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At least 379 records · Page 21Linked to original sources
PubMed2006

Human fertility protein PUMILIO2 interacts in vitro with testis mRNA encoding Cdc42 effector 3 (CEP3).

PUMILIO protein regulates translation of specific mRNAs in morphogenesis and in development of the germ-line of model organisms such as flies and worms. Given that a human homologue (PUMILIO2) was recently identified in the germ-line stem cells, the question was raised whether it regulates translation of fertility mRNAs similarly to Drosophila Pumilio. Here, we describe a candidate mRNA encoding Cdc42 effector protein 3 (CEP3), however, a function for this protein in reproduction has previously not been reported. We detected three CEP3 transcripts in the testis tissue including one which was highly expressed and testis specific by northern blotting. We found that CEP mRNA contains GUUGU (A) and AUUGUA (B) motifs (ABB) within the 3' untranslated region (3'UTR), which are also present in mRNA targets of Pumilio in Drosophila. Interaction of PUMILIO2 with the fragment of CEP3 transcript containing the ABB array was tested by mobility shift assay and we found that PUMILIO2 binds the 3' untranslated region of the CEP3 mRNA. These results support the hypothesis that CEP3 mRNA may be a target of PUMILIO2 protein in the human male gonad and be under translational control mediated by specific nucleotide motifs within the 3'UTR.

3' Untranslated Regions↗
PubMed2006

Redox proteomics in some age-related neurodegenerative disorders or models thereof.

Neurodegenerative diseases cause memory loss and cognitive impairment. Results from basic and clinical scientific research suggest a complex network of mechanisms involved in the process of neurodegeneration. Progress in treatment of such disorders requires researchers to better understand the functions of proteins involved in neurodegenerative diseases, to characterize their role in pathogenic disease mechanisms, and to explore their roles in the diagnosis, treatment, and prevention of neurodegenerative diseases. A variety of conditions of neurodegenerative diseases often lead to post-translational modifications of proteins, including oxidation and nitration, which might be involved in the pathogenesis of neurodegenerative diseases. Redox proteomics, a subset of proteomics, has made possible the identification of specifically oxidized proteins in neurodegenerative disorders, providing insight into a multitude of pathways that govern behavior and cognition and the response of the nervous system to injury and disease. Proteomic analyses are particularly suitable to elucidate post-translational modifications, expression levels, and protein-protein interactions of thousands of proteins at a time. Complementing the valuable information generated through the integrative knowledge of protein expression and function should enable the development of more efficient diagnostic tools and therapeutic modalities. Here we review redox proteomic studies of some neurodegenerative diseases.

Aged↗
PubMed1991

Retinoylation of the cAMP-binding regulatory subunits of type I and type II cAMP-dependent protein kinases in HL60 cells.

Retinoylation (retinoic acid acylation) is a post-translational modification of proteins occurring in a variety of eukaryotic cell lines. There are at least 20 retinoylated proteins in the human myeloid leukemia cell line HL60 (N. Takahashi and T.R. Breitman (1990) J. Biol. Chem. 265, 19, 158-19, 162). Here we found that some retinoylated proteins may be cAMP-binding proteins. Five proteins, covalently labeled by 8-azido-[32P]cAMP which specifically reacts with the regulatory subunits of cAMP-dependent protein kinase, comigrated on two-dimensional polyacrylamide gel electrophoresis with retinoylated proteins of Mr 37,000 (p37RA), 47,000 (p47RA), and 51,000 (p51RA) labeled by [3H]retinoic acid treatment of intact cells. Furthermore, p47RA coeluted on Mono Q anion exchange chromatography with the type I cAMP-dependent protein kinase holoenzyme and p51RA coeluted on Mono Q anion exchange chromatography with the type II cAMP-dependent protein kinase holoenzyme. An antiserum specific to RI, the cAMP-binding regulatory subunit of type I cAMP-dependent protein kinase, immunoprecipitated p47RA. An antiserum specific to RII, the cAMP-binding regulatory subunit of type II cAMP-dependent protein kinase, immunoprecipitated p51RA. These results indicate that both the RI and the RII regulatory subunits of cAMP-dependent protein kinase are retinoylated. Thus, an early event in RA-induced differentiation of HL60 cells may be the retinoylation of subpopulations of both RI and RII.

