ubiquitin c-terminal hydrolase-l1: Topics by WorldWideScience.org

Ubiquitin over-expression phenotypes and ubiquitin gene molecular misreading during aging in Drosophila melanogaster

Arf GTPases control vesicle formation from different intracellular membranes and are regulated by Arf guanine nucleotide exchange factors (GEFs). Outside of their conserved catalytic domains, known...Full Text Available

2008-12-05

Ubiquitin-Dependent Degradation of p53 Protein despite Phosphorylation at its N-Terminus by Acetaminophen

Molecular Misreading (MM) is the inaccurate conversion of genomic information into aberrant proteins. For example, when RNA polymerase II transcribes a GAGAG motif it synthesizes at low frequency RNA...Full Text Available

Proteasomal chymotrypsin-like peptidase activity is required for essential functions of human monocyte-derived dendritic cells

We previously reported that acetaminophen (APAP) caused apoptosis of C6 glioma cells. Therefore, we hypothesized that the level of p53, which usually stimulates apoptosis, might be increased...Full Text Available

2006-04-01

ATG12 Conjugation to ATG3 Regulates Mitochondrial Homeostasis and Cell Death

The ubiquitin–proteasome pathway is the principal system for extralysosomal protein degradation in eukaryotic cells, and is essential for the regulation and maintenance of basic cellular processes,...Full Text Available

2007-01-01

PAS Domain of the Aer Redox Sensor Requires C-Terminal Residues for Native-Fold Formation and Flavin Adenine Dinucleotide Binding

SUMMARYATG12, an ubiquitin-like modifier required for macroautophagy, has a single known conjugation target, another autophagy regulator called ATG5. Here, we identify ATG3 as...Full Text Available

2010-08-20

Competition by inhibitory oligonucleotides prevents binding of CpG to C-terminal TLR9

The Aer protein in Escherichia coli is a membrane-bound, FAD-containing aerotaxis and energy sensor that putatively monitors the redox state of the electron transport system. Binding...Full Text Available

2004-10-01

British Library Electronic Table of Contents (United Kingdom)

Abstract TLR9 recognizes unmethylated CpG-containing DNA commonly found in bacteria. Synthetic oligonucleotides containing CpG-motifs (CpG ODNs) recapitulate the activation of TLR9 by microbial DNA, whereas inversion of the CG dinucleotide within the CpG motif to GC (GpC ODNs) renders such ODNs inactive. This difference cannot be attributed to binding of ODNs to the full-length TLR9 ectodomain, as both CpG and GpC ODNs bind comparably. Activation of murine TLR9 requires cleavage into an active C-terminal fragment, which binds CpG robustly. We therefore compared the ability of CpG and GpC ODNs to bind to full-length and C-terminal TLR9, and their impact on the cleavage of TLR9. We found that CpG binds better to C-terminal TLR9 when compared with GpC, despite comparably low binding of both O...

2011-01-01

Amino acid analysis at the picomole level. Application to the C-terminal sequence analysis of polypeptides.

NMR analysis of the structure of synaptobrevin and of its interaction with syntaxin

Amino acids labelled with dimethylaminoazobenzenesulphonyl chloride can be separated by reversed-phase high-pressure liquid chromatography and detected in the visible region (436 nm). All 19 naturally...Full Text Available

1981-12-01

A novel glioblastoma cancer gene therapy using AAV-mediated long-term expression of human TERT C-terminal polypeptide

Synaptobrevin is a synaptic vesicle protein that has an essential role in exocytosis and forms the SNARE complex with syntaxin and SNAP-25. We have analyzed the structure of isolated synaptobrevin and its binary interaction with syntaxin using NMR spectroscopy. Our results demonstrate that isolated synaptobrevin is largely unfolded in solution. The entire SNARE motif of synaptobrevin is capable of interacting with the isolated C-terminal SNARE motif of syntaxin but only a few residues bind to the full-length cytoplasmic region of syntaxin. This result suggests an interaction between the N- and C-terminal regions of syntaxin that competes with core complex assembly.

