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At least 325 records · Page 18Linked to original sources
PubMed1992

Sec23p and a novel 105-kDa protein function as a multimeric complex to promote vesicle budding and protein transport from the endoplasmic reticulum.

A cell-free protein transport reaction has been used to monitor the purification of a functional form of the Sec23 protein, a SEC gene product required for the formation or stability of protein transport vesicles that bud from the endoplasmic reticulum (ER). Previously, we reported that Sec23p is an 84-kDa peripheral membrane protein that is released from a sedimentable fraction by vigorous mechanical agitation of yeast cells and is required for ER to Golgi transport assayed in vitro. We have purified soluble Sec23p by complementation of an in vitro ER to Golgi transport reaction reconstituted with components from sec23 mutant cells. Sec23p overproduced in yeast exists in two forms: a monomeric species and a species that behaves as a 250- to 300-kDa complex that contains Sec23p and a distinct 105-kDa polypeptide (p105). Sec23p purified from cells containing one SEC23 gene exists solely in the large multimeric form. A stable association between Sec23p and p105 is confirmed by cofractionation of the two proteins throughout the purification. p105 is a novel yeast protein involved in ER to Golgi transport. Like Sec23p, it is required for vesicle budding from the ER because p105 antiserum completely inhibits transport vesicle formation in vitro.

Animals↗
PubMed2006

PspB and PspC of Yersinia enterocolitica are dual function proteins: regulators and effectors of the phage-shock-protein response.

The phage-shock-protein (Psp) stress-response system is conserved in many bacteria and has been linked to important phenotypes in Escherichia coli, Salmonella enterica and also Yersinia enterocolitica, where it is essential for virulence. It is activated by specific extracytoplasmic stress events such as the mislocalization of secretin proteins. From studies of the Psp system in E. coli, the cytoplasmic membrane proteins PspB and PspC have only been proposed to act as positive regulators of psp gene expression. However, in this study we show that PspB and PspC of Y. enterocolitica are dual function proteins, acting both as regulators and effectors of the Psp system. Consistent with the current model, they positively control psp gene expression in response to diverse inducing cues. PspB and PspC must work together to achieve this regulatory function, and bacterial two-hybrid (BACTH) analysis demonstrated a specific interaction between them, which was confirmed by in vivo cross-linking. We also show that PspB and PspC play a second role in supporting growth when a secretin protein is overexpressed. This function is independent from their role as regulators of psp gene expression. Furthermore, whereas PspB and PspC must work together for their regulatory function, they can apparently act independently to support growth during secretin production. This study expands the current understanding of the roles played by PspB and PspC, and demonstrates that they cannot be considered only as positive regulators of psp gene expression in Y. enterocolitica.

Bacterial Proteins↗
PubMed2000

An additional transcript of the cdc25C gene from A431 cells encodes a functional protein.

Human p53 protein was found to be functional in fission yeast in terms of growth repression and checkpoint control. Expression of wild-type p53 or the hot spot mutant p53His273 results in dramatic morphological changes and loss of viability of recipient yeast cells. Overexpression of cdc25C phosphatase, the mitotic activator of cdc2, results in suppression of a p53-induced growth arrest. In order to understand the interplay between p53 and cdc25C in mammalian cells we isolated and sequenced cdc25C cDNA from the epidermoid carcinoma cell line A431, which is known to carry the p53His273 mutation. Two different transcripts of the human cdc25C gene were detected by RT-PCR analysis - one full-length transcript and a shortened version (cdc25Cdm) that carries two deletions in the 5'-region of the gene. In normal human skin fibroblasts only one full-length cdc25C transcript was detected. The two different transcripts code for proteins with a molecular weight of 55 kDa and 46 kDa, respectively. Both cdc25C cDNAs from A431 cells were found to complement a conditional lethal cdc25.22 mutant strain as well as a cdc25 deletion strain of Schizosaccharomyces pombe indicating that functional proteins were translated. Expression of cdc25Cdm variant leads to a stronger uncoupling of DNA replication from mitosis than expression of cdc25C suggesting that the deletion within the amino-terminus of cdc25C leads to a protein which might contribute some potential for oncogenic transformation. As with cdc25C, uncoupling of the DNA synthesis checkpoint by cdc25Cdm was reversed by coexpression of wild-type p53.