Azides↗
PubMed2005

A window of opportunity: timing protein degradation by trimming of sugars and ubiquitins.

Of the many post-translational modifications of proteins, ubiquitination and N-glycosylation stand out because they are polymeric additions. In contrast to single-unit modifications, the fate of the modified protein is determined by the dynamic equilibrium of polymerization versus depolymerization, rather than by the initial addition itself. Notably, it is the trimming of sugar chains and elongation of polyubiquitin that target the protein to degradation. Recent research suggests that, for each process, special receptors recognize chains that reach an appropriate length and commit the conjugated substrate for proteasomal disposal. We propose that the 'magic numbers' are loss of at least three mannose residues from the initial chain, or extension to at least four ubiquitins. Although these processes are compartmentalized to either side of the endoplasmic reticulum (ER) membrane, some proteins are sequentially subjected to both because they transverse this membrane for ER-associated degradation.

Calnexin↗
PubMed2006

Post-translational modifications in the context of therapeutic proteins.

The majority of protein-based biopharmaceuticals approved or in clinical trials bear some form of post-translational modification (PTM), which can profoundly affect protein properties relevant to their therapeutic application. Whereas glycosylation represents the most common modification, additional PTMs, including carboxylation, hydroxylation, sulfation and amidation, are characteristic of some products. The relationship between structure and function is understood for many PTMs but remains incomplete for others, particularly in the case of complex PTMs, such as glycosylation. A better understanding of such structural-functional relationships will facilitate the development of second-generation products displaying a PTM profile engineered to optimize therapeutic usefulness.

Glycoproteins↗
PubMed2003

Post-translational modification of betaH-crystallin of bovine lens with aging.

This study investigates post-translational modification of proteins of bovine lens with aging (3 year old vs. 6 month old cows). After water-soluble proteins were submitted to gel and ion exchange chromatography, betaH-crystallin, a subunit of beta-crystallin, and modified materials were isolated. These materials were then submitted to two dimensional polyacrylamide gel electrophoresis (2D-SDS PAGE) to detect and isolate the new spots. Results for lens proteins from 3 year old animals were compared to those from 6 month old animals. All spots were digested in gel with trypsin and the molecular masses of tryptic digests were measured by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOFMS). Peptides peaks obtained from mass mapping were identified using the protein database of the MS-Fit program in the Protein prospector program of the University of California, San Francisco. We found that two post translational modifications of betaH-crystallin, acetylation and phosphorylation occurred with aging.

Acetylation↗
PubMed2005

Strategies for revealing lower abundance proteins in two-dimensional protein maps.

One of the most challenging contemporary research endeavors is the mapping of proteins and establishing their linkages to normal and pathological conditions. The availability of current proteomics technologies has greatly facilitated the separation and identification of proteins in a complex protein mixture by standard two-dimensional gel electrophoresis and subsequent MALDI-TOF mass spectrometry. Due to the huge differences in the distribution of proteins in complex proteomes of humans, the detection and identification of proteins expressed in low copy number is a major challenge. The low abundance of important physiologically relevant proteins has rendered their analyses almost impossible without some means of prior purification and enrichment from tissue lysates or biological fluids. It is the current limits of detection of the methods that are used that prevents the detection of these proteins not the proteins themselves. More importantly, considering the frequency at which post-translational modifications of proteins occur, the separation of protein isoforms is essential to understand biological changes, and two-dimensional gel electrophoresis remains the only technique that can offer sufficient resolution to address this issue at a functional level. Cellular fractionation techniques followed by specific affinity probes for tracking target proteins have been developed to deplete the proteome of high abundance proteins in order to increase the sample loading for achieving greater sensitivity for proteins present in low abundance. Those applications can entail the removal of one protein or a class of proteins that interferes with the resolution of proteins in a 2-DE map. Moreover, the use of better solubilizing detergents in combination with an overlapping narrow immobilized pH gradients, results in higher resolution by stretching the protein pattern in the first dimension. In this review we will discuss strategies to remove high abundance proteins that can result in the visualization and detection of low abundance proteins in biological samples. The potential use of these strategies, as a means of developing diagnostic tools for early screening of diseases and identification of drug targets for therapeutic intervention, will also be discussed.