1999-07-15

British Library Electronic Table of Contents (United Kingdom)

Glioblastoma multiforme is the most aggressive form of human brain tumor, which has no effective cure. Previously, we have demonstrated that overexpression of the C-terminal fragment of the human telomerase reverse transcriptase (hTERTC27) inhibits the growth and tumorigenicity of human cervical cancer HeLa cells. In this study, the therapeutic effect and molecular mechanisms of hTERTC27-mediated cancer gene therapy were further explored in vivo in established human glioblastoma xenografts in nude mice. We showed that intratumoral injection of adeno-associated virus carrying hTERTC27 (rAAV-hTERTC27) is highly effective in reducing the growth of the subcutaneously transplanted glioblastoma tumors. Histological analyses showed that rAAV-hTERTC27 treatment leads to profound necrosis, apoptosi...

2007-01-01

A System-Based Comparison of Gene Expression Reveals Alterations in Oxidative Stress, Disruption of Ubiquitin-Proteasome System and Altered Cell Cycle Regulation after Exposure to Cadmium and Methylmercury in Mouse Embryonic Fibroblast

Rin-like, a novel regulator of endocytosis, acts as guanine nucleotide exchange factor for Rab5a and Rab22

Environmental and occupational exposures to heavy metals such as methylmercury (MeHg) and cadmium (Cd) pose significant health risks to humans, including neurotoxicity. The underlying mechanisms of...Full Text Available

2010-04-01

Inhibition of Melanoma Growth by Subcutaneous Administration of hTERTC27 Viral Cocktail in C57BL/6 Mice

RIN proteins serve as guanine nucleotide exchange factors for Rab5a. They are characterized by the presence of a RIN homology domain and a C-terminal Vps9 domain. Currently three family members have...Full Text Available

2011-06-01

Hepatitis C Virus RNA Replication Requires a Conserved Structural Motif within the Transmembrane Domain of the NS5B RNA-Dependent RNA Polymerase ?

BackgroundhTERTC27 is a 27 kDa C-terminal polypeptide of human telomerase reverse transcriptase that has previously been shown to reduce tumorigenicity of HeLa cells and suppress...Full Text Available

Recognition and Detoxification of the Insecticide DDT by Drosophila melanogaster Glutathione S-Transferase D1

Hepatitis C virus (HCV) nonstructural protein 5B (NS5B), the viral RNA-dependent RNA polymerase (RdRp), is a tail-anchored protein with a highly conserved C-terminal transmembrane domain (TMD) that...Full Text Available

2010-11-01

A human TERT C-terminal polypeptide sensitizes HeLa cells to H2O2-induced senescence without affecting telomerase enzymatic activity.

GSTD1 is one of several insect glutathione S-transferases capable of metabolizing the insecticide DDT. Here we use crystallography and NMR to elucidate the binding of DDT and glutathione to GSTD1. The crystal structure of Drosophila melanogaster GSTD1 has been determined to 1.1 {angstrom} resolution, which reveals that the enzyme adopts the canonical GST fold but with a partially occluded active site caused by the packing of a C-terminal helix against one wall of the binding site for substrates. This helix would need to unwind or be displaced to enable catalysis. When the C-terminal helix is removed from the model of the crystal structure, DDT can be computationally docked into the active site in an orientation favoring catalysis. Two-dimensional {sup 1}H,{sup 15}N heteronuclear single-quantum coherence NMR experiments of GSTD1 indicate that conformational changes occur upon glutathione and DDT binding and the residues that broaden upon DDT ...

2010-06-14

Alterations in heart looping induced by overexpression of the tight junction protein Claudin-1 are dependent on its C-terminal cytoplasmic tail.

We have constructed a 27-kDa hTERT C-terminal polypeptide (hTERTC27) devoid of domains required for telomerase activity and demonstrated that it is capable of nuclear translocation/telomere-end targeting. Here we showed that expression of a low level of hTERTC27 renders hTERT positive HeLa cells sensitive to H(2)O(2)-induced oxidative stress and subsequent cell senescence. The senescence-associated gene, the cyclin/cdk inhibitor p21(Waf1), was up-regulated. This occurs without changing the expression of endogenous hTERT, causing significant telomere shortening or inhibiting telomerase activity. Results from this study suggest for the first time that in addition to telomerase activity, the C-terminus of hTERT also plays a role in hTERT-mediated cellular resistance to oxidative stress. PMID:12565825

