein fusions to promote soluble expression, but MBP was most effective as a C-terminal fusion. 63 of 95 mammalian proteins expressed at soluble levels of greater than 1 mg/l as N-terminal H10-MBP fusions and those that failed possessed, on average, a higher molecular weight and greater number of contiguous hydrophobic amino acids and low complexity regions. Conclusions By analysis of the protein features identified here, this study will help predict which mammalian proteins and domains can be successfully expressed in E. coli as soluble product and also which are best targeted for a eukaryotic expression system. In some cases

Identification of surface proteins in Enterococcus faecalis V583

Directory of Open Access Journals (Sweden)

Eijsink Vincent GH

2011-03-01

Full Text Available Abstract Background Surface proteins are a key to a deeper understanding of the behaviour of Gram-positive bacteria interacting with the human gastro-intestinal tract. Such proteins contribute to cell wall synthesis and maintenance and are important for interactions between the bacterial cell and the human host. Since they are exposed and may play roles in pathogenicity, surface proteins are interesting targets for drug design. Results Using methods based on proteolytic "shaving" of bacterial cells and subsequent mass spectrometry-based protein identification, we have identified surface-located proteins in Enterococcus faecalis V583. In total 69 unique proteins were identified, few of which have been identified and characterized previously. 33 of these proteins are predicted to be cytoplasmic, whereas the other 36 are predicted to have surface locations (31 or to be secreted (5. Lipid-anchored proteins were the most dominant among the identified surface proteins. The seemingly most abundant surface proteins included a membrane protein with a potentially shedded extracellular sulfatase domain that could act on the sulfate groups in mucin and a lipid-anchored fumarate reductase that could contribute to generation of reactive oxygen species. Conclusions The present proteome analysis gives an experimental impression of the protein landscape on the cell surface of the pathogenic bacterium E. faecalis. The 36 identified secreted (5 and surface (31 proteins included several proteins involved in cell wall synthesis, pheromone-regulated processes, and transport of solutes, as well as proteins with unknown function. These proteins stand out as interesting targets for further investigation of the interaction between E. faecalis and its environment.

CRISPR/Cas9-mediated mutation revealed cytoplasmic tail is dispensable for IZUMO1 function and male fertility

DEFF Research Database (Denmark)

Young, Samantha A M; Miyata, Haruhiko; Satouh, Yuhkoh

2016-01-01

manipulation system of CRISPR/Cas9 to generate a point mutation resulting in a premature stop codon, producing mice with truncated IZUMO1. Mice without the cytoplasmic tail of IZUMO1 showed normal fertility but decreased the amount of protein, indicating that whilst this region is important for the expression...

A novel link between Sus1 and the cytoplasmic mRNA decay machinery suggests a broad role in mRNA metabolism

Directory of Open Access Journals (Sweden)

Llopis Ana

2010-03-01

Full Text Available Abstract Background Gene expression is achieved by the coordinated action of multiple factors to ensure a perfect synchrony from chromatin epigenetic regulation through to mRNA export. Sus1 is a conserved mRNA export/transcription factor and is a key player in coupling transcription initiation, elongation and mRNA export. In the nucleus, Sus1 is associated to the transcriptional co-activator SAGA and to the NPC associated complex termed TREX2/THSC. Through these associations, Sus1 mediates the nuclear dynamics of different gene loci and facilitate the export of the new transcripts. Results In this study, we have investigated whether the yeast Sus1 protein is linked to factors involved in mRNA degradation pathways. We provide evidence for genetic interactions between SUS1 and genes coding for components of P-bodies such as PAT1, LSM1, LSM6 and DHH1. We demonstrate that SUS1 deletion is synthetic lethal with 5'→3' decay machinery components LSM1 and PAT1 and has a strong genetic interaction with LSM6 and DHH1. Interestingly, Sus1 overexpression led to an accumulation of Sus1 in cytoplasmic granules, which can co-localise with components of P-bodies and stress granules. In addition, we have identified novel physical interactions between Sus1 and factors associated to P-bodies/stress granules. Finally, absence of LSM1 and PAT1 slightly promotes the Sus1-TREX2 association. Conclusions In this study, we found genetic and biochemical association between Sus1 and components responsible for cytoplasmic mRNA metabolism. Moreover, Sus1 accumulates in discrete cytoplasmic granules, which partially co-localise with P-bodies and stress granules under specific conditions. These interactions suggest a role for Sus1 in gene expression during cytoplasmic mRNA metabolism in addition to its nuclear function.

Cytoplasmic ATR Activation Promotes Vaccinia Virus Genome Replication

Directory of Open Access Journals (Sweden)

Antonio Postigo

2017-05-01

Full Text Available In contrast to most DNA viruses, poxviruses replicate their genomes in the cytoplasm without host involvement. We find that vaccinia virus induces cytoplasmic activation of ATR early during infection, before genome uncoating, which is unexpected because ATR plays a fundamental nuclear role in maintaining host genome integrity. ATR, RPA, INTS7, and Chk1 are recruited to cytoplasmic DNA viral factories, suggesting canonical ATR pathway activation. Consistent with this, pharmacological and RNAi-mediated inhibition of canonical ATR signaling suppresses genome replication. RPA and the sliding clamp PCNA interact with the viral polymerase E9 and are required for DNA replication. Moreover, the ATR activator TOPBP1 promotes genome replication and associates with the viral replisome component H5. Our study suggests that, in contrast to long-held beliefs, vaccinia recruits conserved components of the eukaryote DNA replication and repair machinery to amplify its genome in the host cytoplasm.

A new fluorescence-based method identifies protein phosphatases regulating lipid droplet metabolism.

Directory of Open Access Journals (Sweden)

Bruno L Bozaquel-Morais

Full Text Available In virtually every cell, neutral lipids are stored in cytoplasmic structures called lipid droplets (LDs and also referred to as lipid bodies or lipid particles. We developed a rapid high-throughput assay based on the recovery of quenched BODIPY-fluorescence that allows to quantify lipid droplets. The method was validated by monitoring lipid droplet turnover during growth of a yeast culture and by screening a group of strains deleted in genes known to be involved in lipid metabolism. In both tests, the fluorimetric assay showed high sensitivity and good agreement with previously reported data using microscopy. We used this method for high-throughput identification of protein phosphatases involved in lipid droplet metabolism. From 65 yeast knockout strains encoding protein phosphatases and its regulatory subunits, 13 strains revealed to have abnormal levels of lipid droplets, 10 of them having high lipid droplet content. Strains deleted for type I protein phosphatases and related regulators (ppz2, gac1, bni4, type 2A phosphatase and its related regulator (pph21 and sap185, type 2C protein phosphatases (ptc1, ptc4, ptc7 and dual phosphatases (pps1, msg5 were catalogued as high-lipid droplet content strains. Only reg1, a targeting subunit of the type 1 phosphatase Glc7p, and members of the nutrient-sensitive TOR pathway (sit4 and the regulatory subunit sap190 were catalogued as low-lipid droplet content strains, which were studied further. We show that Snf1, the homologue of the mammalian AMP-activated kinase, is constitutively phosphorylated (hyperactive in sit4 and sap190 strains leading to a reduction of acetyl-CoA carboxylase activity. In conclusion, our fast and highly sensitive method permitted us to catalogue protein phosphatases involved in the regulation of LD metabolism and present evidence indicating that the TOR pathway and the SNF1/AMPK pathway are connected through the Sit4p-Sap190p pair in the control of lipid droplet biogenesis.

Tritium Suicide Selection Identifies Proteins Involved in the Uptake and Intracellular Transport of Sterols in Saccharomyces cerevisiae▿

Science.gov (United States)

Sullivan, David P.; Georgiev, Alexander; Menon, Anant K.

2009-01-01

Sterol transport between the plasma membrane (PM) and the endoplasmic reticulum (ER) occurs by a nonvesicular mechanism that is poorly understood. To identify proteins required for this process, we isolated Saccharomyces cerevisiae mutants with defects in sterol transport. We used Upc2-1 cells that have the ability to take up sterols under aerobic conditions and exploited the observation that intracellular accumulation of exogenously supplied [3H]cholesterol in the form of [3H]cholesteryl ester requires an intact PM-ER sterol transport pathway. Upc2-1 cells were mutagenized using a transposon library, incubated with [3H]cholesterol, and subjected to tritium suicide selection to isolate mutants with a decreased ability to accumulate [3H]cholesterol. Many of the mutants had defects in the expression and trafficking of Aus1 and Pdr11, PM-localized ABC transporters that are required for sterol uptake. Through characterization of one of the mutants, a new role was uncovered for the transcription factor Mot3 in controlling expression of Aus1 and Pdr11. A number of mutants had transposon insertions in the uncharacterized Ydr051c gene, which we now refer to as DET1 (decreased ergosterol transport). These mutants expressed Aus1 and Pdr11 normally but were severely defective in the ability to accumulate exogenously supplied cholesterol. The transport of newly synthesized sterols from the ER to the PM was also defective in det1Δ cells. These data indicate that the cytoplasmic protein encoded by DET1 is involved in intracellular sterol transport. PMID:19060182

A novel nucleo-cytoplasmic hybrid clone formed via androgenesis in polyploid gibel carp

Directory of Open Access Journals (Sweden)

Zhou Li

2011-03-01

Full Text Available Abstract Background Unisexual vertebrates have been demonstrated to reproduce by gynogenesis, hybridogenesis, parthenogenesis, or kleptogenesis, however, it is uncertain how the reproduction mode contributes to the clonal diversity. Recently, polyploid gibel carp has been revealed to possess coexisting dual modes of unisexual gynogenesis and sexual reproduction and to have numerous various clones. Using sexual reproduction mating between clone D female and clone A male and subsequent 7 generation multiplying of unisexual gynogenesis, we have created a novel clone strain with more than several hundred millions of individuals. Here, we attempt to identify genetic background of the novel clone and to explore the significant implication for clonal diversity contribution. Methods Several nuclear genome markers and one cytoplasmic marker, the mitochondrial genome sequence, were used to identify the genetic organization of the randomly sampled individuals from different generations of the novel clone. Results Chromosome number, Cot-1 repetitive DNA banded karyotype, microsatellite patterns, AFLP profiles and transferrin alleles uniformly indicated that nuclear genome of the novel clone is identical to that of clone A, and significantly different from that of clone D. However, the cytoplasmic marker, its complete mtDNA genome sequence, is same to that of clone D, and different from that of clone A. Conclusions The present data indicate that the novel clone is a nucleo-cytoplasmic hybrid between the known clones A and D, because it originates from the offspring of gonochoristic sexual reproduction mating between clone D female and clone A male, and contains an entire nuclear genome from the paternal clone A and a mtDNA genome (cytoplasm from the maternal clone D. It is suggested to arise via androgenesis by a mechanism of ploidy doubling of clone A sperm in clone D ooplasm through inhibiting the first mitotic division. Significantly, the selected nucleo-cytoplasmic

A novel nucleo-cytoplasmic hybrid clone formed via androgenesis in polyploid gibel carp

Science.gov (United States)

2011-01-01

Background Unisexual vertebrates have been demonstrated to reproduce by gynogenesis, hybridogenesis, parthenogenesis, or kleptogenesis, however, it is uncertain how the reproduction mode contributes to the clonal diversity. Recently, polyploid gibel carp has been revealed to possess coexisting dual modes of unisexual gynogenesis and sexual reproduction and to have numerous various clones. Using sexual reproduction mating between clone D female and clone A male and subsequent 7 generation multiplying of unisexual gynogenesis, we have created a novel clone strain with more than several hundred millions of individuals. Here, we attempt to identify genetic background of the novel clone and to explore the significant implication for clonal diversity contribution. Methods Several nuclear genome markers and one cytoplasmic marker, the mitochondrial genome sequence, were used to identify the genetic organization of the randomly sampled individuals from different generations of the novel clone. Results Chromosome number, Cot-1 repetitive DNA banded karyotype, microsatellite patterns, AFLP profiles and transferrin alleles uniformly indicated that nuclear genome of the novel clone is identical to that of clone A, and significantly different from that of clone D. However, the cytoplasmic marker, its complete mtDNA genome sequence, is same to that of clone D, and different from that of clone A. Conclusions The present data indicate that the novel clone is a nucleo-cytoplasmic hybrid between the known clones A and D, because it originates from the offspring of gonochoristic sexual reproduction mating between clone D female and clone A male, and contains an entire nuclear genome from the paternal clone A and a mtDNA genome (cytoplasm) from the maternal clone D. It is suggested to arise via androgenesis by a mechanism of ploidy doubling of clone A sperm in clone D ooplasm through inhibiting the first mitotic division. Significantly, the selected nucleo-cytoplasmic hybrid female

Identifying protein phosphorylation sites with kinase substrate specificity on human viruses.