Animals↗
PubMed2001

Glycosylation analysis of gel-separated proteins.

Beyond the identification of proteins involved in a particular physiological situation, many aspects of proteomics require more detailed characterization of the proteins involved. Post-translational modifications (PTMs) of proteins are a common means to target proteins, regulate their activities and to mediate communication between proteins and cells. Owing to the much higher analytical complexity of glycan analysis compared to e.g. protein identification, PTM analysis in general and glycosylation analysis in particular is largely neglected in proteomics. In this review, the current technological status of global and site-specific glycosylation analysis of gel-separated proteins is described and the way in which the available technology can be employed in proteomics is critically discussed.

Animals↗
PubMed1996

Characterization of prenyl protein transferase enzymes in a human keratinocyte cell line.

Prenylation is a post-translational modification of proteins that involves the attachment of an isoprenoid group derived from mevalonic acid, either 15-carbon farnesyl or 20-carbon geranylgeranyl, to a specific carboxy-terminal domain of acceptor proteins. Three prenyl transferase enzymes have been identified so far. In this paper we report the presence of two prenyl transferases in the HaCaT human keratinocyte cell line. Chromatography of a cytosolic extract from these cells resolved a farnesyl protein transferase (FPT) and geranylgeranyl protein transferase-I (GGPT-I) whose activities were measured using a novel peptide-based assay. Both enzymes were inhibited dose dependently by zaragozic acids A and C. Zaragozic acid C was more active towards the FPT than GGPT-I while zaragozic acid A inhibited both enzymes with similar potency. Incubation of HaCaT cell homogenates with [3H] prenyl precursors resulted in the labelling of a number of proteins which was increased when the cells were pretreated with an inhibitor of hydroxymethylglutaryl CoA reductase. Given the role of prenylated proteins in proliferative and inflammatory processes, our finding that prenyl transferases capable of prenylating endogenous substrates are also present in keratinocytes suggests that these enzymes might provide novel therapeutic targets of dermatological importance.

Alkyl and Aryl Transferases↗
PubMed2006

Minimotif Miner: a tool for investigating protein function.

In addition to large domains, many short motifs mediate functional post-translational modification of proteins as well as protein-protein interactions and protein trafficking functions. We have constructed a motif database comprising 312 unique motifs and a web-based tool for identifying motifs in proteins. Functional motifs predicted by MnM can be ranked by several approaches, and we validated these scores by analyzing thousands of confirmed examples and by confirming prediction of previously unidentified 14-3-3 motifs in EFF-1.

14-3-3 Proteins↗
PubMed1996

Molecular characterization of MPT83: a seroreactive antigen of Mycobacterium tuberculosis with homology to MPT70.

The Mycobacterium bovis antigens MPB70 and MPB83 are homologous cross-reactive proteins. It has been reported previously that MPB83 is glycosylated and exists in two forms with apparent molecular masses of 23kDa and 25kDa, whereas the apparent molecular mass of MPB70 is 22kDa. Using a monoclonal antibody, SB10, which recognizes an epitope common to both MPB70 and MPB83, we compared the expression of these proteins in M. bovis BCG, virulent M. bovis and virulent Mycobacterium tuberculosis by Western blotting of bacterial lysates. The previously described pattern of high and low producing substrains of BCG for MPB70 was also applicable for MPB83. Virulent M. bovis was found to express high levels of MPB70 and MPB83. Immunoblotting experiments using sera from Balb/c mice infected with live M. tuberculosis H37Rv revealed that although the MPB83 homologue of M. tuberculosis, MPT83, is expressed at low levels in M. tuberculosis when grown in vitro, the protein is highly immunogenic during infection with live bacteria. A clone from a mycobacterial shuttle cosmid library of M. tuberculosis H37Rv was isolated which expressed both MPT70 and MPT83. Genetic analysis of this cosmid revealed that MPT70 and MPT83 were encoded by separate genes with the gene encoding MPT83 situated 2.4kb upstream of mpt70. Both genes are transcribed in the same direction. The gene encoding MPT83 was cloned and DNA sequencing revealed an open reading frame of 660bp encoding a protein with a predicted molecular mass of 22kDa. Recombinant MPT83 was expressed in Escherichia coli from the native AUG initiation codon by translational coupling. In E. coli MPT83 was expressed as a 23kDa antigen whereas in the rapid growing mycobacterium Mycobacterium smegmatis the protein was expressed as a 25kDa protein indicating post-translational modification of the protein by M. smegmatis. In recombinant M. smegmatis MPT83 was predominantly cell associated whereas MPT70 was secreted into the culture medium. Amino acid sequence comparison between MPT83 and MPT70 revealed a 61% identity between the proteins, although little homology was apparent at the amino terminus. In MPT83 this region contained a typical lipoprotein signal peptide cleavage motif and a putative signal motif for O glycosylation. Both these motifs were absent from the amino acid sequence of MPT70.