2003-02-14

Inhibition of the heterotetrameric K++ channel KCNQ1/KCNE1 by the AMP-activated protein kinase

In vertebrates, the positioning of the internal organs relative to the midline is asymmetric and evolutionarily conserved. A number of molecules have been shown to play critical roles in left-right patterning. Using representational difference analysis to identify genes that are differentially expressed on the left and right sides of the chick embryo, we cloned chick Claudin-1, an integral component of epithelial tight junctions. Here, we demonstrate that retroviral overexpression of Claudin-1, but not Claudin-3, on the right side of the chick embryo between HH stages 4 and 7 randomizes the direction of heart looping. This effect was not observed when Claudin-1 was overexpressed on the left side of the embryo. A small, but reproducible, induction of Nodal expression in the perinodal region on the right side of the embryo was noted in embryos that were injected with Claudin-1 retroviral particles on their right sides. However, no changes in Lefty,Pitx2 or cSnR expression were observed. ...

2006-02-24

British Library Electronic Table of Contents (United Kingdom)

Abstract The heterotetrameric K++-channel KCNQ1/KCNE1 is expressed in heart, skeletal muscle, liver and several epithelia including the renal proximal tubule. In the heart, it contributes to the repolarization of cardiomyocytes. The repolarization is impaired in ischemia. Ischemia stimulates the AMP-activated protein kinase (AMPK), a serine/threonine kinase, sensing energy depletion and stimulating several cellular mechanisms to enhance energy production and to limit energy utilization. AMPK has previously been shown to downregulate the epithelial Na++ channel ENaC, an effect mediated by the ubiquitin ligase Nedd4-2. The present study explored whether AMPK regulates KCNQ1/KCNE1. To this end, cRNA encoding KCNQ1/KCNE1 was injected into Xenopus oocytes with and without additional injection o...

2011-01-01

Assessment of GABARAP self-association by its diffusion properties

Mechanism of Dephosphorylation of the SR Protein ASF/SF2 by Protein Phosphatase 1

Gamma-aminobutyric acid type A receptor-associated protein (GABARAP) belongs to a family of small ubiquitin-like adaptor proteins implicated in intracellular vesicle trafficking and autophagy. We have used diffusion-ordered nuclear magnetic resonance spectroscopy to study the temperature and concentration dependence of the diffusion properties of GABARAP. Our data suggest the presence of distinct conformational states and provide support for self-association of GABARAP molecules. Assuming a monomer-dimer equilibrium, a temperature-dependent dissociation constant could be derived. Based on a temperature series of {sup 1}H{sup 15}N heteronuclear single quantum coherence nuclear magnetic resonance spectra, we propose residues potentially involved in GABARAP self-interaction. The possible biological significance of these observations is discussed with respect to alternative scenarios of oligomerization.

2010-09-15

British Library Electronic Table of Contents (United Kingdom)

SR proteins are essential splicing factors whose function is controlled by multi-site phosphorylation of a C-terminal domain rich in arginine-serine repeats (RS domain). The protein kinase SRPK1 has been shown to polyphosphorylate the N-terminal portion of the RS domain (RS1) of the SR protein ASF/SF2, a modification that promotes nuclear entry of this splicing factor and engagement in splicing function. Later, dephosphorylation is required for maturation of the spliceosome and other RNA processing steps. While phosphates are attached to RS1 in a sequential manner by SRPK1, little is known about how they are removed. To investigate factors that control dephosphorylation, we monitored region-specific mapping of phosphorylation sites in ASF/SF2 as a function of the protein phosphatase PP1. W...

2010-01-01

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Mapping a nucleolar targeting sequence of an RNA binding nucleolar protein, Nop25

A novel glioblastoma cancer gene therapy using AAV-mediated long-term expression of human TERT C-terminal polypeptide.

Nop25 is a putative RNA binding nucleolar protein associated with rRNA transcription. The present study was undertaken to determine the mechanism of Nop25 localization in the nucleolus. Deletion experiments of Nop25 amino acid sequence showed Nop25 to contain a nuclear targeting sequence in the N-terminal and a nucleolar targeting sequence in the C-terminal. By expressing derivative peptides from the C-terminal as GFP-fusion proteins in the cells, a lysine and arginine residue-enriched peptide (KRKHPRRAQDSTKKPPSATRTSKTQRRRR) allowed a GFP-fusion protein to be transported and fully retained in the nucleolus. When the peptide was fused with cMyc epitope and expressed in the cells, a cMyc epitope was then detected in the nucleolus. Nop25 did not localize in the nucleolus by deletion of the peptide from Nop25. Furthermore, deletion of a subdomain (KRKHPRRAQ) in the peptide or amino acid substitution of lysine and arginine residues in the subdomain ...