Directory of Open Access Journals (Sweden)

Neil Arvin Bretaña

Full Text Available Viruses infect humans and progress inside the body leading to various diseases and complications. The phosphorylation of viral proteins catalyzed by host kinases plays crucial regulatory roles in enhancing replication and inhibition of normal host-cell functions. Due to its biological importance, there is a desire to identify the protein phosphorylation sites on human viruses. However, the use of mass spectrometry-based experiments is proven to be expensive and labor-intensive. Furthermore, previous studies which have identified phosphorylation sites in human viruses do not include the investigation of the responsible kinases. Thus, we are motivated to propose a new method to identify protein phosphorylation sites with its kinase substrate specificity on human viruses. The experimentally verified phosphorylation data were extracted from virPTM--a database containing 301 experimentally verified phosphorylation data on 104 human kinase-phosphorylated virus proteins. In an attempt to investigate kinase substrate specificities in viral protein phosphorylation sites, maximal dependence decomposition (MDD is employed to cluster a large set of phosphorylation data into subgroups containing significantly conserved motifs. The experimental human phosphorylation sites are collected from Phospho.ELM, grouped according to its kinase annotation, and compared with the virus MDD clusters. This investigation identifies human kinases such as CK2, PKB, CDK, and MAPK as potential kinases for catalyzing virus protein substrates as confirmed by published literature. Profile hidden Markov model is then applied to learn a predictive model for each subgroup. A five-fold cross validation evaluation on the MDD-clustered HMMs yields an average accuracy of 84.93% for Serine, and 78.05% for Threonine. Furthermore, an independent testing data collected from UniProtKB and Phospho.ELM is used to make a comparison of predictive performance on three popular kinase

Identifying protein phosphorylation sites with kinase substrate specificity on human viruses.

Science.gov (United States)

Bretaña, Neil Arvin; Lu, Cheng-Tsung; Chiang, Chiu-Yun; Su, Min-Gang; Huang, Kai-Yao; Lee, Tzong-Yi; Weng, Shun-Long

2012-01-01

Viruses infect humans and progress inside the body leading to various diseases and complications. The phosphorylation of viral proteins catalyzed by host kinases plays crucial regulatory roles in enhancing replication and inhibition of normal host-cell functions. Due to its biological importance, there is a desire to identify the protein phosphorylation sites on human viruses. However, the use of mass spectrometry-based experiments is proven to be expensive and labor-intensive. Furthermore, previous studies which have identified phosphorylation sites in human viruses do not include the investigation of the responsible kinases. Thus, we are motivated to propose a new method to identify protein phosphorylation sites with its kinase substrate specificity on human viruses. The experimentally verified phosphorylation data were extracted from virPTM--a database containing 301 experimentally verified phosphorylation data on 104 human kinase-phosphorylated virus proteins. In an attempt to investigate kinase substrate specificities in viral protein phosphorylation sites, maximal dependence decomposition (MDD) is employed to cluster a large set of phosphorylation data into subgroups containing significantly conserved motifs. The experimental human phosphorylation sites are collected from Phospho.ELM, grouped according to its kinase annotation, and compared with the virus MDD clusters. This investigation identifies human kinases such as CK2, PKB, CDK, and MAPK as potential kinases for catalyzing virus protein substrates as confirmed by published literature. Profile hidden Markov model is then applied to learn a predictive model for each subgroup. A five-fold cross validation evaluation on the MDD-clustered HMMs yields an average accuracy of 84.93% for Serine, and 78.05% for Threonine. Furthermore, an independent testing data collected from UniProtKB and Phospho.ELM is used to make a comparison of predictive performance on three popular kinase-specific phosphorylation site

Effect of mitochondrial complex I inhibition on Fe-S cluster protein activity

Energy Technology Data Exchange (ETDEWEB)

Mena, Natalia P. [Department of Biology, Faculty of Sciences, Universidad de Chile, Las Palmeras 3425, Santiago (Chile); Millennium Institute of Cell Dynamics and Biotechnology, Santiago (Chile); Bulteau, Anne Laure [UPMC Univ Paris 06, UMRS 975 - UMR 7725, Centre de Recherche en Neurosciences, ICM, Therapeutique Experimentale de la Neurodegenerescence, Hopital de la Salpetriere, F-75005 Paris (France); Inserm, U 975, Centre de Recherche en Neurosciences, ICM, Therapeutique Experimentale de la Neurodegenerescence, Hopital de la Salpetriere, F-75005 Paris (France); CNRS, UMR 7225, Centre de Recherche en Neurosciences, ICM, Therapeutique Experimentale de la Neurodegenerescence, Hopital de la Salpetriere, F-75005 Paris (France); ICM, Therapeutique Experimentale de la Neurodegenerescence, Hopital de la Salpetriere, Paris 75013 (France); Salazar, Julio [Millennium Institute of Cell Dynamics and Biotechnology, Santiago (Chile); Hirsch, Etienne C. [UPMC Univ Paris 06, UMRS 975 - UMR 7725, Centre de Recherche en Neurosciences, ICM, Therapeutique Experimentale de la Neurodegenerescence, Hopital de la Salpetriere, F-75005 Paris (France); Inserm, U 975, Centre de Recherche en Neurosciences, ICM, Therapeutique Experimentale de la Neurodegenerescence, Hopital de la Salpetriere, F-75005 Paris (France); CNRS, UMR 7225, Centre de Recherche en Neurosciences, ICM, Therapeutique Experimentale de la Neurodegenerescence, Hopital de la Salpetriere, F-75005 Paris (France); ICM, Therapeutique Experimentale de la Neurodegenerescence, Hopital de la Salpetriere, Paris 75013 (France); Nunez, Marco T., E-mail: mnunez@uchile.cl [Department of Biology, Faculty of Sciences, Universidad de Chile, Las Palmeras 3425, Santiago (Chile); Millennium Institute of Cell Dynamics and Biotechnology, Santiago (Chile)

2011-06-03

Highlights: {yields} Mitochondrial complex I inhibition resulted in decreased activity of Fe-S containing enzymes mitochondrial aconitase and cytoplasmic aconitase and xanthine oxidase. {yields} Complex I inhibition resulted in the loss of Fe-S clusters in cytoplasmic aconitase and of glutamine phosphoribosyl pyrophosphate amidotransferase. {yields} Consistent with loss of cytoplasmic aconitase activity, an increase in iron regulatory protein 1 activity was found. {yields} Complex I inhibition resulted in an increase in the labile cytoplasmic iron pool. -- Abstract: Iron-sulfur (Fe-S) clusters are small inorganic cofactors formed by tetrahedral coordination of iron atoms with sulfur groups. Present in numerous proteins, these clusters are involved in key biological processes such as electron transfer, metabolic and regulatory processes, DNA synthesis and repair and protein structure stabilization. Fe-S clusters are synthesized mainly in the mitochondrion, where they are directly incorporated into mitochondrial Fe-S cluster-containing proteins or exported for cytoplasmic and nuclear cluster-protein assembly. In this study, we tested the hypothesis that inhibition of mitochondrial complex I by rotenone decreases Fe-S cluster synthesis and cluster content and activity of Fe-S cluster-containing enzymes. Inhibition of complex I resulted in decreased activity of three Fe-S cluster-containing enzymes: mitochondrial and cytosolic aconitases and xanthine oxidase. In addition, the Fe-S cluster content of glutamine phosphoribosyl pyrophosphate amidotransferase and mitochondrial aconitase was dramatically decreased. The reduction in cytosolic aconitase activity was associated with an increase in iron regulatory protein (IRP) mRNA binding activity and with an increase in the cytoplasmic labile iron pool. Since IRP activity post-transcriptionally regulates the expression of iron import proteins, Fe-S cluster inhibition may result in a false iron deficiency signal. Given that

Effect of mitochondrial complex I inhibition on Fe-S cluster protein activity

International Nuclear Information System (INIS)

Mena, Natalia P.; Bulteau, Anne Laure; Salazar, Julio; Hirsch, Etienne C.; Nunez, Marco T.

2011-01-01

Highlights: → Mitochondrial complex I inhibition resulted in decreased activity of Fe-S containing enzymes mitochondrial aconitase and cytoplasmic aconitase and xanthine oxidase. → Complex I inhibition resulted in the loss of Fe-S clusters in cytoplasmic aconitase and of glutamine phosphoribosyl pyrophosphate amidotransferase. → Consistent with loss of cytoplasmic aconitase activity, an increase in iron regulatory protein 1 activity was found. → Complex I inhibition resulted in an increase in the labile cytoplasmic iron pool. -- Abstract: Iron-sulfur (Fe-S) clusters are small inorganic cofactors formed by tetrahedral coordination of iron atoms with sulfur groups. Present in numerous proteins, these clusters are involved in key biological processes such as electron transfer, metabolic and regulatory processes, DNA synthesis and repair and protein structure stabilization. Fe-S clusters are synthesized mainly in the mitochondrion, where they are directly incorporated into mitochondrial Fe-S cluster-containing proteins or exported for cytoplasmic and nuclear cluster-protein assembly. In this study, we tested the hypothesis that inhibition of mitochondrial complex I by rotenone decreases Fe-S cluster synthesis and cluster content and activity of Fe-S cluster-containing enzymes. Inhibition of complex I resulted in decreased activity of three Fe-S cluster-containing enzymes: mitochondrial and cytosolic aconitases and xanthine oxidase. In addition, the Fe-S cluster content of glutamine phosphoribosyl pyrophosphate amidotransferase and mitochondrial aconitase was dramatically decreased. The reduction in cytosolic aconitase activity was associated with an increase in iron regulatory protein (IRP) mRNA binding activity and with an increase in the cytoplasmic labile iron pool. Since IRP activity post-transcriptionally regulates the expression of iron import proteins, Fe-S cluster inhibition may result in a false iron deficiency signal. Given that inhibition of complex

Plant vegetative and animal cytoplasmic actins share functional competence for spatial development with protists.

Science.gov (United States)

Kandasamy, Muthugapatti K; McKinney, Elizabeth C; Roy, Eileen; Meagher, Richard B

2012-05-01

Actin is an essential multifunctional protein encoded by two distinct ancient classes of genes in animals (cytoplasmic and muscle) and plants (vegetative and reproductive). The prevailing view is that each class of actin variants is functionally distinct. However, we propose that the vegetative plant and cytoplasmic animal variants have conserved functional competence for spatial development inherited from an ancestral protist actin sequence. To test this idea, we ectopically expressed animal and protist actins in Arabidopsis thaliana double vegetative actin mutants that are dramatically altered in cell and organ morphologies. We found that expression of cytoplasmic actins from humans and even a highly divergent invertebrate Ciona intestinalis qualitatively and quantitatively suppressed the root cell polarity and organ defects of act8 act7 mutants and moderately suppressed the root-hairless phenotype of act2 act8 mutants. By contrast, human muscle actins were unable to support prominently any aspect of plant development. Furthermore, actins from three protists representing Choanozoa, Archamoeba, and green algae efficiently suppressed all the phenotypes of both the plant mutants. Remarkably, these data imply that actin's competence to carry out a complex suite of processes essential for multicellular development was already fully developed in single-celled protists and evolved nonprogressively from protists to plants and animals.

Microdomain-forming proteins and the role of the reggies/flotillins during axon regeneration in zebrafish

OpenAIRE

Stürmer, Claudia

2011-01-01

The two proteins reggie-1 and reggie-2 (flotillins) were identified in axon-regenerating neurons in the central nervous system and shown to be essential for neurite growth and regeneration in fish and mammals. Reggies/flotillins are microdomain scaffolding proteins sharing biochemical properties with lipid raft molecules, form clusters at the cytoplasmic face of the plasma membrane and interact with signaling molecules in a cell type specific manner. In this review, reggie microdomains, lipid...

Sequencing and annotation of the chloroplast DNAs and identification of polymorphisms distinguishing normal male-fertile and male-sterile cytoplasms of onion.

