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PubMed1996

[Microtubules: functional polymorphisms of tubulin and associated proteins (structural and motor MAP's)].

In neuronal cells, microtubules are built from a very large number of alpha- and beta-tubulin variants. This diversity is due to the expression of a multigene family and to a combination of several original posttranslational modifications. Similarly, structural and motor microtubule-associated proteins, which regulate the assembly of microtubules, the modeling of their network and the mediation of their functions, are also very heterogeneous. As a consequence, mixing of these two protein polymorphisms leads to the formation of functionally-distinct microtubules. We have shown that polyglutamylation, the major posttranslational modification of neuronal tubulin, was used as a progressive regulator in the binding of structural and motor microtubule-associated proteins, in modulating gradually the conformation of the tubulin carboxy-terminal domain, playing thus a crucial role in microtubule dynamics.

Animals↗
PubMed2003

Plasma protein synthesis measurements using a proteomics strategy.

The analysis of the synthesis of proteins has been the subject of many studies in animals and humans. Plasma proteins can be used as an easy accessible source of specific proteins. In this paper, an innovative method to study the synthetic rate of plasma proteins is described. This methodology, based on the proteomics approach, enables the direct observation of the effects of posttranslational modifications of protein synthesis and/or degradation. The methodology is based on 1D or 2D electrophoresis and subsequent electrospray ionization liquid chromatography mass spectrometry (ESI-LC-MS). Protein synthesis is measured in isotopically labeled peptides of the identified proteins. This innovative method can be used to assess amino acid adequacy and safety by studying protein synthesis and posttranslational modification of plasma proteins in more detail.

Amino Acids↗
PubMed2004

High-throughput identification of proteins and unanticipated sequence modifications using a mass-based alignment algorithm for MS/MS de novo sequencing results.

With the increasing availability of de novo sequencing algorithms for interpreting high-mass accuracy tandem mass spectrometry (MS/MS) data, there is a growing need for programs that accurately identify proteins from de novo sequencing results. De novo sequences derived from tandem mass spectra of peptides often contain ambiguous regions where the exact amino acid order cannot be determined. One problem this poses for sequence alignment algorithms is the difficulty in distinguishing discrepancies due to de novo sequencing errors from actual genomic sequence variation and posttranslational modifications. We present a novel, mass-based approach to sequence alignment, implemented as a program called OpenSea, to resolve these problems. In this approach, de novo and database sequences are interpreted as masses of residues, and the masses, rather than the amino acid codes, are compared. To provide further flexibility, the masses can be aligned in groups, which can resolve many de novo sequencing errors. The performance of OpenSea was tested with three types of data: a mixture of known proteins, a mixture of unknown proteins that commonly contain sequence variations, and a mixture of posttranslationally modified known proteins. In all three cases, we demonstrate that OpenSea can identify more peptides and proteins than commonly used database-searching programs (SEQUEST and ProteinLynx) while accurately locating sequence variation sites and unanticipated posttranslational modifications in a high-throughput environment.

Algorithms↗
PubMed2000

Heterogeneity in human cardiac troponin I standards.

The LC-MS analysis of recombinant cardiac troponin I (cTnI) and cTnI extracted from human hearts showed a high degree of structural heterogeneity among all samples. The examined recombinant cTnI samples indicated posttranslational modifications, presumably due to their purification (i.e., 2-mercaptoethanol adducts and carbamylation) and related to their expression (i.e., an N-terminal expression tag). The extracted cTnI samples, while having a higher degree of structural heterogeneity, showed less structural variance between samples than the recombinant proteins. The LC-MS analysis of the extracted cTnI samples provided evidence of posttranslational modification by phosphorylation, acetylation, proteolytic cleavage, and intrachain disulfide bond formation.

Amino Acid Sequence↗
PubMed1989

Structural and functional diversity in vertebrate skeletal muscle nicotinic acetylcholine receptors.