2006-06-10

UHRF1, a modular multi-domain protein, regulates replication-coupled crosstalk between DNA methylation and histone modifications

2007-03-23

Rice RING protein OsBBI1 with E3 ligase activity confers broad-spectrum resistance against Magnaporthe oryzae by modifying the cell wall defence

Cytosine methylation in DNA is a major epigenetic signal, and plays a central role in propagating chromatin status during cell division. However the mechanistic links between DNA methylation and histone methylation are poorly understood. A multi-domain protein UHRF1 (ubiquitin-like, containing PHD and RING finger domains 1) is required for DNA CpG maintenance methylation at replication forks, and mouse UHRF1-null cells show enhanced susceptibility to DNA replication arrest and DNA damaging agents. Recent data demonstrated that the SET and RING associated (SRA) domain of UHRF1 binds hemimethylated CpG and flips 5-methylcytosine out of the DNA helix, whereas its tandom tudor domain and PHD domain bind the tail of histone H3 in a highly methylation sensitive manner. We hypothesize that UHRF1 brings the two components (histones and DNA) carrying appropriate markers (on the tails of H3 and hemimethylated CpG sites) ready to be assembled into a nucleosome after ...

2009-01-01

British Library Electronic Table of Contents (United Kingdom)

Emerging evidence suggests that E3 ligases play critical roles in diverse biological processes, including innate immune responses in plants. However, the mechanism of the E3 ligase involvement in plant innate immunity is unclear. We report that a rice gene, OsBBI1, encoding a RING finger protein with E3 ligase activity, mediates broad-spectrum disease resistance. The expression of OsBBI1 was induced by rice blast fungus Magnaporthe oryzae, as well as chemical inducers, benzothiadiazole and salicylic acid. Biochemical analysis revealed that OsBBI1 protein possesses E3 ubiquitin ligase activity in vitro. Genetic analysis revealed that the loss of OsBBI1 function in a Tos17-insertion line increased susceptibility, while the overexpression of OsBBI1 in transgenic plants conferred enhanced resi...

2011-01-01

A novel small-molecule inhibitor of NF-#kappa#B signaling

NMR studies of Borrelia burgdorferi OspA, a 28 kDa protein containing a single-layer #beta#-sheet

The inducible transcription factor NF-#kappa#B regulates divergent signaling pathways including inflammatory response and cancer development. Selective inhibitors for NF-#kappa#B signaling are potentially useful for treatment of inflammation and cancer. NF-#kappa#B is canonically activated by preferential disposal of its inhibitory protein; I#kappa#B, which suppresses the nuclear translocation of NF-#kappa#B. I#kappa#B#alpha# (a major member of I#kappa#B family proteins) is phosphorylated with an I#kappa#B kinase (IKK) and subsequently polyubiquitylated by SCF"#beta#"T"r"C"P"1 ubiquitin-ligase in the presence of E1 and E2 prior to proteasomal degradation. Here, we describe a novel inhibitor termed GS143, which suppressed I#kappa#B#alpha# ubiquitylation, but not I#kappa#B#alpha# phosphorylation, MDM2-directed p53 ubiquitylation, and proteasome activity in vitro. GS143 markedly suppressed the destruction of I#kappa#B#alpha# stimulated by TNF#alpha# and a set of ...

2008-04-18

FMRFamide-like immunoreactivity in rat brain: development of a radioimmunoassay and its application in studies of distribution and chromatographic properties

The crystal structure of outer surface protein A (OspA) from Borrelia burgdorferi contains a single-layer #beta#-sheet connecting the N- and C-terminal globular domains. The central #beta#-sheet consists largely of polar amino acids and it is solvent-exposed on both faces, which so far appears to be unique among known protein structures. We have accomplished nearly complete backbone H, C and N and C";/H"#beta# assignments of OspA (28 kDa) using standard triple resonance techniques without perdeuteration. This was made possible by recording spectra at a high temperature (45 "oC ). The chemical shift index and "1"5N T_1/T_2 ratios show that both the secondary structure and the global conformation of OspA in solution are similar to the crystal structure, suggesting that the unique central #beta#-sheet is fairly rigid.