Science.gov (United States)

von Kohn, Christopher; Kiełkowska, Agnieszka; Havey, Michael J

2013-12-01

Male-sterile (S) cytoplasm of onion is an alien cytoplasm introgressed into onion in antiquity and is widely used for hybrid seed production. Owing to the biennial generation time of onion, classical crossing takes at least 4 years to classify cytoplasms as S or normal (N) male-fertile. Molecular markers in the organellar DNAs that distinguish N and S cytoplasms are useful to reduce the time required to classify onion cytoplasms. In this research, we completed next-generation sequencing of the chloroplast DNAs of N- and S-cytoplasmic onions; we assembled and annotated the genomes in addition to identifying polymorphisms that distinguish these cytoplasms. The sizes (153 538 and 153 355 base pairs) and GC contents (36.8%) were very similar for the chloroplast DNAs of N and S cytoplasms, respectively, as expected given their close phylogenetic relationship. The size difference was primarily due to small indels in intergenic regions and a deletion in the accD gene of N-cytoplasmic onion. The structures of the onion chloroplast DNAs were similar to those of most land plants with large and small single copy regions separated by inverted repeats. Twenty-eight single nucleotide polymorphisms, two polymorphic restriction-enzyme sites, and one indel distributed across 20 chloroplast genes in the large and small single copy regions were selected and validated using diverse onion populations previously classified as N or S cytoplasmic using restriction fragment length polymorphisms. Although cytoplasmic male sterility is likely associated with the mitochondrial DNA, maternal transmission of the mitochondrial and chloroplast DNAs allows for polymorphisms in either genome to be useful for classifying onion cytoplasms to aid the development of hybrid onion cultivars.

Cytoplasmic tail of Coronavirus spike protein has intracellular ...

Indian Academy of Sciences (India)

58

1Department of Biological Sciences, Indian Institute of Science Education and ... Introduction .... A major effort is underway to understand the interplay of .... For protein analysis, cells were plated on 100 mm tissue culture dishes and transfected.

A 39-kD plasma membrane protein (IP39) is an anchor for the unusual membrane skeleton of Euglena gracilis

International Nuclear Information System (INIS)

Rosiere, T.K.; Marrs, J.A.; Bouck, G.B.

1990-01-01

The major integral plasma membrane protein (IP39) of Euglena gracilis was radiolabeled, peptide mapped, and dissected with proteases to identify cytoplasmic domains that bind and anchor proteins of the cell surface. When plasma membranes were radioiodinated and extracted with octyl glucoside, 98% of the extracted label was found in IP39 or the 68- and 110-kD oligomers of IP39. The octyl glucoside extracts were incubated with unlabeled cell surface proteins immobilized on nitrocellulose (overlays). Radiolabel from the membrane extract bound one (80 kD) of the two (80 and 86 kD) major membrane skeletal protein bands. Resolubilization of the bound label yielded a radiolabeled polypeptide identical in Mr to IP39. Intact plasma membranes were also digested with papain before or after radioiodination, thereby producing a cytoplasmically truncated IP39. The octyl glucoside extract of truncated IP39 no longer bound to the 80-kD membrane skeletal protein in the nitrocellulose overlays. EM of intact or trypsin digested plasma membranes incubated with membrane skeletal proteins under stringent conditions similar to those used in the nitrocellulose overlays revealed a partially reformed membrane skeletal layer. Little evidence of a membrane skeletal layer was found, however, when plasma membranes were predigested with papain before reassociation. A candidate 80-kD binding domain of IP39 has been tentatively identified as a peptide fragment that was present after trypsin digestion of plasma membranes, but was absent after papain digestion in two-dimensional peptide maps of IP39. Together, these data suggest that the unique peripheral membrane skeleton of Euglena binds to the plasma membrane through noncovalent interactions between the major 80-kD membrane skeletal protein and a small, papain sensitive cytoplasmic domain of IP39

Genomes2Drugs: identifies target proteins and lead drugs from proteome data.

LENUS (Irish Health Repository)

Toomey, David

2009-01-01

BACKGROUND: Genome sequencing and bioinformatics have provided the full hypothetical proteome of many pathogenic organisms. Advances in microarray and mass spectrometry have also yielded large output datasets of possible target proteins\\/genes. However, the challenge remains to identify new targets for drug discovery from this wealth of information. Further analysis includes bioinformatics and\\/or molecular biology tools to validate the findings. This is time consuming and expensive, and could fail to yield novel drugs if protein purification and crystallography is impossible. To pre-empt this, a researcher may want to rapidly filter the output datasets for proteins that show good homology to proteins that have already been structurally characterised or proteins that are already targets for known drugs. Critically, those researchers developing novel antibiotics need to select out the proteins that show close homology to any human proteins, as future inhibitors are likely to cross-react with the host protein, causing off-target toxicity effects later in clinical trials. METHODOLOGY\\/PRINCIPAL FINDINGS: To solve many of these issues, we have developed a free online resource called Genomes2Drugs which ranks sequences to identify proteins that are (i) homologous to previously crystallized proteins or (ii) targets of known drugs, but are (iii) not homologous to human proteins. When tested using the Plasmodium falciparum malarial genome the program correctly enriched the ranked list of proteins with known drug target proteins. CONCLUSIONS\\/SIGNIFICANCE: Genomes2Drugs rapidly identifies proteins that are likely to succeed in drug discovery pipelines. This free online resource helps in the identification of potential drug targets. Importantly, the program further highlights proteins that are likely to be inhibited by FDA-approved drugs. These drugs can then be rapidly moved into Phase IV clinical studies under \\'change-of-application\\' patents.

Genomes2Drugs: identifies target proteins and lead drugs from proteome data.

Directory of Open Access Journals (Sweden)

David Toomey

Full Text Available BACKGROUND: Genome sequencing and bioinformatics have provided the full hypothetical proteome of many pathogenic organisms. Advances in microarray and mass spectrometry have also yielded large output datasets of possible target proteins/genes. However, the challenge remains to identify new targets for drug discovery from this wealth of information. Further analysis includes bioinformatics and/or molecular biology tools to validate the findings. This is time consuming and expensive, and could fail to yield novel drugs if protein purification and crystallography is impossible. To pre-empt this, a researcher may want to rapidly filter the output datasets for proteins that show good homology to proteins that have already been structurally characterised or proteins that are already targets for known drugs. Critically, those researchers developing novel antibiotics need to select out the proteins that show close homology to any human proteins, as future inhibitors are likely to cross-react with the host protein, causing off-target toxicity effects later in clinical trials. METHODOLOGY/PRINCIPAL FINDINGS: To solve many of these issues, we have developed a free online resource called Genomes2Drugs which ranks sequences to identify proteins that are (i homologous to previously crystallized proteins or (ii targets of known drugs, but are (iii not homologous to human proteins. When tested using the Plasmodium falciparum malarial genome the program correctly enriched the ranked list of proteins with known drug target proteins. CONCLUSIONS/SIGNIFICANCE: Genomes2Drugs rapidly identifies proteins that are likely to succeed in drug discovery pipelines. This free online resource helps in the identification of potential drug targets. Importantly, the program further highlights proteins that are likely to be inhibited by FDA-approved drugs. These drugs can then be rapidly moved into Phase IV clinical studies under 'change-of-application' patents.

Identification and characterization of cytosolic Hansenula polymorpha proteins belonging to the Hsp70 protein family

NARCIS (Netherlands)

Titorenko, Vladimir I.; Evers, Melchior E.; Diesel, Andre; Samyn, Bart; Beeumen, Josef van; Roggenkamp, Rainer; Kiel, Jan A.K.W.; Klei, Ida J. van der; Veenhuis, Marten

We have isolated two members of the Hsp70 protein family from the yeast Hansenula polymorpha using affinity chromatography. Both proteins were located in the cytoplasm. One of these, designated Hsp72, was inducible in nature (e.g. by heat shock). The second protein (designated Hsc74) was

Analysis of cytoplasmic effects and fine-mapping of a genic male sterile line in rice.

Directory of Open Access Journals (Sweden)

Peng Qin

Full Text Available Cytoplasm has substantial genetic effects on progeny and is important for yield improvement in rice breeding. Studies on the cytoplasmic effects of cytoplasmic male sterility (CMS show that most types of CMS have negative effects on yield-related traits and that these negative effects vary among CMS. Some types of genic male sterility (GMS, including photo-thermo sensitive male sterility (PTMS, have been widely used in rice breeding, but the cytoplasmic effects of GMS remain unknown. Here, we identified a GMS mutant line, h2s, which exhibited small, white anthers and failed to produce mature pollen. Unlike CMS, the h2s had significant positive cytoplasmic effects on the seed set rate, weight per panicle, yield, and general combining ability (GCA for plant height, seed set rate, weight per panicle, and yield. These effects indicated that h2s cytoplasm may show promise for the improvement of rice yield. Genetic analysis suggested that the phenotype of h2s was controlled by a single recessive locus. We mapped h2s to a 152 kb region on chromosome 6, where 22 candidate genes were predicted. None of the 22 genes had previously been reported to be responsible for the phenotypes of h2s. Sequencing analysis showed a 12 bp deletion in the sixth exon of Loc_Os06g40550 in h2s in comparison to wild type, suggesting that Loc_Os06g40550 is the best candidate gene. These results lay a strong foundation for cloning of the H2S gene to elucidate the molecular mechanism of male reproduction.

Identification of poly(rC) binding protein 2 (PCBP2) as a target protein of immunosuppressive agent 15-deoxyspergualin

Energy Technology Data Exchange (ETDEWEB)

Murahashi, Masataka; Simizu, Siro; Morioka, Masahiko [Department of Applied Chemistry, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama 223-8522 (Japan); Umezawa, Kazuo, E-mail: umezawa@aichi-med-u.ac.jp [Department of Molecular Target Medicine, Aichi Medical University School of Medicine, 1-1 Yazako-Karimata, Nagakute 480-1195 (Japan)

2016-08-05

15-Deoxyspergualin (DSG) is an immunosuppressive agent being clinically used. Unlike tacrolimus and cyclosporine A, it does not inhibit the calcineurin pathway, and its mechanism of action and target molecule have not been elucidated. Therefore, we previously prepared biotinylated derivative of DSG (BDSG) to fish up the target protein. In the present research, we identified poly(rC) binding protein 2 (PCBP2) as a DSG-binding protein using this probe. DSG was confirmed to bind to PCBP2 by pull-down assay. Intracellular localization of PCBP2 was changed from the nucleus to the cytoplasm by DSG treatment. DSG inhibited the cell growth, and over-expression of PCBP2 reduced the anti-proliferative activity of DSG. PCBP2 is known to regulate various proteins including STAT1/2. Thus, we found PCBP2 as the first target protein of DSG that can explain the immunosuppressive activity. -- Highlights: •Fifteen-deoxyspergualin (DSG) is an immunosuppressive agent clinically used. •We have identified PCBP2, an RNA-binding protein, as a molecular target of DSG. •Alteration of PCBP2 activity may explain the immunosuppressive activity of DSG.

Constitutively expressed Protocadherin-α regulates the coalescence and elimination of homotypic olfactory axons through its cytoplasmic region

Directory of Open Access Journals (Sweden)

Sonoko eHasegawa

2012-10-01

Full Text Available Olfactory sensory neuron (OSN axons coalesce into specific glomeruli in the olfactory bulb (OB according to their odorant receptor (OR expression. Several guidance molecules enhance the coalescence of homotypic OSN projections, in an OR-specific- and neural-activity-dependent manner. However, the mechanism by which homotypic OSN axons are organized into glomeruli is unsolved. We previously reported that the clustered protocadherin-α (Pcdh-α family of diverse cadherin-related molecules plays roles in the coalescence and elimination of homotypic OSN axons throughout development. Here we showed that the elimination of small ectopic homotypic glomeruli required the constitutive expression of a Pcdh-α isoform and Pcdh-α’s cytoplasmic region, but not OR specificity or neural activity. These results suggest that Pcdh-α proteins provide a cytoplasmic signal to regulate repulsive activity for homotypic OSN axons independently of OR expression and neural activity. The counterbalancing effect of Pcdh-α proteins for the axonal coalescence mechanisms mediated by other olfactory guidance molecules indicate a possible mechanism for the organization of homotypic OSN axons into glomeruli during development.