Overall, the data support the conclusion that the gamma and epsilon subunits explain major features of the functional diversity of vertebrate muscle nAChR. They cannot account, however, for all observed functional diversity. It is not clear whether additional subunits exist or whether some posttranslational modifications produce the functional differences. Work is underway to correlate structural features of the muscle nicotinic AChR with functional properties. To date, the results demonstrate that each known subunit can affect receptor kinetic parameters and/or channel conductance. Two remaining sets of questions must be answered. The first lies within the field of cell biology: What subunits are expressed, do posttranslational modifications underlie functional modifications, and what structural changes are associated with physiologically relevant changes in function? The second is more strictly of structure-function relationships: What are the functional parameters of structurally defined nAChRs and can structural features be correlated with function?

Animals↗
PubMed2002

Inhibitors of post-translational modifications of G-proteins as probes to study the pancreatic beta cell function: potential therapeutic implications.

It is well established that glucose-induced insulin secretion involves generation of intracellular second messengers. Using specific inhibitors of guanosine triphosphate [GTP] biosynthesis [e.g., mycophenolic acid; MPA], we have identified a permissive role for GTP in glucose-stimulated insulin secretion. While the exact site of action for GTP within the islet beta cell remains to be identified and defined, recent evidence from several laboratories, including our own, indicate that it could involve activation of GTP-binding proteins [G-proteins]. These studies have identified both trimeric and monomeric forms of G-proteins within the pancreatic beta cell. Recent data also indicate that these G-proteins, specifically the monomeric G-proteins and the gamma subunits of trimeric G-proteins undergo a series of posttranslational modifications at their C-terminal cysteine. Such modifications include, isoprenylation, carboxyl methylation and palmitoylation. These modification steps appear to be essential for translocation of these proteins to the membrane sites for interaction with their respective effector proteins. This review primarily focuses on recent findings that clearly support the viewpoint that these posttranslational modification steps not only play obligatory roles in fuel-induced insulin secretion, but also in cytokine-mediated apoptotic demise of the beta cell. In this review, we also attempted to describe those findings involving the use of specific inhibitors for each of these pathways, and it is our hope that these aspects of beta cell metabolism and function generate interest in development of therapeutic intervention modalities to states of perturbed insulin release.

Apoptosis↗
PubMed2005

[Variations on the topic of the "histone code"].

Histones are the fundamental structural proteins intimately associated with eukaryotic DNA to form a highly ordered and condensed nucleoproteic complex termed chromatin. They are the targets of various posttranslational modifications including acetylation, methylation, phosphorylation and ubiquitination that modulate the structure/function of chromatin. The combinatorial nature of histone modifications is hypothesized to define a "histone code" that considerably extends the information potential of the genetic code, giving rise to epigenetic information. Moreover, most core histones consist of several nonallelic variants that can mark specific loci and could play an important role in establishment and maintenance of epigenetic memory. Here we will briefly present our current knowledge about histone posttranslational modifications and their implications in the regulation of epigenetic information. We will next describe core histone variants, insisting on their mode of incorporation into chromatin to discuss their epigenetic function and inheritance.

Histones↗
PubMed2006

E4F1 is an atypical ubiquitin ligase that modulates p53 effector functions independently of degradation.

p53 is regulated by multiple posttranslational modifications, including Hdm2-mediated ubiquitylation that drives its proteasomal degradation. Here, we identify the p53-associated factor E4F1, a ubiquitously expressed zinc-finger protein first identified as a cellular target of the viral oncoprotein E1A, as an atypical ubiquitin E3 ligase for p53 that modulates its effector functions without promoting proteolysis. E4F1 stimulates oligo-ubiquitylation in the hinge region of p53 on lysine residues distinct from those targeted by Hdm2 and previously described to be acetylated by the acetyltransferase PCAF. E4F1 and PCAF mediate mutually exclusive posttranslational modifications of p53. E4F1-dependent Ub-p53 conjugates are associated with chromatin, and their stimulation coincides with the induction of a p53-dependent transcriptional program specifically involved in cell cycle arrest, and not apoptosis. Collectively, our data reveal that E4F1 is a key posttranslational regulator of p53, which modulates its effector functions involved in alternative cell fates: growth arrest or apoptosis.

Acetylation↗
PubMed1992

Cloning and sequence analysis of the tyrosinase gene from a patient with tyrosinase-positive oculocutaneous albinism.