Amino Acid Sequence↗
PubMed2005

The protein stability and transcriptional activity of p63alpha are regulated by SUMO-1 conjugation.

Post-translational modification of proteins by the ubiquitin-like molecule SUMO-1 regulates their stability and activity with crucial implications for many cellular processes. Here we show that p63alpha, but not p63beta and gamma, is sumoylated in vitro and in vivo at a single lysine residue, K637, in the post-SAM domain. SUMO-1 attachment targets DeltaNp63alpha for proteasome mediated degradation while it does not influence p63alpha intracellular localization, as wild-type protein and a mutant carring the K637 mutated into arginine (K637R), have the same nuclear localization. Four natural p63 mutations, falling within the SAM and post-SAM domain of p63alpha, were found to be altered in their sumoylation capacity. The transcriptional activities of the natural mutants and of K637R were strongly increased compared to that of wild type p63, suggesting that sumoylation has a negative effect on p63 driven transcription. The findings that DeltaNp63alpha protein levels are regulated by SUMO-1 and that this regulation is altered in natural p63 mutants, suggest that SUMO conjugation to p63 plays a critical role in regulating the biological activity of p63.

Arginine↗
PubMed2006

Semisynthetic proteins in mechanistic studies: using chemistry to go where nature can't.

The manipulation of the chemical structure of proteins beyond what is feasible with standard genetics, offers a powerful strategy to investigate protein mechanisms. By allowing the incorporation of biophysical probes, unnatural amino acids, and post-translational modifications in proteins, chemistry-driven approaches have greatly contributed to the understanding of the molecular basis of protein function.

Amino Acids↗
PubMedDate not supplied

[Proteolytic splitting of proteins from the cortex and nuclear zones of the crystalline lens].

The proteolytic attacking capacity by pronase, a proteolytic wide-range enzyme, has been studied in soluble and insoluble proteins from the cortex and nuclear lens zones of young (aged 1.5-2 years) and old (aged 6-12 years) animals. It is established that proteolysis stability of proteins from the cortex lens zone in young and old animals is the same. The soluble proteins of the lens nucleus are attacked by the enzyme considerably weaker in old animals than in young animals; insoluble proteins in the process of ageing get more sensitive to the pronase action. Post-translation modifications of proteins--deamidation, intra- and intermolecular crosslinkage--may be the reason of changes in the pronase attacking capacity of proteins in the nuclear lens zone of old animals.

Aging↗
PubMed2005

Quantitative polysome analysis identifies limitations in bacterial cell-free protein synthesis.

Cell-free protein synthesis (CFPS) is becoming increasingly used for protein production as yields increase and costs decrease. CFPS optimization efforts have focused primarily on energy supply and small molecule metabolism, though little is known about the protein synthesis machinery or what limits protein synthesis rates. Here, quantitative polysome profile analysis was used to characterize cell-free translation, thereby elucidating many kinetic parameters. The ribosome concentration in Escherichia coli-based CFPS reactions was 1.6 +/- 0.1 microM, with 72 +/- 4% actively translating at maximal protein synthesis rate. A translation elongation rate of 1.5 +/- 0.2 amino acids per second per ribosome and an initiation rate of 8.2 x 10(-9) +/- 0.3 x 10(-9) M/s, which correlates to, on average, one initiation every 60 +/- 9 s per mRNA, were determined. The measured CFPS initiation and elongation rates are an order of magnitude lower than the in vivo rates and further analysis identified elongation as the major limitation. Adding purified elongation factors (EFs) to CFPS reactions increased the ribosome elongation rate and protein synthesis rates and yields, as well as the translation initiation rate, indicating a possible coupling between initiation and elongation. Further examination of translation initiation in the cell-free system showed that the first initiation on an mRNA is slower than subsequent initiations. Our results demonstrate that polysome analysis is a valid tool to characterize cell-free translation and to identify limiting steps, that dilution of translation factors is a limitation of CFPS, and that CFPS is a useful platform for making novel observations about translation.