Antineutrophil Cytoplasmic Antibodies Associated With Infective Endocarditis

Science.gov (United States)

Langlois, Vincent; Lesourd, Anais; Girszyn, Nicolas; Ménard, Jean-Francois; Levesque, Hervé; Caron, Francois; Marie, Isabelle

2016-01-01

Abstract To determine the prevalence of antineutrophil cytoplasmic antibodies (ANCA) in patients with infective endocarditis (IE) in internal medicine; and to compare clinical and biochemical features and outcome between patients exhibiting IE with and without ANCA. Fifty consecutive patients with IE underwent ANCA testing. The medical records of these patients were reviewed. Of the 50 patients with IE, 12 exhibited ANCA (24%). ANCA-positive patients with IE exhibited: longer duration between the onset of first symptoms and IE diagnosis (P = 0.02); and more frequently: weight loss (P = 0.017) and renal impairment (P = 0.08), lower levels of C-reactive protein (P = 0.0009) and serum albumin (P = 0.0032), involvement of both aortic and mitral valves (P = 0.009), and longer hospital stay (P = 0.016). Under multivariate analysis, significant factors for ANCA-associated IE were: longer hospital stay (P = 0.004), lower level of serum albumin (P = 0.02), and multiple valve involvement (P = 0.04). Mortality rate was 25% in ANCA patients; death was because of IE complications in all these patients. Our study identifies a high prevalence of ANCA in unselected patients with IE in internal medicine (24%). Our findings further underscore that ANCA may be associated with a subacute form of IE leading to multiple valve involvement and more frequent renal impairment. Because death was due to IE complications in all patients, our data suggest that aggressive therapy may be required to improve such patients’ outcome. PMID:26817911

Discrete nuclear structures in actively growing neuroblastoma cells are revealed by antibodies raised against phosphorylated neurofilament proteins

Directory of Open Access Journals (Sweden)

Raabe Timothy D

2003-04-01

Full Text Available Abstract Background Nuclear objects that have in common the property of being recognized by monoclonal antibodies specific for phosphoprotein epitopes and cytoplasmic intermediate filaments (in particular, SMI-31 and RT-97 have been reported in glial and neuronal cells, in situ and in vitro. Since neurofilament and glial filaments are generally considered to be restricted to the cytoplasm, we were interested in exploring the identity of the structures labeled in the nucleus as well as the conditions under which they could be found there. Results Using confocal microscopy and western analysis techniques, we determined 1 the immunolabeled structures are truly within the nucleus; 2 the phosphoepitope labeled by SMI-31 and RT-97 is not specific to neurofilaments (NFs and it can be identified on other intermediate filament proteins (IFs in other cell types; and 3 there is a close relationship between DNA synthesis and the amount of nuclear staining by these antibodies thought to be specific for cytoplasmic proteins. Searches of protein data bases for putative phosphorylation motifs revealed that lamins, NF-H, and GFAP each contain a single tyrosine phosphorylation motif with nearly identical amino acid sequence. Conclusion We therefore suggest that this sequence may be the epitope recognized by SMI-31 and RT-97 mABs, and that the nuclear structures previously reported and shown here are likely phosphorylated lamin intermediate filaments, while the cytoplasmic labeling revealed by the same mABs indicates phosphorylated NFs in neurons or GFAP in glia.

The nuclear import of ribosomal proteins is regulated by mTOR

Science.gov (United States)

Kazyken, Dubek; Kaz, Yelimbek; Kiyan, Vladimir; Zhylkibayev, Assylbek A.; Chen, Chien-Hung; Agarwal, Nitin K.; Sarbassov, Dos D.

2014-01-01

Mechanistic target of rapamycin (mTOR) is a central component of the essential signaling pathway that regulates cell growth and proliferation by controlling anabolic processes in cells. mTOR exists in two distinct mTOR complexes known as mTORC1 and mTORC2 that reside mostly in cytoplasm. In our study, the biochemical characterization of mTOR led to discovery of its novel localization on nuclear envelope where it associates with a critical regulator of nuclear import Ran Binding Protein 2 (RanBP2). We show that association of mTOR with RanBP2 is dependent on the mTOR kinase activity that regulates the nuclear import of ribosomal proteins. The mTOR kinase inhibitors within thirty minutes caused a substantial decrease of ribosomal proteins in the nuclear but not cytoplasmic fraction. Detection of a nuclear accumulation of the GFP-tagged ribosomal protein rpL7a also indicated its dependence on the mTOR kinase activity. The nuclear abundance of ribosomal proteins was not affected by inhibition of mTOR Complex 1 (mTORC1) by rapamycin or deficiency of mTORC2, suggesting a distinctive role of the nuclear envelope mTOR complex in the nuclear import. Thus, we identified that mTOR in association with RanBP2 mediates the active nuclear import of ribosomal proteins. PMID:25294810

Endoplasmic-reticulum-mediated microtubule alignment governs cytoplasmic streaming.

Science.gov (United States)

Kimura, Kenji; Mamane, Alexandre; Sasaki, Tohru; Sato, Kohta; Takagi, Jun; Niwayama, Ritsuya; Hufnagel, Lars; Shimamoto, Yuta; Joanny, Jean-François; Uchida, Seiichi; Kimura, Akatsuki

2017-04-01

Cytoplasmic streaming refers to a collective movement of cytoplasm observed in many cell types. The mechanism of meiotic cytoplasmic streaming (MeiCS) in Caenorhabditis elegans zygotes is puzzling as the direction of the flow is not predefined by cell polarity and occasionally reverses. Here, we demonstrate that the endoplasmic reticulum (ER) network structure is required for the collective flow. Using a combination of RNAi, microscopy and image processing of C. elegans zygotes, we devise a theoretical model, which reproduces and predicts the emergence and reversal of the flow. We propose a positive-feedback mechanism, where a local flow generated along a microtubule is transmitted to neighbouring regions through the ER. This, in turn, aligns microtubules over a broader area to self-organize the collective flow. The proposed model could be applicable to various cytoplasmic streaming phenomena in the absence of predefined polarity. The increased mobility of cortical granules by MeiCS correlates with the efficient exocytosis of the granules to protect the zygotes from osmotic and mechanical stresses.

Pericentriolar Targeting of the Mouse Mammary Tumor Virus GAG Protein.

Directory of Open Access Journals (Sweden)

Guangzhi Zhang

Full Text Available The Gag protein of the mouse mammary tumor virus (MMTV is the chief determinant of subcellular targeting. Electron microscopy studies show that MMTV Gag forms capsids within the cytoplasm and assembles as immature particles with MMTV RNA and the Y box binding protein-1, required for centrosome maturation. Other betaretroviruses, such as Mason-Pfizer monkey retrovirus (M-PMV, assemble adjacent to the pericentriolar region because of a cytoplasmic targeting and retention signal in the Matrix protein. Previous studies suggest that the MMTV Matrix protein may also harbor a similar cytoplasmic targeting and retention signal. Herein, we show that a substantial fraction of MMTV Gag localizes to the pericentriolar region. This was observed in HEK293T, HeLa human cell lines and the mouse derived NMuMG mammary gland cells. Moreover, MMTV capsids were observed adjacent to centrioles when expressed from plasmids encoding either MMTV Gag alone, Gag-Pro-Pol or full-length virus. We found that the cytoplasmic targeting and retention signal in the MMTV Matrix protein was sufficient for pericentriolar targeting, whereas mutation of the glutamine to alanine at position 56 (D56/A resulted in plasma membrane localization, similar to previous observations from mutational studies of M-PMV Gag. Furthermore, transmission electron microscopy studies showed that MMTV capsids accumulate around centrioles suggesting that, similar to M-PMV, the pericentriolar region may be a site for MMTV assembly. Together, the data imply that MMTV Gag targets the pericentriolar region as a result of the MMTV cytoplasmic targeting and retention signal, possibly aided by the Y box protein-1 required for the assembly of centrosomal microtubules.

In-cell intrabody selection from a diverse human library identifies C12orf4 protein as a new player in rodent mast cell degranulation.

Directory of Open Access Journals (Sweden)

Elsa Mazuc

Full Text Available The high specificity of antibodies for their antigen allows a fine discrimination of target conformations and post-translational modifications, making antibodies the first choice tool to interrogate the proteome. We describe here an approach based on a large-scale intracellular expression and selection of antibody fragments in eukaryotic cells, so-called intrabodies, and the subsequent identification of their natural target within living cell. Starting from a phenotypic trait, this integrated system allows the identification of new therapeutic targets together with their companion inhibitory intrabody. We applied this system in a model of allergy and inflammation. We first cloned a large and highly diverse intrabody library both in a plasmid and a retroviral eukaryotic expression vector. After transfection in the RBL-2H3 rat basophilic leukemia cell line, we performed seven rounds of selection to isolate cells displaying a defect in FcεRI-induced degranulation. We used high throughput sequencing to identify intrabody sequences enriched during the course of selection. Only one intrabody was common to both plasmid and retroviral selections, and was used to capture and identify its target from cell extracts. Mass spectrometry analysis identified protein RGD1311164 (C12orf4, with no previously described function. Our data demonstrate that RGD1311164 is a cytoplasmic protein implicated in the early signaling events following FcεRI-induced cell activation. This work illustrates the strength of the intrabody-based in-cell selection, which allowed the identification of a new player in mast cell activation together with its specific inhibitor intrabody.

Interaction of nucleosome assembly proteins abolishes nuclear localization of DGKζ by attenuating its association with importins

International Nuclear Information System (INIS)

Okada, Masashi; Hozumi, Yasukazu; Ichimura, Tohru; Tanaka, Toshiaki; Hasegawa, Hiroshi; Yamamoto, Masakazu; Takahashi, Nobuya; Iseki, Ken; Yagisawa, Hitoshi; Shinkawa, Takashi; Isobe, Toshiaki; Goto, Kaoru

2011-01-01

Diacylglycerol kinase (DGK) is involved in the regulation of lipid-mediated signal transduction through the metabolism of a second messenger diacylglycerol. Of the DGK family, DGKζ, which contains a nuclear localization signal, localizes mainly to the nucleus but translocates to the cytoplasm under pathological conditions. However, the detailed mechanism of translocation and its functional significance remain unclear. To elucidate these issues, we used a proteomic approach to search for protein targets that interact with DGKζ. Results show that nucleosome assembly protein (NAP) 1-like 1 (NAP1L1) and NAP1-like 4 (NAP1L4) are identified as novel DGKζ binding partners. NAP1Ls constitutively shuttle between the nucleus and the cytoplasm in transfected HEK293 cells. The molecular interaction of DGKζ and NAP1Ls prohibits nuclear import of DGKζ because binding of NAP1Ls to DGKζ blocks import carrier proteins, Qip1 and NPI1, to interact with DGKζ, leading to cytoplasmic tethering of DGKζ. In addition, overexpression of NAP1Ls exerts a protective effect against doxorubicin-induced cytotoxicity. These findings suggest that NAP1Ls are involved in a novel molecular basis for the regulation of nucleocytoplasmic shuttling of DGKζ and provide a clue to examine functional significance of its translocation under pathological conditions.

Molecular cloning of the common acute lymphoblastic leukemia antigen (CALLA) identifies a type II integral membrane protein

International Nuclear Information System (INIS)

Shipp, M.A.; Richardson, N.E.; Sayre, P.H.; Brown, N.R.; Masteller, E.L.; Clayton, L.K.; Ritz, J.; Reinherz, E.L.

1988-01-01

Common acute lymphoblastic leukemia antigen (CALLA) is a 100-kDa cell-surface glycoprotein expressed on most acute lymphoblastic leukemias and certain other immature lymphoid malignancies and on normal lymphoid progenitors. The latter are either uncommitted to B- or T-cell lineage or committed to only the earliest stages of B- or T-lymphocyte maturation. To elucidate the primary structure of CALLA, the authors purified the protein to homogeneity, obtained the NH 2 -terminal sequence from both the intact protein and derived tryptic and V8 protease peptides and isolated CALLA cDNAs from a Nalm-6 cell line λgt10 library using redundant oligonucleotide probes. The CALLA cDNA sequence predicts a 750-amino acid integral membrane protein with a single 24-amino acid hydrophobic segment that could function as both a transmembrane region and a signal peptide. The COOH-terminal 700 amino acids, including six potential N-linked glycosylation sites compose the extracellular protein segment, whereas the 25 NM 2 -terminal amino acids remaining after cleavage of the initiation methionine form the cytoplasmic tail. CALLA + cells contain CALLA transcripts of 2.7 to 5.7 kilobases with the major 5.7- and 3.7-kilobase mRNAs being preferentially expressed in specific cell types

Juvenile hormone-binding proteins of Melanoplus bivittatus identified by EFDA photoaffinity labeling

International Nuclear Information System (INIS)

Winder, B.S.