Tyrosinase is synthesized on membrane-bound ribosomes and transported into melanosomes through smooth endoplasmic reticulum and Golgi apparatus. Melanin polymers are produced only in melanosomes but never in smooth endoplasmic reticulum or Golgi apparatus, indicating that posttranslational modifications of tyrosinase are completed with melanosomes where tyrosinase becomes an active form. Based on a working hypothesis that tyrosinase-positive oculocutaneous albinism is a consequence of the structurally altered tyrosinase due to a point mutation in the gene of its gene coding for a glycosylation site or a membrane-binding site, which leads to the impairment in the posttranslational modification of tyrosinase and its catalytic activity, we have cloned the tyrosinase gene of one patient affected with tyrosinase-positive oculocutaneous albinism and determined its nucleotide sequence. Thus demonstrated all exons' nucleotide sequence of the patient's tyrosinase gene was found to be identical to that of the wild-type gene. The results indicate that the patient's tyrosinase itself is not altered. We therefore propose that the molecular basis for the development of tyrosinase-positive oculocutaneous albinism exists as a defect in other proteins required for the activation of tyrosinase or in other regions of the tyrosinase gene.

Adult↗
PubMed1984

Tubulin proteins and RNA during the myxamoeba-flagellate transformation of Physarum polycephalum.

Physarum myxamoebae can be reversibly induced to become flagellates. Physarum flagellates contain a new form of tubulin, alpha 3, that is not found in nonflagellated cells. Evidence is presented that suggests that alpha 3 tubulin arises through posttranslational modification of a preexisting alpha tubulin. Pulse-chase experiments showed that labeled alpha 3 tubulin could be detected when flagellates formed after a chase. RNA was isolated from myxamoebae at different times after induction of flagellum formation. When this RNA was translated in vitro, the resulting products contained no alpha 3 tubulin, also consistent with alpha 3 being made by posttranslational modification. Levels of alpha and beta tubulin RNA increased with the proportion of flagellates in the culture. These elevated tubulin RNA levels declined after the number of flagellates in the population achieved plateau values.

Actins↗
PubMed1992

Recombinant human complement subcomponent C1s lacking beta-hydroxyasparagine, sialic acid, and one of its two carbohydrate chains still reassembles with C1q and C1r to form a functional C1 complex.

In contrast to the human serum protein which is approximately one-half erythro-beta-hydroxyasparagine at asparagine 134 [Theilens et al. (1990) Biochemistry 29, 3570-3578], recombinant C1s expressed by insect cells after infection with recombinant baculovirus entirely lacks posttranslational modification at asparagine 134. It is also incompletely glycosylated, lacking, at least, sialic acid. Site-directed mutagenesis of one of the two sites of carbohydrate attachment (Asn 159 to Gln 159) yields a faster migrating recombinant C1s still abundantly secreted. Furthermore, the mutated protein displays good hemolytic activity when reassembled with C1q and either human serum or recombinant C1r, demonstrating that these posttranslational modifications are not critical for any of the multiple interactions between C1s and C1q, C1r, C2, and C4 required for reassembly of the C1 complex, activation, and initiation of the classical complement pathway. The 4.0S recombinant C1s dimerizes to yield 5.6S C1s2 in the presence of Ca2+ and forms the 9.1S C1s-C1r-C1r-C1s tetramer upon the addition of human serum C1r and the 15.6S C1 complex upon the addition of C1q to the tetramer. The recombinant C1s and human serum C1s have identical N-terminal amino acid sequences, indicating proper recognition by the insect signal peptidase. The recombinant C1s is secreted and isolated as the unactivated zymogen, and it may be activated by human serum C1r which cleaves at Arg422-Ile423 to yield the characteristic heavy and light chains. A very tight complex is formed between C1-inhibitor and the light chain of recombinant C1s.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Characteristics of phenol oxidase of Schistosoma mansoni and its functional implications in eggshell synthesis.