Cell-Free System↗
PubMed2006

Annotating the human proteome: beyond establishing a parts list.

The completion of the human genome has shifted the attention from deciphering the sequence to the identification and characterisation of the functional components, including genes. Improved gene prediction algorithms, together with the existing transcript and protein information, have enabled the identification of most exons in a genome. Availability of the 'parts list' has fostered the development of experimental approaches to systematically interrogate gene function on the genome, transcriptome and proteome level. Studying gene function at the protein level is vital to the understanding of how cells perform their functions as variations in protein isoforms and protein quantity which may underlie a change in phenotype can often not be deduced from sequence or transcript level genomics experiments alone. Recent advancements in proteomics have afforded technologies capable of measuring protein expression, post-translational modifications of these proteins, their subcellular localisation and assembly into complexes and pathways. Although an enormous amount of data already exists on the function of many human proteins, much of it is scattered over multiple resources. Public domain databases are therefore required to manage and collate this information and present it to the user community in both a human and machine readable manner. Of special importance here is the integration of heterogeneous data to facilitate the creation of resources that go beyond a mere parts list.

Humans↗
PubMed2004

Identification of the protein acetyltransferase (Pat) enzyme that acetylates acetyl-CoA synthetase in Salmonella enterica.

Post-translational modification of proteins is an efficient way cells use to control the activity of structural proteins, gene expression regulatory proteins, and enzymes. In eukaryotes, the Sir2-dependent system of protein acetylation/deacetylation controls a number of processes that affect cell longevity. Sir2 proteins have NAD(+)-dependent protein deacetylase activity and are found in all forms of life. Although the identity of the acetyltransferases that partner with Sir2 enzymes is known in eukaryotes, the identity of the prokaryotic acetyltransferases is not. We report the identification of the gene of Salmonella enterica serovar Typhimurium LT2 encoding the major protein acetyltransferase (Pat) enzyme that, in concert with the CobB sirtuin of this bacterium, regulates the activity of the central metabolic enzyme acetyl-coenzyme A synthetase (Acs). The Pat enzyme uses acetyl-CoA as substrate to modify residue Lys609 of Acs. The Pat/CobB system of S.enterica should serve as the paradigm to further investigate the contributions of this system to the physiology of prokaryotes.

Acetate-CoA Ligase↗
PubMed2006

Phosphorylation and activation of a transducible recombinant form of human HSP20 in Escherichia coli.

Protein-based cellular therapeutics have been limited by getting molecules into cells and the fact that many proteins require post-translational modifications for activation. Protein transduction domains (PTDs), including that from the HIV TAT protein (TAT), are small arginine rich peptides that carry molecules across the cell membrane. We have shown that the heat shock-related protein, HSP20 is a downstream-mediator of cyclic nucleotide-dependent relaxation of vascular smooth muscle and is activated by phosphorylation. In this study, we co-expressed in Escherichia coli the cDNAs encoding the catalytic subunit of protein kinase G and a TAT-HSP20 fusion protein composed of the TAT PTD (-YGRKKRRQRRR-) fused to the N-terminus of human HSP20. Immunoblot and HPLC-ESI-MS/MS analysis of the purified TAT-HSP20 demonstrated that it was phosphorylated at serine 40 (equivalent to serine 16 in wild-type human HSP20). This phosphorylated TAT-HSP20 was physiologically active in intact smooth muscles in that it inhibited 5-hydroxytryptamine-induced contractions by 57%+/-4.5. The recombinant phosphorylated protein also led to changes in actin cytoskeletal morphology in 3T3 cells. These results delineate strategies for the expression and activation of therapeutic molecules for intracellular protein based therapeutics.

3T3 Cells↗