1988-01-01

Proteins that bind juvenile hormone in the hemolymph and fat body of the grasshopper, Melanoplus bivittatus were identified by photoaffinity labeling with radiolabeled epoxyfarnesyl diazoacetate ( 3 H-EFDA), and were characterized by electrophoretic analysis. A protocol was developed which allowed detection of 3 H-EFDA that was covalently linked to proteins upon exposure to ultraviolet light at 254 nm. Quantification of protein-linked 3 H-EFDA by liquid scintillation spectrometry took advantage of the differential solubility of unlinked 3 H-EFDA in toluene alone, and of the protein-linked 3 H-EFDA in toluene plus the detergent, Triton X-100. Competition between EFDA and juvenile hormone (JH) for binding to JH-specific binding sites was measured by hydroxyapatite protein binding assays in the presence of radiolabeled JH or EFDA and competing non-radiolabeled hormone. The protein-linked EFDA was detected on fluorograms of SDS or nondenaturing polyacrylamide gels (PAGE), and by liquid scintillation spectrometry of membranes to which the proteins had been electrophoretically transferred. Proteins which specifically bound JH were identified by photolabeling proteins in the presence and absence of nonlabeled JH-III

Xcat, a novel mouse model for Nance-Horan syndrome inhibits expression of the cytoplasmic-targeted Nhs1 isoform.

Science.gov (United States)

Huang, Kristen M; Wu, Junhua; Duncan, Melinda K; Moy, Chris; Dutra, Amalia; Favor, Jack; Da, Tong; Stambolian, Dwight

2006-01-15

Nance-Horan syndrome (NHS) is an X-linked disorder characterized by congenital cataracts, dental anomalies, dysmorphic features and mental retardation. A recent report suggests that the novel gene NHS1 is involved in this disorder due to the presence of point mutations in NHS patients. A possible mouse model for NHS, Xcat, was mapped to a 2.11 Mb interval on the X-chromosome. Sequence and FISH analysis of the X-chromosome region containing the Xcat mutation reveal a large insertion between exons 1 and 2 of the mouse Nhs1 gene. The insertion inhibits the expression of the Nhs1 isoform containing exon 1 and results in exclusive expression of the alternative isoform containing exon 1A. Quantitative RT-PCR of Xcat cDNA shows reduced levels of Nhs1 transcripts. The Nhs1 protein is strongly expressed within the cytoplasm of elongating lens fiber cells from wild-type neonate lens, but is significantly reduced within the Xcat lens. Transient transfection studies of CHO cells with Nhs1-GFP fusion proteins were done to determine whether the amino acids encoded by exon 1 were critical for protein localization. We found the presence of Nhs1 exon 1 critical for localization of the fusion protein to the cytoplasm, whereas fusion proteins lacking Nhs1 exon 1 are predominantly nuclear. These results indicate that the first exon of Nhs1 contains crucial information required for the proper expression and localization of Nhs1 protein. Inhibition of expression of the exon 1 containing isoform results in the abnormal phenotype of Xcat.

Autoselection of cytoplasmic yeast virus like elements encoding toxin/antitoxin systems involves a nuclear barrier for immunity gene expression.

Science.gov (United States)

Kast, Alene; Voges, Raphael; Schroth, Michael; Schaffrath, Raffael; Klassen, Roland; Meinhardt, Friedhelm

2015-05-01

Cytoplasmic virus like elements (VLEs) from Kluyveromyces lactis (Kl), Pichia acaciae (Pa) and Debaryomyces robertsiae (Dr) are extremely A/T-rich (>75%) and encode toxic anticodon nucleases (ACNases) along with specific immunity proteins. Here we show that nuclear, not cytoplasmic expression of either immunity gene (PaORF4, KlORF3 or DrORF5) results in transcript fragmentation and is insufficient to establish immunity to the cognate ACNase. Since rapid amplification of 3' ends (RACE) as well as linker ligation of immunity transcripts expressed in the nucleus revealed polyadenylation to occur along with fragmentation, ORF-internal poly(A) site cleavage due to the high A/T content is likely to prevent functional expression of the immunity genes. Consistently, lowering the A/T content of PaORF4 to 55% and KlORF3 to 46% by gene synthesis entirely prevented transcript cleavage and permitted functional nuclear expression leading to full immunity against the respective ACNase toxin. Consistent with a specific adaptation of the immunity proteins to the cognate ACNases, cross-immunity to non-cognate ACNases is neither conferred by PaOrf4 nor KlOrf3. Thus, the high A/T content of cytoplasmic VLEs minimizes the potential of functional nuclear recruitment of VLE encoded genes, in particular those involved in autoselection of the VLEs via a toxin/antitoxin principle.

Autoselection of cytoplasmic yeast virus like elements encoding toxin/antitoxin systems involves a nuclear barrier for immunity gene expression.

Directory of Open Access Journals (Sweden)

Alene Kast

2015-05-01

Full Text Available Cytoplasmic virus like elements (VLEs from Kluyveromyces lactis (Kl, Pichia acaciae (Pa and Debaryomyces robertsiae (Dr are extremely A/T-rich (>75% and encode toxic anticodon nucleases (ACNases along with specific immunity proteins. Here we show that nuclear, not cytoplasmic expression of either immunity gene (PaORF4, KlORF3 or DrORF5 results in transcript fragmentation and is insufficient to establish immunity to the cognate ACNase. Since rapid amplification of 3' ends (RACE as well as linker ligation of immunity transcripts expressed in the nucleus revealed polyadenylation to occur along with fragmentation, ORF-internal poly(A site cleavage due to the high A/T content is likely to prevent functional expression of the immunity genes. Consistently, lowering the A/T content of PaORF4 to 55% and KlORF3 to 46% by gene synthesis entirely prevented transcript cleavage and permitted functional nuclear expression leading to full immunity against the respective ACNase toxin. Consistent with a specific adaptation of the immunity proteins to the cognate ACNases, cross-immunity to non-cognate ACNases is neither conferred by PaOrf4 nor KlOrf3. Thus, the high A/T content of cytoplasmic VLEs minimizes the potential of functional nuclear recruitment of VLE encoded genes, in particular those involved in autoselection of the VLEs via a toxin/antitoxin principle.

Molecular Characterization of Bombyx mori Cytoplasmic Polyhedrosis Virus Genome Segment 4

Science.gov (United States)

Ikeda, Keiko; Nagaoka, Sumiharu; Winkler, Stefan; Kotani, Kumiko; Yagi, Hiroaki; Nakanishi, Kae; Miyajima, Shigetoshi; Kobayashi, Jun; Mori, Hajime

2001-01-01

The complete nucleotide sequence of the genome segment 4 (S4) of Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) was determined. The 3,259-nucleotide sequence contains a single long open reading frame which spans nucleotides 14 to 3187 and which is predicted to encode a protein with a molecular mass of about 130 kDa. Western blot analysis showed that S4 encodes BmCPV protein VP3, which is one of the outer components of the BmCPV virion. Sequence analysis of the deduced amino acid sequence of BmCPV VP3 revealed possible sequence homology with proteins from rice ragged stunt virus (RRSV) S2, Nilaparvata lugens reovirus S4, and Fiji disease fijivirus S4. This may suggest that plant reoviruses originated from insect viruses and that RRSV emerged more recently than other plant reoviruses. A chimeric protein consisting of BmCPV VP3 and green fluorescent protein (GFP) was constructed and expressed with BmCPV polyhedrin using a baculovirus expression vector. The VP3-GFP chimera was incorporated into BmCPV polyhedra and released under alkaline conditions. The results indicate that specific interactions occur between BmCPV polyhedrin and VP3 which might facilitate BmCPV virion occlusion into the polyhedra. PMID:11134312

The nuclear protein Sam68 is cleaved by the FMDV 3C protease redistributing Sam68 to the cytoplasm during FMDV infection of host cells

International Nuclear Information System (INIS)

Lawrence, Paul; Schafer, Elizabeth A.; Rieder, Elizabeth

2012-01-01

Picornavirus infection can lead to disruption of nuclear pore traffic, shut-off of cell translation machinery, and cleavage of proteins involved in cellular signal transduction and the innate response to infection. Here, we demonstrated that the FMDV 3C pro induced the cleavage of nuclear RNA-binding protein Sam68 C-terminus containing the nuclear localization sequence (NLS). Consequently, it stimulated the redistribution of Sam68 to the cytoplasm. The siRNA knockdown of Sam68 resulted in a 1000-fold reduction in viral titers, which prompted us to study the effect of Sam68 on FMDV post-entry events. Interestingly, Sam68 interacts with the internal ribosomal entry site within the 5′ non-translated region of the FMDV genome, and Sam68 knockdown decreased FMDV IRES-driven activity in vitro suggesting that it could modulate translation of the viral genome. The results uncover a novel role for Sam68 in the context of picornaviruses and the proteolysis of a new cellular target of the FMDV 3C pro .

Hierarchical protein export mechanism of the bacterial flagellar type III protein export apparatus.

Science.gov (United States)

Minamino, Tohru

2018-06-01

The bacterial flagellum is supramolecular motility machinery consisting of the basal body, the hook and the filament. Flagellar proteins are translocated across the cytoplasmic membrane via a type III protein export apparatus, diffuse down the central channel of the growing structure and assemble at the distal end. Flagellar assembly begins with the basal body, followed by the hook and finally the filament. The completion of hook assembly is the most important morphological checkpoint of the sequential flagellar assembly process. When the hook reaches its mature length of about 55 nm in Salmonella enterica, the type III protein export apparatus switches export specificity from proteins required for the structure and assembly of the hook to those responsible for filament assembly, thereby terminating hook assembly and initiating filament assembly. Three flagellar proteins, namely FliK, FlhB and FlhA, are responsible for this substrate specificity switching. Upon completion of the switching event, interactions among FlhA, the cytoplasmic ATPase complex and flagellar type III export chaperones establish the assembly order of the filament at the hook tip. Here, we describe our current understanding of a hierarchical protein export mechanism used in flagellar type III protein export.

Heterologous gln/asn-rich proteins impede the propagation of yeast prions by altering chaperone availability.

Directory of Open Access Journals (Sweden)

Zi Yang

Full Text Available Prions are self-propagating conformations of proteins that can cause heritable phenotypic traits. Most yeast prions contain glutamine (Q/asparagine (N-rich domains that facilitate the accumulation of the protein into amyloid-like aggregates. Efficient transmission of these infectious aggregates to daughter cells requires that chaperones, including Hsp104 and Sis1, continually sever the aggregates into smaller "seeds." We previously identified 11 proteins with Q/N-rich domains that, when overproduced, facilitate the de novo aggregation of the Sup35 protein into the [PSI(+] prion state. Here, we show that overexpression of many of the same 11 Q/N-rich proteins can also destabilize pre-existing [PSI(+] or [URE3] prions. We explore in detail the events leading to the loss (curing of [PSI(+] by the overexpression of one of these proteins, the Q/N-rich domain of Pin4, which causes Sup35 aggregates to increase in size and decrease in transmissibility to daughter cells. We show that the Pin4 Q/N-rich domain sequesters Hsp104 and Sis1 chaperones away from the diffuse cytoplasmic pool. Thus, a mechanism by which heterologous Q/N-rich proteins impair prion propagation appears to be the loss of cytoplasmic Hsp104 and Sis1 available to sever [PSI(+].

vProtein: identifying optimal amino acid complements from plant-based foods.

Directory of Open Access Journals (Sweden)

Peter J Woolf

Full Text Available BACKGROUND: Indispensible amino acids (IAAs are used by the body in different proportions. Most animal-based foods provide these IAAs in roughly the needed proportions, but many plant-based foods provide different proportions of IAAs. To explore how these plant-based foods can be better used in human nutrition, we have created the computational tool vProtein to identify optimal food complements to satisfy human protein needs. METHODS: vProtein uses 1251 plant-based foods listed in the United States Department of Agriculture standard release 22 database to determine the quantity of each food or pair of foods required to satisfy human IAA needs as determined by the 2005 daily recommended intake. The quantity of food in a pair is found using a linear programming approach that minimizes total calories, total excess IAAs, or the total weight of the combination. RESULTS: For single foods, vProtein identifies foods with particularly balanced IAA patterns such as wheat germ, quinoa, and cauliflower. vProtein also identifies foods with particularly unbalanced IAA patterns such as macadamia nuts, degermed corn products, and wakame seaweed. Although less useful alone, some unbalanced foods provide unusually good complements, such as Brazil nuts to legumes. Interestingly, vProtein finds no statistically significant bias toward grain/legume pairings for protein complementation. These analyses suggest that pairings of plant-based foods should be based on the individual foods themselves instead of based on broader food group-food group pairings. Overall, the most efficient pairings include sweet corn/tomatoes, apple/coconut, and sweet corn/cherry. The top pairings also highlight the utility of less common protein sources such as the seaweeds laver and spirulina, pumpkin leaves, and lambsquarters. From a public health perspective, many of the food pairings represent novel, low cost food sources to combat malnutrition. Full analysis results are available online

Pollen mitochondria in cytoplasmically male sterile tobacco zygotic and embryonic cells

International Nuclear Information System (INIS)

Symillides, Y.