Alternative Splicing↗
PubMed2000

Chemical strategies for the global analysis of protein function.

As the human genome project nears completion, biological research is entering a new era in which experimental focus will shift from identifying novel genes to determining the function of gene products. Rising to this challenge, several technologies have emerged that aim to characterise genes and/or proteins collectively rather than individually. Of particular interest is a new breed of strategies that employs synthetic chemistry to enrich our understanding of protein function on a global scale.

Animals↗
PubMed2005

Protein function prediction using local 3D templates.

The prediction of a protein's function from its 3D structure is becoming more and more important as the worldwide structural genomics initiatives gather pace and continue to solve 3D structures, many of which are of proteins of unknown function. Here, we present a methodology for predicting function from structure that shows great promise. It is based on 3D templates that are defined as specific 3D conformations of small numbers of residues. We use four types of template, covering enzyme active sites, ligand-binding residues, DNA-binding residues and reverse templates. The latter are templates generated from the target structure itself and scanned against a representative subset of all known protein structures. Together, the templates provide a fairly thorough coverage of the known structures and ensure that if there is a match to a known structure it is unlikely to be missed. A new scoring scheme provides a highly sensitive means of discriminating between true positive and false positive template matches. In all, the methodology provides a powerful new tool for function prediction to complement those already in use.

Models, Molecular↗
PubMed2005

Inhibition of papillomavirus protein function in cervical cancer cells by intrabody targeting.

Papillomaviruses (HPVs) are a major cause of human disease, and are responsible for approximately half a million cases of cervical cancer each year. HPVs also cause genital warts, and are the most common sexually transmitted disease in many countries. Despite their importance, there are currently no specific antivirals that are active against HPVs. Papillomavirus protein function is mediated largely by protein-protein interactions, which are difficult to inhibit using conventional approaches. To circumvent these problems, we have prepared an scFv library, and have used this to isolate high-affinity binding molecules that may stearically hinder the association of E6 with p53 and prevent E6-mediated p53 degradation in cervical cancer cells. One of the molecules isolated from the library (GTE6-1), had an affinity for 16E6 of 60nM, and bound within the first zinc finger of the protein. GTE6-1 was able to associate with non-denatured E6 following expression in mammalian cells and could inhibit E6-mediated p53 degradation in in vitro assays. E6-mediated p53 degradation is essential for the continuous growth of cervical cancer cells caused by HPV16. To examine the potential of GTE6-1 as an inhibitor of E6 function in such cells, the molecule was expressed in scFv, diabody and triabody formats in a number of cell lines that are driven to proliferate by the HPV16 oncogenes E6 and E7, including the cervical cancer cell line SiHa. In contrast to small E6-binding peptides containing the ELLG E6-binding motif, GTE6-1 expression lead to changes in nuclear structure, the appearance of apoptosis markers, and an elevation in the levels of p53. No effects were seen with a control scFv molecule, or when GTE6-1 was expressed in cells that are driven to proliferate by simian virus 40 (SV40) T-antigen. Given the accessibility of HPV-associated lesions to topical therapy, our results suggest that large interfering molecules such as intrabodies may be useful inhibitors of viral protein-protein interactions and be particularly appropriate for the treatment of HPV-associated disease.

Amino Acid Sequence↗
PubMed1998

[Peptide synthesis aiming at elucidation and creation of protein functions].