1998-05-01

Development of recombinant adeno-associated virus and adenovirus cocktail system for efficient hTERTC27 polypeptide-mediated cancer gene therapy

A radioimmunoassay is described for the molluscan neuropeptide, Phe-Met-Arg-Phe-NH"2(FMRFamide). The antibody used is C-terminal-specific and shows slight but significant (1-2%) cross-reactivity with chicken pancreatic polypeptide (APP). The assay has been used to identify in rat brain extracts a pair of molecules that may represent mammalian counterparts of FMRFamide. Their concentrations were highest in spinal cord and hypothalamus (>10 pmol.g"-_1) and lowest in cerebellum and striatum (<3.5 pmol.g"-_1). The two immunoreactive peptides were separated on CM ion-exchange chromatography where they appeared to be less basic than FMRFamide. On Sephadex G50 gel filtration one eluted in a similar position to FMRFamide and the other slightly earlier suggesting it may be of higher molecular weight. The rat immunoreactive components do not correspond to previously described neuropeptides or hormones, and may be members of a new group of mammalian neuropeptides with ...

British Library Electronic Table of Contents (United Kingdom)

The low in vivo transduction efficiency of recombinant adeno-associated virus (rAAV) and the undesirably strong immunogenicity of adenovirus (rAdv) have limited their clinical utilization in cancer gene therapy. We have previously demonstrated that intratumoral injection of rAAV expressing a C-terminal polypeptide of human telomerase reverse transcriptase (rAAV-hTERTC27) effectively inhibits the growth of glioblastoma xenografts in nude mice. To further improve its efficacy, we combined rAAV-hTERTC27 with rAdv and investigated the efficiency of the cocktail vectors in vivo. At a nontherapeutic dose (1 x 108 plaque-forming units (PFUs)), rAdv-null and rAdv-hTERTC27 were equipotent in enhancing the therapeutic efficacy of rAAV-hTERTC27 (1.5 x 1011?v.g.), and complete tumor regression w...

2008-01-01

Artificial receptor for peptide recognition in protic media: The role of metal ion coordination

High-mobility group box 1 release and redox regulation accompany regeneration and remodeling of skeletal muscle.

The production of molecularly imprinted polymers (MIPs) for the recognition of C-terminal cholecystokinin pentapeptide (CCK-5) in the presence of metal ion is reported. The MIPs were produced under the same molar ratio of template to monomers (acrylamide, N,N'-methylene bisacrylamide) in the presence or absence of nitrilotriacetic acid-nickel (Ni-NTA) complex. Scanning electron microscopy images of MIPs were obtained in an attempt to correlate the adsorption characteristics with polymer's morphology. Subsequently Ni2+ was removed and substituted by other divalent ions such as Mg2+, Fe2+, Zn2+, Co2+ and Cu2+. It was found that polymers containing the metal ion complex with the order Fe-NTA, Ni-NTA and Cu-NTA presented lower dissociation constant values than the rest thus exhibiting stronger guest binding activity. The percentage of theoretical maximum binding sites Bmax was almost the same for these ions, indicating that the ion-template coordination is responsible ...

2008-08-25

Glial inflammation and neurodegeneration induced by candoxin, a novel neurotoxin from Bungarus candidus venom: global gene expression analysis using microarray.

Abstract High-mobility group box 1 (HMGB1), a damage-associated molecular pattern (DAMP) molecules, favors tissue regeneration via recruitment and activation of leukocytes and stem cells. Here we demonstrate, in a model of acute sterile muscle injury, that regeneration is accompanied by active reactive oxygen species (ROS) production counterbalanced and overcome by the generation of antioxidant moieties. Mitochondria are initially responsible for ROS formation. However, they undergo rapid disruption with almost complete disappearance. Twenty-four hours after injury, we observed a strong induction of MURF1 and atrogin-1 ubiquitin ligases, key signals in activation of the proteasome system and induction of muscle atrophy. At later time points, ROS generation is maintained by nonmitochondrial sources. The antioxidant response occurs in both regenerating fibers and leukocytes that express high levels of free thiols and antioxidant enzymes, such as superoxide dismutase ...