1985-09-01

An attempt is being made to establish cytoplasmic organelles transmission during the process of fertilization, by using tobacco grain pollen labelled with leucine 14 C and tritiated thymidine. Through autoradiography the fate of pollen germination and its entry into the embryo sac has been studied. A few days after fertilization, labelled cytoplasmic organelles - mainly mitochondria - were detected in the embryo sac. However, labelling was not observed in cytoplasmic organelles by using tritiated thymidine. For more conclusive results labelled DNA incorporated in cytoplasmic organelles have to be traced during the embryo and endosperm development

Hormone signaling linked to silkmoth sex pheromone biosynthesis involves Ca2+/calmodulin-dependent protein kinase II-mediated phosphorylation of the insect PAT family protein Bombyx mori lipid storage droplet protein-1(BmLsd)

Science.gov (United States)

The structurally-related members of the PAT family of proteins, which are so name based on similarity amongst perilipin, adipophilin/adipocyte differentiation-related protein (ADRP), and tail-interacting protein of 47 kilodaltons (TIP47), are cytoplasmic lipid droplet (LD)-associated proteins charac...

Development of Novel Cytoplasmic Male Sterile Source from Dongxiang Wild Rice (Oryza rufipogon

Directory of Open Access Journals (Sweden)

Xian-hua SHEN

2013-09-01

Full Text Available This study was conducted to develop and characterize a novel cytoplasmic male sterile (CMS source which was identified from Dongxiang wild rice (Oryza rufipogon by crossing Dongxiang wild rice as female with Zhongzao 35, an indica inbred variety, as male and continuous backcrossing with Zhongzao 35. Observation under optical microscope manifested that this novel CMS belonged to typical abortion type with less pollen compared with wild abortive type cytoplasm (CMS-WA. Sequential planting showed that this novel CMS has complete and stable male sterility. Testcross experiment showed that all the 24 tested materials including maintainer and restorer lines of CMS-WA and Honglian type cytoplasm (CMS-HL and other indica inbred varieties are the maintainers with complete maintaining ability, suggesting that this novel CMS has fertility restoration totally different from CMS-WA and CMS-HL and belongs to a novel type of CMS. So far, we only discovered a unique fertility restoration source for this novel CMS. Inheritance analysis showed that the fertility restoration of this CMS was governed by three pairs of independent dominant genes. Prospect for application of this novel CMS system in hybrid rice breeding was also discussed.

Subcellular localization and logistics of integral membrane protein biogenesis in Escherichia coli.

Science.gov (United States)

Bogdanov, Mikhail; Aboulwafa, Mohammad; Saier, Milton H

2013-01-01

Transporters catalyze entry and exit of molecules into and out of cells and organelles, and protein-lipid interactions influence their activities. The bacterial phosphoenolpyruvate: sugar phosphotransferase system (PTS) catalyzes transport-coupled sugar phosphorylation as well as nonvectorial sugar phosphorylation in the cytoplasm. The vectorial process is much more sensitive to the lipid environment than the nonvectorial process. Moreover, cytoplasmic micellar forms of these enzyme-porters have been identified, and non-PTS permeases have similarly been shown to exist in 'soluble' forms. The latter porters exhibit lipid-dependent activities and can adopt altered topologies by simply changing the lipid composition. Finally, intracellular membranes and vesicles exist in Escherichia coli leading to the following unanswered questions: (1) what determines whether a PTS permease catalyzes vectorial or nonvectorial sugar phosphorylation? (2) How do phospholipids influence relative amounts of the plasma membrane, intracellular membrane, inner membrane-derived vesicles and cytoplasmic micelles? (3) What regulates the route(s) of permease insertion and transfer into and between the different subcellular sites? (4) Do these various membranous forms have distinct physiological functions? (5) What methods should be utilized to study the biogenesis and interconversion of these membranous structures? While research concerning these questions is still in its infancy, answers will greatly enhance our understanding of protein-lipid interactions and how they control the activities, conformations, cellular locations and biogenesis of integral membrane proteins. Copyright © 2013 S. Karger AG, Basel.

Cytoplasmic localization of alteration/deficiency in activation 3 (ADA3) predicts poor clinical outcome in breast cancer patients.

Science.gov (United States)

Mirza, Sameer; Rakha, Emad A; Alshareeda, Alaa; Mohibi, Shakur; Zhao, Xiangshan; Katafiasz, Bryan J; Wang, Jun; Gurumurthy, Channabasavaiah Basavaraju; Bele, Aditya; Ellis, Ian O; Green, Andrew R; Band, Hamid; Band, Vimla

2013-02-01

ErbB2+/EGFR+ expression and together is a marker of poor prognosis. Thus, ADA3 cytoplasmic localization together with ErbB2+/EGFR+ status may serve as better prognostic marker than individual proteins to predict survival of patients.

Roles of the conserved cytoplasmic region and non-conserved carboxy-terminal region of SecE in Escherichia coli protein translocase.

Science.gov (United States)

Kontinen, V P; Yamanaka, M; Nishiyama, K; Tokuda, H

1996-06-01

SecE, an essential membrane component of the Escherichia coli protein translocase, consists of 127 amino acid residues. Only a part of the second putative cytoplasmic region comprising some 13 residues is essential for the SecE function as long as the proper topological arrangement is retained. The Trp84 and Pro85 residues of this region are conserved in all eubacterial SecE homologues. The conservation of positively charged residues corresponding to Arg80 and Lys81 is also substantial. We deleted or replaced these residues to assess their roles in the SecE function. Deletion of the Arg80-Lys81 dipeptide did not abolish the SecE function whereas that of Trp84 or Pro85 caused a loss of the function. Strikingly, however, replacement of Pro85 with either Gly, Ser, or Ala, and that of Trp84 with Lys did not abolish the SecE function. These results indicate that the strong conservation of these residues does not reflect their obligatory requirement for the SecE function. A chimeric SecE possessing the cytoplasmic region of the E. coli SecE and the following region of the Bacillus subtilis SecE was able to form the translocation machinery together with SecA, SecY, and SecG. Although a Leu to Arg mutation at position 108 has been thought to cause a loss of signal recognition fidelity and thereby suppress a signal sequence defect, the same mutation at position 111 caused a complete loss of the function. The levels of SecY and SecG in the secEcsE501 mutant, which expresses SecE at a decreased level and is sensitive to low temperature, increased upon the expression of functional SecE derivatives, irrespective of the site of mutation, suggesting that the levels of SecY and SecG are co-operatively determined by the level of functional, but not non-functional, SecE. Based on these results, the SecE function in the translocase is discussed.

Evidence for the interaction of the regulatory protein Ki-1/57 with p53 and its interacting proteins

International Nuclear Information System (INIS)

Nery, Flavia C.; Rui, Edmilson; Kuniyoshi, Tais M.; Kobarg, Joerg

2006-01-01

Ki-1/57 is a cytoplasmic and nuclear phospho-protein of 57 kDa and interacts with the adaptor protein RACK1, the transcription factor MEF2C, and the chromatin remodeling factor CHD3, suggesting that it might be involved in the regulation of transcription. Here, we describe yeast two-hybrid studies that identified a total of 11 proteins interacting with Ki-1/57, all of which interact or are functionally associated with p53 or other members of the p53 family of proteins. We further found that Ki-1/57 is able to interact with p53 itself in the yeast two-hybrid system when the interaction was tested directly. This interaction could be confirmed by pull down assays with purified proteins in vitro and by reciprocal co-immunoprecipitation assays from the human Hodgkin analogous lymphoma cell line L540. Furthermore, we found that the phosphorylation of p53 by PKC abolishes its interaction with Ki-1/57 in vitro

Isolation of cell nuclei using inert macromolecules to mimic the crowded cytoplasm.

Directory of Open Access Journals (Sweden)

Ronald Hancock

Full Text Available Cell nuclei are commonly isolated and studied in media which include millimolar concentrations of cations, which conserve the nuclear volume by screening the negative charges on chromatin and maintaining its compaction. However, two factors question if these ionic conditions correctly reproduce the environment of nuclei in vivo: the small-scale motion and conformation of chromatin in vivo are not reproduced in isolated nuclei, and experiments and theory suggest that small ions in the cytoplasm are not free in the soluble phase but are predominantly bound to macromolecules. We studied the possible role in maintaining the structure and functions of nuclei in vivo of a further but frequently overlooked property of the cytoplasm, the crowding or osmotic effects caused by diffusible macromolecules whose concentration, measured in several studies, is in the range of 130 mg/ml. Nuclei which conserved their volume in the cell and their ultrastructure seen by electron microscopy were released from K562 cells in media containing the inert polymer 70 kDa Ficoll (50% w/v or 70 kDa dextran (35% w/v to replace the diffusible cytoplasmic molecules which were dispersed on cell lysis with digitonin, with 100 microM K-Hepes buffer as the only source of ions. Immunofluorescence labelling and experiments using cells expressing GFP-fusion proteins showed that internal compartments (nucleoli, PML and coiled bodies, foci of RNA polymerase II were conserved in these nuclei, and nascent RNA transcripts could be elongated. Our observations are consistent with the hypothesis that crowding by diffusible cytoplasmic macromolecules is a crucial but overlooked factor which supports the nucleus in vivo by equilibrating the opposing osmotic pressure cause by the high concentration of macromolecules in the nucleus, and suggest that crowded media provide more physiological conditions to study nuclear structure and functions. They may also help to resolve the long-standing paradox

Soluble expression of recombinant proteins in the cytoplasm of Escherichia coli

DEFF Research Database (Denmark)

Sørensen, Hans; Mortensen, Kim

2005-01-01

Pure, soluble and functional proteins are of high demand in modern biotechnology. Natural protein sources rarely meet the requirements for quantity, ease of isolation or price and hence recombinant technology is often the method of choice. Recombinant cell factories are constantly employed...... molecular tools available. In spite of all these qualities, expression of recombinant proteins with E. coli as the host often results in insoluble and/or nonfunctional proteins. Here we review new approaches to overcome these obstacles by strategies that focus on either controlled expression of target...... for the production of protein preparations bound for downstream purification and processing. Eschericia coli is a frequently used host, since it facilitates protein expression by its relative simplicity, its inexpensive and fast high density cultivation, the well known genetics and the large number of compatible...

Proteomic profiling of human plasma exosomes identifies PPARγ as an exosome-associated protein

International Nuclear Information System (INIS)

Looze, Christopher; Yui, David; Leung, Lester; Ingham, Matthew; Kaler, Maryann; Yao, Xianglan; Wu, Wells W.; Shen Rongfong; Daniels, Mathew P.; Levine, Stewart J.

2009-01-01

Exosomes are nanovesicles that are released from cells as a mechanism of cell-free intercellular communication. Only a limited number of proteins have been identified from the plasma exosome proteome. Here, we developed a multi-step fractionation scheme incorporating gel exclusion chromatography, rate zonal centrifugation through continuous sucrose gradients, and high-speed centrifugation to purify exosomes from human plasma. Exosome-associated proteins were separated by SDS-PAGE and 66 proteins were identified by LC-MS/MS, which included both cellular and extracellular proteins. Furthermore, we identified and characterized peroxisome proliferator-activated receptor-γ (PPARγ), a nuclear receptor that regulates adipocyte differentiation and proliferation, as well as immune and inflammatory cell functions, as a novel component of plasma-derived exosomes. Given the important role of exosomes as intercellular messengers, the discovery of PPARγ as a component of human plasma exosomes identifies a potential new pathway for the paracrine transfer of nuclear receptors.

The Structure of the ZMYND8/Drebrin Complex Suggests a Cytoplasmic Sequestering Mechanism of ZMYND8 by Drebrin.