The recent development of molecular biology has been elucidating outlines of the cross-talk of biomolecules. The understanding of the function of these biomolecules from the viewpoint of chemistry is now demanded not only for the understanding of biological systems but also for the creation of novel functional molecules. Here two topics are described about peptide synthesis aiming at the elucidation and the creation of protein functions. The first topic is the development of approaches for the synthesis of Tyr (SO3H)-containing peptides. Tyrosine sulfation is one of the most popular protein post-translational modifications. Synthetic peptides are of great help for the elucidation of the biological significance of tyrosine sulfation. We have developed two approaches for the efficient synthesis of tyrosine sulfate [Tyr (SO3H)]-containing peptides. The first approach employs a dimethylformamide-sulfur trioxide (DMF-SO3) complex as a sulfating agent and safety-catch protecting groups for the selective sulfation of tyrosine in the presence of serine. The second approach employs the direct introduction of Tyr(SO3H) into the peptide chain in the form of Fmoc-Tyr(SO3Na) followed by deprotection at 4 degrees C in trifluoroacetic acid. These approaches were successfully applied for the synthesis of cholecystokinin (CCK)-related peptides. The second topic deals with new approaches for the creation of artificial proteins through assembling alpha-helical peptides via selective disulfide or thioether formation. Approaches to assemble individual peptide segments on a peptide template were also developed. Four peptides corresponding to the transmembrane segments of the sodium channel (S4 in repeat I-IV) were assembled on a peptide template to give a protein having ion channel activity with rectification.

Amino Acid Sequence↗
PubMed2004

An interactive network of zinc-finger proteins contributes to regionalization of the Drosophila embryo and establishes the domains of HOM-C protein function.

During animal development, the HOM-C/HOX proteins direct axial patterning by regulating region-specific expression of downstream target genes. Though much is known about these pathways, significant questions remain regarding the mechanisms of specific target gene recognition and regulation, and the role of co-factors. From our studies of the gnathal and trunk-specification proteins Disconnected (DISCO) and Teashirt (TSH), respectively, we present evidence for a network of zinc-finger transcription factors that regionalize the Drosophila embryo. Not only do these proteins establish specific regions within the embryo, but their distribution also establishes where specific HOM-C proteins can function. In this manner, these factors function in parallel to the HOM-C proteins during axial specification. We also show that in tsh mutants, disco is expressed in the trunk segments, probably explaining the partial trunk to head transformation reported in these mutants, but more importantly demonstrating interactions between members of this regionalization network. We conclude that a combination of regionalizing factors, in concert with the HOM-C proteins, promotes the specification of individual segment identity.

Animals↗
PubMed2005

Role of the extracytoplasmic function protein family sigma factor RpoE in metal resistance of Escherichia coli.

RpoE of Escherichia coli is a sigma factor of the extracytoplasmic function protein family and is required for the expression of proteins involved in maintaining the integrity of periplasmic and outer membrane components. RpoE of E. coli was needed for full resistance to Zn(II), Cd(II), and Cu(II). Promoter gene fusion and quantitative real time reverse transcription (RT)-PCR (qRT-PCR) assays demonstrated that expression of RpoE was induced by metals. Global gene expression profiles upon metal treatment of a DeltarpoE mutant strain and its wild-type strain were analyzed with microarrays, and selected genes were confirmed by qRT-PCR. The absolute number of genes that were changed in their expression upon metal stress was similar in both strains, but the increase or decrease in transcript levels upon metal treatment was smaller in the DeltarpoE mutant strain than in the wild type. Genes showing increased expression in the DeltarpoE mutant strain encoded proteins that belong to general defense systems against protein-denaturing agents. Genes showing decreased expression were part of the RpoE modulon itself plus the ompC gene, encoding a major outer membrane protein. A DeltaompC deletion strain was as sensitive to Cu(II) and Cd(II) as the DeltarpoE mutant or a DeltarpoE DeltaompC double mutant strain. In the case of Zn(II), the double mutant was more sensitive than either single mutant. This indicates that increased expression of OmpC contributes to the RpoE modulon-mediated response to metals.

Cadmium↗
PubMed1987

Rapid localization and characterization of random mutations within the 2 micron circle site-specific recombinase: a general strategy for analysis of protein function.