2011-05-09

A Wheat Homolog of MOTHER OF FT AND TFL1 Acts in the Regulation of Germination.

Candoxin (PDB #1JGK), a three-finger neurotoxin from Bungarus candidus venom, inhibits post-synaptic neuromuscular and neuronal alpha7nACh-receptors, and induces delayed cell-death throughout the glial population. When applied to cultured human glial cell lines, candoxin (CDX) induced cell death in a concentration (EC(50) approximately 1muM) and time dependent manner. Results of TUNEL-histochemistry further confirm CDX-induced brain (hippocampus, frontal cortex, and temporal regions) damage when administered intracerebroventricularly (i.c.v) in adult mice. In this study, we explored differential gene expression profiles following exposure of human glial (Hs 683) cell lines to CDX at various time intervals using Affymetrix-GeneChips. By means of MAS and GeneSpring analyses, 105 genes whose expression was significantly (P<0.01) altered by at least 3-fold were selected. Results of the genome analysis reveal that the potential role of CDX at molecular level involves the regulation of ...

2005-11-23

Yeast allosteric chorismate mutase is locked in the activated state by a single amino acid substitution

Seed dormancy is an adaptive mechanism and an important agronomic trait. Temperature during seed development strongly affects seed dormancy in wheat (Triticum aestivum) with lower temperatures producing higher levels of seed dormancy. To identify genes important for seed dormancy, we used a wheat microarray to analyze gene expression in embryos from mature seeds grown at lower and higher temperatures. We found that a wheat homolog of MOTHER OF FT AND TFL1 (MFT) was upregulated after physiological maturity in dormant seeds grown at the lower temperature. In situ hybridization analysis indicated that MFT was exclusively expressed in the scutellum and coleorhiza. Mapping analysis showed that MFT on chromosome 3A (MFT-3A) colocalized with the seed dormancy quantitative trait locus (QTL) QPhs.ocs-3A.1. MFT-3A expression levels in a dormant cultivar used for the detection of the QTL were higher after physiological maturity; this increased expression correlated with a single nucleotide ...

2011-09-01

Structures of trihydroxynaphthalene reductase-fungicide complexes: implications for structure-based design and catalysis

Chorismate mutase, a branch-point enzyme in the aromatic amino acid pathway of Saccharomyces cerevisiae, and also a mutant chorismate mutase with a single amino acid substitution in the C-terminal part of the protein have been purified approximately 20-fold and 64-fold from overproducing strains, respectively. The wild-type enzyme is activated by tryptophan and subject to feedback inhibition by tyrosine, whereas the mutant enzyme does not respond to activation by tryptophan nor inhibition by tyrosine. Both enzymes are dimers consisting of two identical subunits of M_r 30,000, each one capable of binding one substrate and one activator molecule. Each subunit of the wild-type enzyme also binds one inhibitor molecule, whereas the mutant enzyme lost this ability. The enzyme reaction was observed by "1H NMR and shows a direct and irreversible conversion of chorismate to prephenate without the accumulation of any enzyme-free intermediates. The kinetic data of the ...

Structure-function studies on inhibitory activity of Bungarus multicinctus protease inhibitor-like protein on matrix metalloprotease-2, and invasion and migration of human neuroblastoma SK-N-SH cells.

Trihydroxynaphthalene reductase catalyzes two intermediate steps in the fungal melanin biosynthetic pathway. The enzyme, a typical short-chain dehydrogenase, is the biochemical target of three commercial fungicides. The fungicides bind preferentially to the NADPH form of the enzyme. Three X-ray structures of the Magnaporthe grisea enzyme complexed with NADPH and two commercial and one experimental fungicide were determined at 1.7 {angstrom} (pyroquilon), 2.0 {angstrom} (2,3-dihydro-4-nitro-1H-inden-1-one, 1), and 2.1 {angstrom} (phthalide) resolutions. The chemically distinct inhibitors occupy similar space within the enzyme's active site. The three inhibitors share hydrogen bonds with the side chain hydroxyls of Ser-164 and Tyr-178 via a carbonyl oxygen (pyroquilon and 1) or via a carbonyl oxygen and a ring oxygen (phthalide). Active site residues occupy similar positions among the three structures. A buried water molecule that is hydrogen bonded to the NZ nitrogen of Lys-182 ...