Science.gov (United States)

Yao, Ningning; Li, Jianchao; Liu, Haiyang; Wan, Jun; Liu, Wei; Zhang, Mingjie

2017-11-07

Malfunctions of the actin binding protein Drebrin have been implicated in various human diseases such as Alzheimer's disease, cognitive impairments, cancer, and digestive disorders, though with poorly understood mechanisms. The ADF-H domain of Drebrin does not contain actin binding and depolymerizing activity. Instead, it binds to a histone marker reader, ZMYND8. Here we present the high-resolution crystal structure of Drebrin ADF-H in complex with the ZMYND8 PHD-BROMO-PWWP tandem, elucidating the mechanistic basis governing the highly specific interaction of the two proteins. The structure reveals that the ZMYND8 PHD-BROMO-PWWP tandem forms a structural supramodule that is necessary for binding to Drebrin ADF-H. Drebrin ADF-H competes with modified histone for binding to ZMYND8. Binding of Drebrin can shuttle ZMYND8 from nucleus to cytoplasm in living cells. Taken together, our study uncovers a non-actin target binding mode for ADF-H domains, and suggests that Drebrin may regulate activities of epigenetic reader ZMYND8 via its cytoplasmic sequestration. Copyright © 2017 Elsevier Ltd. All rights reserved.

Amyotrophic lateral sclerosis mutant vesicle-associated membrane protein-associated protein-B transgenic mice develop TAR-DNA-binding protein-43 pathology.

LENUS (Irish Health Repository)

Tudor, E L

2010-05-19

Cytoplasmic ubiquitin-positive inclusions containing TAR-DNA-binding protein-43 (TDP-43) within motor neurons are the hallmark pathology of sporadic amyotrophic lateral sclerosis (ALS). TDP-43 is a nuclear protein and the mechanisms by which it becomes mislocalized and aggregated in ALS are not properly understood. A mutation in the vesicle-associated membrane protein-associated protein-B (VAPB) involving a proline to serine substitution at position 56 (VAPBP56S) is the cause of familial ALS type-8. To gain insight into the molecular mechanisms by which VAPBP56S induces disease, we created transgenic mice that express either wild-type VAPB (VAPBwt) or VAPBP56S in the nervous system. Analyses of both sets of mice revealed no overt motor phenotype nor alterations in survival. However, VAPBP56S but not VAPBwt transgenic mice develop cytoplasmic TDP-43 accumulations within spinal cord motor neurons that were first detected at 18 months of age. Our results suggest a link between abnormal VAPBP56S function and TDP-43 mislocalization.

A Mechanism for Cytoplasmic Streaming: Kinesin-Driven Alignment of Microtubules and Fast Fluid Flows.

Science.gov (United States)

Monteith, Corey E; Brunner, Matthew E; Djagaeva, Inna; Bielecki, Anthony M; Deutsch, Joshua M; Saxton, William M

2016-05-10

The transport of cytoplasmic components can be profoundly affected by hydrodynamics. Cytoplasmic streaming in Drosophila oocytes offers a striking example. Forces on fluid from kinesin-1 are initially directed by a disordered meshwork of microtubules, generating minor slow cytoplasmic flows. Subsequently, to mix incoming nurse cell cytoplasm with ooplasm, a subcortical layer of microtubules forms parallel arrays that support long-range, fast flows. To analyze the streaming mechanism, we combined observations of microtubule and organelle motions with detailed mathematical modeling. In the fast state, microtubules tethered to the cortex form a thin subcortical layer and undergo correlated sinusoidal bending. Organelles moving in flows along the arrays show velocities that are slow near the cortex and fast on the inward side of the subcortical microtubule layer. Starting with fundamental physical principles suggested by qualitative hypotheses, and with published values for microtubule stiffness, kinesin velocity, and cytoplasmic viscosity, we developed a quantitative coupled hydrodynamic model for streaming. The fully detailed mathematical model and its simulations identify key variables that can shift the system between disordered (slow) and ordered (fast) states. Measurements of array curvature, wave period, and the effects of diminished kinesin velocity on flow rates, as well as prior observations on f-actin perturbation, support the model. This establishes a concrete mechanistic framework for the ooplasmic streaming process. The self-organizing fast phase is a result of viscous drag on kinesin-driven cargoes that mediates equal and opposite forces on cytoplasmic fluid and on microtubules whose minus ends are tethered to the cortex. Fluid moves toward plus ends and microtubules are forced backward toward their minus ends, resulting in buckling. Under certain conditions, the buckling microtubules self-organize into parallel bending arrays, guiding varying directions

Modulation of integrin-linked kinase nucleo-cytoplasmic shuttling by ILKAP and CRM1.

Science.gov (United States)

Nakrieko, Kerry-Ann; Vespa, Alisa; Mason, David; Irvine, Timothy S; D'Souza, Sudhir J A; Dagnino, Lina

2008-07-15

Integrin-linked kinase (ILK) plays key roles in a variety of cell functions, including cell proliferation, adhesion and migration. Within the cell, ILK localizes to multiple sites, including the cytoplasm, focal adhesion complexes that mediate cell adhesion to extracellular substrates, as well as cell-cell junctions in epidermal keratinocytes. Central to understanding ILK function is the elucidation of the mechanisms that regulate its subcellular localization. We now demonstrate that ILK is imported into the nucleus through sequences in its N-terminus, via active transport mechanisms that involve nuclear pore complexes. In addition, nuclear ILK can be rapidly exported into the cytoplasm through a CRM1-dependent pathway, and its export is enhanced by the type 2C protein phosphatase ILKAP. Nuclear localization of ILK in epidermal keratinocytes is associated with increased DNA synthesis, which is sensitive to inhibition by ILKAP. Our studies demonstrate the importance for keratinocyte proliferation of ILK regulation through changes in its subcellular localization, and establish ILKAP and CRM1 as pivotal modulators of ILK subcellular distribution and activity in these cells.

The autoantigen Ro52 is an E3 ligase resident in the cytoplasm but enters the nucleus upon cellular exposure to nitric oxide

International Nuclear Information System (INIS)

Espinosa, Alexander; Oke, Vilija; Elfving, Ase; Nyberg, Filippa; Covacu, Ruxandra; Wahren-Herlenius, Marie

2008-01-01

Patients with the systemic autoimmune diseases Sjoegrens's syndrome and systemic lupus erythematosus often have autoantibodies against the intracellular protein Ro52. Ro52 is an E3 ligase dependent on the ubiquitin conjugation enzymes UBE2D1 and UBE2E1. While Ro52 and UBE2D1 are cytoplasmic proteins, UBE2E1 is localized to the nucleus. Here, we investigate how domains of human Ro52 regulate its intracellular localization. By expressing fluorescently labeled Ro52 and Ro52 mutants in HeLa cells, an intact coiled-coil domain was found to be necessary for the cytoplasmic localization of Ro52. The amino acids 381-470 of the B30.2 region were essential for translocation into the nucleus. Furthermore, after exposure of HeLa cells to the inflammatory mediator nitric oxide (NO), Ro52 translocated to the nucleus. A nuclear localization of Ro52 in inflamed tissue expressing inducible NO synthetase (iNOS) from cutaneous lupus patients was observed by immunohistochemistry and verified in NO-treated cultures of patient-derived primary keratinocytes. Our results show that the localization of Ro52 is regulated by endogenous sequences, and that nuclear translocation is induced by an inflammatory mediator. This suggests that Ro52 has both cytoplasmic and nuclear substrates, and that Ro52 mediates ubiquitination through UBE2D1 in the cytoplasm and through UBE2E1 in the nucleus

Induction of cytoplasmic male sterility by gamma-ray and chemical mutagens in sugar beets

Energy Technology Data Exchange (ETDEWEB)

Kinoshita, Toshiro [Hokkaido Univ., Sapporo (Japan). Faculty of Agriculture

1982-03-01

Male sterile plants appeared in the population of N cytoplasm sugar beet strains, H-19 and H-2002, when their dry seeds were exposed to 50 kR gamma-ray, and the male sterility was maintained up to the M/sub 4/ generation through the mother plants. Cytoplasmic inheritance was confirmed by the reciprocal crossings between plants with normal phenotype from gamma-strains (progeneis of the male mutants which transmitted male sterility through the mother plants) and H-19 or H-1001. The crossing experiments suggested that various kinds of cytoplasm were induced by gamma-ray irradiation, and that different nuclear genes were responsible for the respective cytoplasms. A specific relationship between the pollen restoring genes and the sterile cytoplasms was established, and was named ''one set of pollen restoring genes for one cytoplasm''. It is probable that the cytoplasmic mutation occurred in normal cytoplasm strains and the specific combination between the altered cytoplasm and the recessive nuclear gene produced male sterility. Ethyl methane sulphonate, ethidium bromide, acriflavine and streptomycin were also effective in inducing cytoplasmic mutation in sugar beets.

Induction of cytoplasmic male sterility by gamma-ray and chemical mutagens in sugar beets

International Nuclear Information System (INIS)

Kinoshita, Toshiro

1982-01-01

Male sterile plants appeared in the population of N cytoplasm sugar beet strains, H-19 and H-2002, when their dry seeds were exposed to 50 kR gamma-ray, and the male sterility was maintained up to the M 4 generation through the mother plants. Cytoplasmic inheritance was confirmed by the reciprocal crossings between plants with normal phenotype from gamma-strains (progeneis of the male mutants which transmitted male sterility through the mother plants) and H-19 or H-1001. The crossing experiments suggested that various kinds of cytoplasm were induced by gamma-ray irradiation, and that different nuclear genes were responsible for the respective cytoplasms. A specific relationship between the pollen restoring genes and the sterile cytoplasms was established, and was named ''one set of pollen restoring genes for one cytoplasm''. It is probable that the cytoplasmic mutation occurred in normal cytoplasm strains and the specific combination between the altered cytoplasm and the recessive nuclear gene produced male sterility. Ethyl methane sulphonate, ethidium bromide, acriflavine and streptomycin were also effective in inducing cytoplasmic mutation in sugar beets. (Kaihara, S.)

Cytoplasmic Flow Enhances Organelle Dispersion in Eukaryotic Cells

Science.gov (United States)

Koslover, Elena; Mogre, Saurabh; Chan, Caleb; Theriot, Julie

The cytoplasm of a living cell is an active environment through which intracellular components move and mix. We explore, using theoretical modeling coupled with microrheological measurements, the efficiency of particle dispersion via different modes of transport within this active environment. In particular, we focus on the role of cytoplasmic flow over different scales in contributing to organelle transport within two different cell types. In motile neutrophil cells, we show that bulk fluid flow associated with rapid cell deformation enhances particle transport to and from the cell periphery. In narrow fungal hyphae, localized flows due to hydrodynamic entrainment are shown to contribute to optimally efficient organelle dispersion. Our results highlight the importance of non-traditional modes of transport associated with flow of the cytoplasmic fluid in the distribution of organelles throughout eukaryotic cells.

Identify High-Quality Protein Structural Models by Enhanced K-Means.

Science.gov (United States)

Wu, Hongjie; Li, Haiou; Jiang, Min; Chen, Cheng; Lv, Qiang; Wu, Chuang

2017-01-01

Background. One critical issue in protein three-dimensional structure prediction using either ab initio or comparative modeling involves identification of high-quality protein structural models from generated decoys. Currently, clustering algorithms are widely used to identify near-native models; however, their performance is dependent upon different conformational decoys, and, for some algorithms, the accuracy declines when the decoy population increases. Results. Here, we proposed two enhanced K -means clustering algorithms capable of robustly identifying high-quality protein structural models. The first one employs the clustering algorithm SPICKER to determine the initial centroids for basic K -means clustering ( SK -means), whereas the other employs squared distance to optimize the initial centroids ( K -means++). Our results showed that SK -means and K -means++ were more robust as compared with SPICKER alone, detecting 33 (59%) and 42 (75%) of 56 targets, respectively, with template modeling scores better than or equal to those of SPICKER. Conclusions. We observed that the classic K -means algorithm showed a similar performance to that of SPICKER, which is a widely used algorithm for protein-structure identification. Both SK -means and K -means++ demonstrated substantial improvements relative to results from SPICKER and classical K -means.