A method for rapidly localizing and characterizing mutations within the 2 micron circle site-specific recombinase enzyme (FLP) is described. The strategy consists of dividing the gene coding for FLP (FLP) by artificially introduced unique restriction enzyme sites (that do not alter the amino acid sequence of the protein) into segments of 150-200 bp, mutagenizing the engineered gene and selecting mutants in vivo, localizing each mutation to one of the gene segments by an in vivo complementation assay, and characterizing the mutation by sequencing of only this DNA segment. The experimental designs embodied by this method should be of wide application in investigations of protein function in general and of DNA-protein interactions in particular.

Amino Acid Sequence↗
PubMed2003

Fusicoccin signaling reveals 14-3-3 protein function as a novel step in left-right patterning during amphibian embryogenesis.

To gain insight into the molecular mechanisms underlying the control of morphogenetic signals by H+ flux during embryogenesis, we tested Fusicoccin-A (FC), a compound produced by the fungus Fusicoccum amygdali Del. In plant cells, FC complexes with 14-3-3 proteins to activate H+ pumping across the plasma membrane. It has long been thought that FC acts on higher plants only; here, we show that exposing frog embryos to FC during early development specifically results in randomization of the asymmetry of the left-right (LR) axis (heterotaxia). Biochemical and molecular-genetic evidence is presented that 14-3-3-family proteins are an obligate component of Xenopus FC receptors and that perturbation of 14-3-3 protein function results in heterotaxia. The subcellular localization of 14-3-3 mRNAs and proteins reveals novel cytoplasmic destinations, and a left-right asymmetry at the first cell division. Using gain-of-function and loss-of-function experiments, we show that 14-3-3E protein is likely to be an endogenous and extremely early aspect of LR patterning. These data highlight a striking conservation of signaling pathways across kingdoms, suggest common mechanisms of polarity establishment between C. elegans and vertebrate embryos, and uncover a novel entry point into the pathway of left-right asymmetry determination.

14-3-3 Proteins↗
PubMed1995

The disease-resistance gene Pto and the fenthion-sensitivity gene fen encode closely related functional protein kinases.

Resistance to bacterial speck in tomato is governed by a gene-for-gene interaction in which a single resistance locus (Pto) in the plant responds to the expression of a specific avirulence gene (avrPto) in the pathogen. Disease susceptibility results if either Pto or avrPto are lacking from the corresponding organisms. Leaves of tomato cultivars that contain the Pto locus also exhibit a hypersensitive-like response upon exposure to an organophosphorous insecticide, fenthion. Recently, the Pto gene was isolated by a map-based cloning approach and was shown to be a member of a clustered multigene family with similarity to various protein-serine/threonine kinases. Another member of this family, termed Fen, was found to confer sensitivity to fenthion. The Pto protein shares 80% identity (87% similarity) with Fen. Here, Pto and Fen are shown to be functional protein kinases that probably participate in the same signal transduction pathway.

Amino Acid Sequence↗
PubMed2002

Accounting for human polymorphisms predicted to affect protein function.

A major interest in human genetics is to determine whether a nonsynonymous single-base nucleotide polymorphism (nsSNP) in a gene affects its protein product and, consequently, impacts the carrier's health. We used the SIFT (Sorting Intolerant From Tolerant) program to predict that 25% of 3084 nsSNPs from dbSNP, a public SNP database, would affect protein function. Some of the nsSNPs predicted to affect function were variants known to be associated with disease. Others were artifacts of SNP discovery. Two reports have indicated that there are thousands of damaging nsSNPs in an individual's human genome; we find the number is likely to be much lower.

Amino Acid Motifs↗
PubMed2002

Role of N-linked glycans in a human immunodeficiency virus envelope glycoprotein: effects on protein function and the neutralizing antibody response.