2010-03-08

Positioning of the Alzheimer A{beta}(1-40) peptide in SDS micelles using NMR and paramagnetic probes

In view of the findings that several Kunitz-type protein inhibitors suppress tumor invasion and metastasis, the aim of the present study is to explore whether Bungarus multicinctus protease inhibitor-like protein-2 (PILP-2) and PILP-3 exhibit anti-tumor activity. Although approximately 28% of amino acid substitutions occurred between PILP-2 and PILP-3, molecular modeling suggested that PILP-2 and PILP-3 shared similar folded structures. Unlike PILP-2, PILP-3 showed a notable activity in abolishing migration and invasion of human neuroblastoma SK-N-SH cells. The ability of PILP-3 to inhibit matrix metalloprotease-2 (MMP-2) activity was higher than that of PILP-2. Pull-down assay revealed protein-protein interaction between PILP-3 and MMP-2. In contrast to mutation on N-terminal region, replacement of amino acids at C-terminus attenuated notably the ability of PILP-3 to inhibit cell invasion, cell migration and MMP-2 activity as well as the binding capability of PILP-3 with MMP-2. ...

2009-08-23

Novel bifunctional natriuretic peptides as potential therapeutics.

NMR spectroscopy combined with paramagnetic relaxation agents was used to study the positioning of the 40-residue Alzheimer Amyloid {beta}-peptide A{beta}(1-40) in SDS micelles. 5-Doxyl stearic acid incorporated into the micelle or Mn{sup 2+} ions in the aqueous solvent were used to determine the position of the peptide relative to the micelle geometry. In SDS solvent, the two {alpha}-helices induced in A{beta}(1-40), comprising residues 15-24, and 29-35, respectively, are surrounded by flexible unstructured regions. NMR signals from these unstructured regions are strongly attenuated in the presence of Mn{sup 2+} showing that these regions are positioned mostly outside the micelle. The central helix (residues 15-24) is significantly affected by 5-doxyl stearic acid however somewhat less for residues 16, 20, 22 and 23. This {alpha}-helix therefore resides in the SDS headgroup region with the face with residues 16, 20, 22 and 23 directed away from the hydrophobic interior of the micelle. ...

2007-09-15

Kinetic, spectroscopic and chemical modification study of iron release from transferrin; iron(III) complexation to adenosine triphosphate

Synthetic atrial natriuretic peptide (carperitide) and B-type natriuretic peptide (BNP; nesiritide) are used to treat congestive heart failure. However, despite beneficial cardiac unloading properties, reductions in renal perfusion pressures limit their clinical effectiveness. Recently, CD-NP, a chimeric peptide composed of C-type natriuretic peptide (CNP) fused to the C-terminal tail of Dendroaspis natriuretic peptide (DNP), was shown to be more glomerular filtration rate-enhancing than BNP in dogs. However, the molecular basis for the increased responsiveness was not determined. Here, we show that the DNP tail has a striking effect on CNP, converting it from a non-agonist to a partial agonist of natriuretic peptide receptor (NPR)-A while maintaining the ability to activate NPR-B. This effect is specific for human receptors because CD-NP was only a slightly better activator of rat NPR-A due to the promiscuous nature of CNP in this species. Interesting, the DNP ...

2008-10-21

In vitro and in vivo activities of T4 endonuclease V mutants altered in the C-terminal aromatic region

Amino acids other than those that serve as ligands have been found to influence the chemical properties of transferrin iron. The catalytic ability of pyrophosphate to mediate transferrin iron release to a terminal acceptor is largely quenched by modification non-liganded histine groups on the protein. The first order rate constants of iron release for several partially histidine modified protein samples were measured. A statistical method was employed to establish that one non-liganded histidine per metal binding domain was responsible for the reduction in rate constant. These results imply that the iron mediated chelator, pyrophosphate, binds directly to a histidine residue on the protein during the iron release process. EPR spectroscopic results are consistent with this interpretation. Kinetic and amino acid sequence studies of ovotransferrin and lactoferrin, in addition to human serum transferrin, have allowed the tentative assignment of His-207 in the N-terminal domain and His-535 ...