A novel whole-cell lysate kinase assay identifies substrates of the p38 MAPK in differentiating myoblasts

Directory of Open Access Journals (Sweden)

Knight James DR

2012-03-01

Full Text Available Abstract Background The p38α mitogen-activated protein kinase (MAPK is a critical mediator of myoblast differentiation, and does so in part through the phosphorylation and regulation of several transcription factors and chromatin remodelling proteins. However, whether p38α is involved in processes other than gene regulation during myogenesis is currently unknown, and why other p38 isoforms cannot compensate for its loss is unclear. Methods To further characterise the involvement of p38α during myoblast differentiation, we developed and applied a simple technique for identifying relevant in vivo kinase substrates and their phosphorylation sites. In addition to identifying substrates for one kinase, the technique can be used in vitro to compare multiple kinases in the same experiment, and we made use of this to study the substrate specificities of the p38α and β isoforms. Results Applying the technique to p38α resulted in the identification of seven in vivo phosphorylation sites on six proteins, four of which are cytoplasmic, in lysate derived from differentiating myoblasts. An in vitro comparison with p38β revealed that substrate specificity does not discriminate these two isoforms, but rather that their distinguishing characteristic appears to be cellular localisation. Conclusion Our results suggest p38α has a novel cytoplasmic role during myogenesis and that its unique cellular localisation may be why p38β and other isoforms cannot compensate for its absence. The substrate-finding approach presented here also provides a necessary tool for studying the hundreds of protein kinases that exist and for uncovering the deeper mechanisms of phosphorylation-dependent cell signalling.

The BRO proteins of Bombyx mori nucleopolyhedrovirus are nucleocytoplasmic shuttling proteins that utilize the CRM1-mediated nuclear export pathway

International Nuclear Information System (INIS)

Kang, Won Kyung; Kurihara, Masaaki; Matsumoto, Shogo

2006-01-01

The BRO proteins of Bombyx mori nucleopolyhedrovirus (BmNPV) display a biphasic pattern of intracellular localization during infection. At early times, they reside in the nucleus but then show both cytoplasmic and nuclear localization as the infection proceeds. Therefore, we examined the possibility of nuclear export. Using inhibitors, we reveal that BmNPV BRO proteins shuttle between the nucleus and cytoplasm. Mutations on the leucine-rich region of BRO proteins resulted in nuclear accumulation of transiently expressed proteins, suggesting that this region functions as a CRM1-dependent nuclear export signal (NES). On the contrary, mutant BRO-D with an altered NES did not show nuclear accumulation in infected cells, although protein production seemed to be reduced. RT-PCR analysis showed that the lower level of protein production was due to a reduction in RNA synthesis. Taken together, our results suggest that BRO proteins are nucleocytoplasmic shuttling proteins that utilize the CRM1-mediated nuclear export pathway

The 'tubulin-like' S1 protein of Spirochaeta is a member of the hsp65 stress protein family

Science.gov (United States)

Munson, D.; Obar, R.; Tzertzinis, G.; Margulis, L.

1993-01-01

A 65-kDa protein (called S1) from Spirochaeta bajacaliforniensis was identified as 'tubulin-like' because it cross-reacted with at least four different antisera raised against tubulin and was isolated, with a co-polymerizing 45-kDa protein, by warm-cold cycling procedures used to purify tubulin from mammalian brain. Furthermore, at least three genera of non-cultivable symbiotic spirochetes (Pillotina, Diplocalyx, and Hollandina) that contain conspicuous 24-nm cytoplasmic tubules displayed a strong fluorescence in situ when treated with polyclonal antisera raised against tubulin. Here we summarize results that lead to the conclusion that this 65-kDa protein has no homology to tubulin. S1 is an hsp65 stress protein homologue. Hsp65 is a highly immunogenic family of hsp60 proteins which includes the 65-kDa antigens of Mycobacterium tuberculosis (an active component of Freund's complete adjuvant), Borrelia, Treponema, Chlamydia, Legionella, and Salmonella. The hsp60s, also known as chaperonins, include E. coli GroEL, mitochondrial and chloroplast chaperonins, the pea aphid 'symbionin' and many other proteins involved in protein folding and the stress response.

Consequences of cytoplasmic irradiation. Studies from microbeam

International Nuclear Information System (INIS)

Zhou, Hongning; Hong, Mei; Chai, Yunfei; Hei, Tom K.

2009-01-01

The prevailing dogma for radiation biology is that genotoxic effects of ionizing radiation such as mutations and carcinogenesis are attributed mainly to direct damage to the nucleus. However, with the development of microbeam that can target precise positions inside the cells, accumulating evidences have shown that energy deposit by radiation in nuclear DNA is not required to trigger the damage, extra-nuclear or extra-cellular radiation could induce the similar biological effects as well. This review will summarize the biological responses after cytoplasm irradiated by microbeam, and the possible mechanisms involved in cytoplasmic irradiation. (author)

Adding biological meaning to human protein-protein interactions identified by yeast two-hybrid screenings: A guide through bioinformatics tools.

Science.gov (United States)

Felgueiras, Juliana; Silva, Joana Vieira; Fardilha, Margarida

2018-01-16

"A man is known by the company he keeps" is a popular expression that perfectly fits proteins. A common approach to characterize the function of a target protein is to identify its interacting partners and thus infer its roles based on the known functions of the interactors. Protein-protein interaction networks (PPINs) have been created for several organisms, including humans, primarily as results of high-throughput screenings, such as yeast two-hybrid (Y2H). Their unequivocal use to understand events underlying human pathophysiology is promising in identifying genes and proteins associated with diseases. Therefore, numerous opportunities have emerged for PPINs as tools for clinical management of diseases: network-based disease classification systems, discovery of biomarkers and identification of therapeutic targets. Despite the great advantages of PPINs, their use is still unrecognised by several researchers who generate high-throughput data to generally characterize interactions in a certain model or to select an interaction to study in detail. We strongly believe that both approaches are not exclusive and that we can use PPINs as a complementary methodology and rich-source of information to the initial study proposal. Here, we suggest a pipeline to deal with Y2H results using bioinformatics tools freely available for academics. Yeast two-hybrid is widely-used to identify protein-protein interactions. Conventionally, the positive clones that result from a yeast two-hybrid screening are sequenced to identify the interactors of the protein of interest (also known as bait protein), and few interactions, thought as potentially relevant for the model in study, are selected for further validation using biochemical methods (e.g. co-immunoprecipitation and co-localization). The huge amount of data that is potentially lost during this conservative approach motivated us to write this tutorial-like review, so that researchers feel encouraged to take advantage of

Affinity resins as new tools for identifying target proteins of ascorbic acid.

Science.gov (United States)

Iwaoka, Yuji; Nishino, Kohei; Ishikawa, Takahiro; Ito, Hideyuki; Sawa, Yoshihiro; Tai, Akihiro

2018-02-12

l-Ascorbic acid (AA) has diverse physiological functions, but little is known about the functional mechanisms of AA. In this study, we synthesized two types of affinity resin on which AA is immobilized in a stable form to identify new AA-targeted proteins, which can provide important clues for elucidating unknown functional mechanisms of AA. To our knowledge, an affinity resin on which AA as a ligand is immobilized has not been prepared, because AA is very unstable and rapidly degraded in an aqueous solution. By using the affinity resins, cytochrome c (cyt c) was identified as an AA-targeted protein, and we showed that oxidized cyt c exhibits specific affinity for AA. These results suggest that two kinds of AA-affinity resin can be powerful tools to identify new target proteins of AA.

Spike protein assembly into the coronavirion: exploring the limits of its sequence requirements

International Nuclear Information System (INIS)

Bosch, Berend Jan; Haan, Cornelis A.M. de; Smits, Saskia L.; Rottier, Peter J.M.

2005-01-01

The coronavirus spike (S) protein, required for receptor binding and membrane fusion, is incorporated into the assembling virion by interactions with the viral membrane (M) protein. Earlier we showed that the ectodomain of the S protein is not involved in this process. Here we further defined the requirements of the S protein for virion incorporation. We show that the cytoplasmic domain, not the transmembrane domain, determines the association with the M protein and suffices to effect the incorporation into viral particles of chimeric spikes as well as of foreign viral glycoproteins. The essential sequence was mapped to the membrane-proximal region of the cytoplasmic domain, which is also known to be of critical importance for the fusion function of the S protein. Consistently, only short C-terminal truncations of the S protein were tolerated when introduced into the virus by targeted recombination. The important role of the about 38-residues cytoplasmic domain in the assembly of and membrane fusion by this approximately 1300 amino acids long protein is discussed

Cytoplasmic BRMS1 expression in malignant melanoma is associated with increased disease-free survival

Directory of Open Access Journals (Sweden)

Slipicevic Ana

2012-02-01

Full Text Available Abstract Background/aims Breast cancer metastasis suppressor 1 (BRMS1 blocks metastasis in melanoma xenografts; however, its usefulness as a biomarker in human melanomas has not been widely studied. The goal was to measure BRMS1 expression in benign nevi, primary and metastatic melanomas and evaluate its impact on disease progression and prognosis. Methods Paraffin-embedded tissue from 155 primary melanomas, 69 metastases and 15 nevi was examined for BRMS1 expression using immunohistochemistry. siRNA mediated BRMS1 down-regulation was used to study impact on invasion and migration in melanoma cell lines. Results A significantly higher percentage of nevi (87%, compared to primary melanomas (20% and metastases (48%, expressed BRMS1 in the nucelus (p Waf1/Cip1 (p = 0.009. Cytoplasmic score index was inversely associated with nuclear p-Akt (p = 0.013 and positively associated with cytoplasmic p-ERK1/2 expression (p = 0.033. Nuclear BRMS1 expression in ≥ 10% of primary melanoma cells was associated with thicker tumors (p = 0.016 and decreased relapse-free period (p = 0.043. Nuclear BRMS1 was associated with expression of fatty acid binding protein 7 (FABP7; p = 0.011, a marker of invasion in melanomas. In line with this, repression of BRMS1 expression reduced the ability of melanoma cells to migrate and invade in vitro. Conclusion Our data suggest that BRMS1 is localized in cytoplasm and nucleus of melanocytic cells and that cellular localization determines its in vivo effect. We hypothesize that cytoplasmic BRMS1 restricts melanoma progression while nuclear BRMS1 possibly promotes melanoma cell invasion. Please see related article: http://www.biomedcentral.com/1741-7015/10/19

Method for early detection of infectious mononucleosis by identifying Inmono proteins

Science.gov (United States)

Willard, Karen E.

1984-01-01

Early detection of infectious mononucleosis is carried out using a sample of human blood by isolating and identifying the presence of Inmono proteins in the sample from a two-dimensional protein map with the proteins being characterized by having isoelectric banding as measured in urea of about -16 to -17 with respect to certain isoelectric point standards and molecular mass of about 70 to 75 K daltons as measured in the presence of sodium dodecylsulfate containing polyacrylamide gels, the presence of the Inmono proteins being correlated with the existence of infectious mononucleosis.

A cataract-causing connexin 50 mutant is mislocalized to the ER due to loss of the fourth transmembrane domain and cytoplasmic domain.

Science.gov (United States)

Somaraju Chalasani, Madhavi Latha; Muppirala, Madhavi; G Ponnam, Surya Prakash; Kannabiran, Chitra; Swarup, Ghanshyam

2013-01-01

Mutations in the eye lens gap junction protein connexin 50 cause cataract. Earlier we identified a frameshift mutant of connexin 50 (c.670insA; p.Thr203AsnfsX47) in a family with autosomal recessive cataract. The mutant protein is smaller and contains 46 aberrant amino acids at the C-terminus after amino acid 202. Here, we have analysed this frameshift mutant and observed that it localized to the endoplasmic reticulum (ER) but not in the plasma membrane. Moreover, overexpression of the mutant resulted in disintegration of the ER-Golgi intermediate compartment (ERGIC), reduction in the level of ERGIC-53 protein and breakdown of the Golgi in many cells. Overexpression of the frameshift mutant partially inhibited the transport of wild type connexin 50 to the plasma membrane. A deletion mutant lacking the aberrant sequence showed predominant localization in the ER and inhibited anterograde protein transport suggesting, therefore, that the aberrant sequence is not responsible for improper localization of the frameshift mutant. Further deletion analysis showed that the fourth transmembrane domain and a membrane proximal region (231-294 amino acids) of the cytoplasmic domain are needed for transport from the ER and localization to the plasma membrane. Our results show that a frameshift mutant of connexin 50 mislocalizes to the ER and causes disintegration of the ERGIC and Golgi. We have also identified a sequence of connexin 50 crucial for transport from the ER and localization to the plasma membrane.