The envelope (Env) glycoprotein of human immunodeficiency virus (HIV) contains 24 N-glycosylation sites covering much of the protein surface. It has been proposed that one role of these carbohydrates is to form a shield that protects the virus from immune recognition. Strong evidence for such a role for glycosylation has been reported for simian immunodeficiency virus (SIV) mutants lacking glycans in the V1 region of Env (J. N. Reitter, R. E. Means, and R. C. Desrosiers, Nat. Med. 4:679-684, 1998). Here we used recombinant vesicular stomatitis viruses (VSVs) expressing HIV Env glycosylation mutants to determine if removal of carbohydrates in the V1 and V2 domains affected protein function and the generation of neutralizing antibodies in mice. Mutations that eliminated one to six of the sites for N-linked glycosylation in the V1 and V2 loops were introduced into a gene encoding the HIV type 1 primary isolate 89.6 envelope glycoprotein with its cytoplasmic domain replaced by that of the VSV G glycoprotein. The membrane fusion activities of the mutant proteins were studied in a syncytium induction assay. The transport and processing of the mutant proteins were studied with recombinant VSVs expressing mutant Env G proteins. We found that HIV Env V1 and V2 glycosylation mutants were no better than wild-type envelope at inducing antibodies neutralizing wild-type Env, although an Env mutant lacking glycans appeared somewhat more sensitive to neutralization by antibodies raised to mutant or wild-type Env. These results indicate significant differences between SIV and HIV with regard to the roles of glycans in the V1 and V2 domains.

AIDS Vaccines↗
PubMed2005

Genome-scale protein function prediction in yeast Saccharomyces cerevisiae through integrating multiple sources of high-throughput data.

As we are moving into the post genome-sequencing era, various high-throughput experimental techniques have been developed to characterize biological systems at the genome scale. Discovering new biological knowledge from high-throughput biological data is a major challenge for bioinformatics today. To address this challenge, we developed a Bayesian statistical method together with Boltzmann machine and simulated annealing for protein function prediction in the yeast Saccharomyces cerevisiae through integrating various high-throughput biological data, including protein binary interactions, protein complexes and microarray gene expression profiles. In our approach, we quantified the relationship between functional similarity and high-throughput data. Based on our method, 1802 out of 2280 unannotated proteins in the yeast were assigned functions systematically. The related computer package is available upon request.

Bayes Theorem↗
PubMed1988

Chemical modification of prothrombin fragment 1: documentation of sequential, two-stage loss of protein function.

The amino groups of prothrombin fragment 1 (amino acids 1-156 of prothrombin) were derivatized by acetylation, amidination, and reductive methylation. Conditions that caused complete acetylation of protein amino groups produced a fragment 1 derivative which no longer displayed a metal ion dependent intrinsic fluorescence change and had lost its membrane binding capability as well. However, when derivatized in the presence of calcium ions, extensive acetylation yielded a product that underwent protein fluorescence quenching at metal ion concentrations similar to those observed for the native protein. This derivative bound to membranes in a calcium-dependent manner with only a small reduction in affinity. Several results showed the existence of a partially functional protein that was characterized by a high degree of calcium-dependent protein fluorescence quenching but which had a requirement for 10-fold higher calcium concentration. This derivative was produced by partial acetylation (greater than 3 equiv) of metal-free protein. This partially acetylated protein had greatly diminished membrane binding. The calcium-protected amino group, therefore, was among the most reactive acetylation sites in the metal-free protein. The second site, responsible for abolishing all metal ion induced fluorescence change, was resistant to acetylation and became derivatized at the last stages of amino group acetylation.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylation↗
PubMed1997

Protein functions in pre-mRNA splicing.

Proteins have been implicated in an expanding variety of functions during pre-mRNA splicing. Molecular cloning has identified genes encoding spliceosomal proteins that potentially act as novel RNA helicases, GTPases, or protein isomerases. Novel protein-protein and protein-RNA interactions that are required for functional spliceosome formation have also been described. Finally, growing evidence suggests that proteins may contribute directly to the spliceosome's active sites.

Fungal Proteins↗