1985-01-01

Development of recombinant adeno-associated virus and adenovirus cocktail system for efficient hTERTC27 polypeptide-mediated cancer gene therapy.

Genes encoding mutants of the thymine photodimer repair enzyme from bacteriophage T4 (T4 endonuclease V) having an amino acid substitution (T127M, W128A, W128S, Y129A, K130L, Y131A, Y132A) were constructed by use of a previously obtained synthetic gene and expressed in Escherichia coli under the control of the E. coli tryptophan promoter. An in vitro assay of partially fractionated mutant proteins for glycosylase activity was performed with chemically synthesized substrates containing a thymine photodimer. T127M and K130L showed almost the same activity as the wild-type protein. Although W128S, Y131A, and Y132A were slightly active, W128A and Y129A lost activity. The results indicated that the aromatic amino acids around position 130 may be important for the glycosylase activity. Mutant T127M was purified, and the Km value was found to be of the same order as that of the wild type (10(-8) M). In vivo activities for all mutants were characterized with UV-sensitive E. coli. The results ...

ATP-dependent partitioning of the DNA template into supercoiled domains by Escherichia coli UvrAB

The low in vivo transduction efficiency of recombinant adeno-associated virus (rAAV) and the undesirably strong immunogenicity of adenovirus (rAdv) have limited their clinical utilization in cancer gene therapy. We have previously demonstrated that intratumoral injection of rAAV expressing a C-terminal polypeptide of human telomerase reverse transcriptase (rAAV-hTERTC27) effectively inhibits the growth of glioblastoma xenografts in nude mice. To further improve its efficacy, we combined rAAV-hTERTC27 with rAdv and investigated the efficiency of the cocktail vectors in vivo. At a nontherapeutic dose (1 x 10(8) plaque-forming units (PFUs)), rAdv-null and rAdv-hTERTC27 were equipotent in enhancing the therapeutic efficacy of rAAV-hTERTC27 (1.5 x 10(11) v.g.), and complete tumor regression was achieved in 25% of the treated animals. Importantly, the combination of rAAV-hTERTC27 and a therapeutic dose (2.5 x 10(9) PFU) of rAdv-hTERTC27 significantly augmented the ...

2008-06-06

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Modulation of neuronal differentiation by CD40 isoforms

The helicase action of the Escherichia coli UvrAB complex on a covalently closed circular DNA template was monitored using bacterial DNA topoisomerase I, which specifically removes negative supercoils. In the presence of E. coli DNA topoisomerase I and ATP, the UvrAB complex gradually introduced positive supercoils into the input relaxed plasmid DNA template. Positive supercoils were not produced when E. coli DNA topoisomerase I was replaced by eukaryotic DNA topoisomerase I or when both E. coli and eukaryotic DNA topoisomerases I were added simultaneously. These results suggest that like other DNA helix-tracking processes, the ATP-dependent action of the UvrAM complex on duplex DNA simultaneously generates both positive and negative supercoils, which are not constrained by protein binding but are torsionally strained. The supercoiling activity of UvrAB on UV-damaged DNA was also studied using UV-damaged plasmid DNA and a mutant UvrA protein that lacks the 40 ...

An ELISA-based high throughput protein truncation test for inherited breast cancer

Neuron differentiation is a complex process involving various cell-cell interactions, and multiple signaling pathways. We showed previously that CD40 is expressed and functional on mouse and human neurons. In neurons, ligation of CD40 protects against serum withdrawal-induced injury and plays a role in survival and differentiation. CD40 deficient mice display neuron dysfunction, aberrant neuron morphologic changes, and associated gross brain abnormalities. Previous studies by Tone and colleagues suggested that five isoforms of CD40 exist with two predominant isoforms expressed in humans: signal-transducible CD40 type I and a C-terminal truncated, non-signal-transducible CD40 type II. We hypothesized that differential expression of CD40 isoform type I and type II in neurons may modulate neuron differentiation. Results show that adult wild-type, and CD40"-"/"- deficient mice predominantly express CD40 type I and II isoforms. Whereas adult wild-type mice express ...

2008-05-02