It is proposed that the phenol oxidase enzyme of schistosomes and other trematodes has a crucial role in the cross-linking of precursor proteins in the formation of the eggshell. However, to date there is no direct evidence to show that the enzyme catalyzes the reactions necessary for the posttranslational modification of eggshell precursor proteins. In this report we demonstrate that an eggshell precursor protein acts as a substrate for a schistosome fraction that catalyzed the 2 steps in the oxidation of tyrosine. This action of the phenol oxidase-containing worm fraction resulted in the tyrosine-dependent insolubilization and aggregation of the protein, suggesting a role for the enzyme in the posttranslational modification and cross-linking of schistosome eggshell proteins. The enzyme-rich fraction from Schistosoma mansoni catalyzed both steps of the reactions necessary for the conversion of tyrosine residues on putative eggshell precursor protein (p48) to quinones. The parasite fraction also catalyzed the hydroxylation of free L-tyrosine to DOPA (monophenol oxidase activity) and the oxidation of L-DOPA to dopaquinone (diphenol oxidase activity). Both activities of the enzyme are avidly bound to membranous structures, are susceptible to agents known to inhibit a functionally related enzyme, tyrosinase, and may reside on the same protein.

Animals↗
PubMed1998

Biochemical characterization of recombinant factor IX.

Mature human factor IX is a 55,000-d glycoprotein with a modular domain structure and numerous posttranslational modifications. A recombinant form of human factor IX (rFIX) has been produced from a Chinese hamster ovary cell line that was engineered for high-level protein processing and expression. To ensure that the recombinant molecule contains the requisite structural and functional features of the plasma-derived form, rFIX was subjected to detailed biochemical and biophysical characterization. The laboratory studies showed that the posttranslational modifications and primary, secondary, and tertiary structures of rFIX were similar to those of plasma-derived factor IX (pdFIX). In addition, rFIX displayed a high degree of purity and a product release specification for specific activity that is > or = 200 IU/mg.

Amino Acid Sequence↗
PubMed1999

Characterization of yeast deoxyhypusine synthase: PKC-dependent phosphorylation in vitro and functional domain identification.

The biosynthesis of hypusine [Nepsilon-(4-amino-2-hydroxybutyl)-lysine] occurs in the eIF-5A precursor protein through two step posttranslational modification involving deoxyhypusine synthase which catalyzes transfer of the butylamine moiety of spermidine to the epsilon-amino group of a designated lysine residue and subsequent hydroxylation of this intermediate. This enzyme is exclusively required for cell viability and growth of yeast (Park, M.H. et al., J. Biol. Chem. 273: 1677-1683, 1998). In an effort to understand structure-function relationship of deoxyhypusine synthase, posttranslational modification(s) of the enzyme by protein kinases were carried out for a possible cellular modulation of this enzyme. And also twelve deletion mutants were constructed, expressed in E. coli system, and enzyme activities were examined. The results showed that deoxyhypusine synthase was phosphorylated by PKC in vitro but not by p56lck and p60c-src. Treatment with PMA specifically increased the relative phosphorylation of the enzyme supporting PKC was involved. Phosphoamino acid analysis of this enzyme revealed that deoxyhypusine synthase is mostly phosphorylated on serine residue and weakly on threonine. Removal of Met1-Glu10 (deltaMet1-Glu10) residues from amino terminal showed no effect on the catalytic activity but further deletion (deltaMet1-Ser20) caused loss of enzyme activity. The enzyme with internal deletion, deltaGln197-Asn212 (residues not present in the human enzyme) was found to be inactive. Removal of 5 residues from carboxyl terminal, deltaLys383-Asn387, retained only slight activity. These results suggested that deoxyhypusine synthase is substrate for PKC dependent phosphorylation and requires most of the polypeptide chains for enzyme activity except the first 15 residues of N-terminal despite of N- and C-terminal residues of the enzyme consist of variable regions.

Amino Acid Motifs↗
PubMed1983

Multiple forms of tubulin in Polytomella and Chlamydomonas: evidence for a precursor of flagellar alpha-tubulin.

The quadriflagellate alga polytomella agilis contains several alpha-tubulins with distinct isoelectric points (McKeithan, T.W., and J.L. Rosenbaum, 1981, J. Cell Biol., 91:352-360). While alpha-3 is the major component in flagella, alpha-1 predominates in cytoskeletal microtubules. For determination of whether the differences in alpha- tubulins are due to distinct genes or to posttranslational modification of a common alpha-tubulin precursor, poly A+ RNA was isolated from deflagellated and control (nonregenerating) cells and translated in vitro. Approximately twice as much alpha-1 was synthesized using RNA from deflagellated as compared to control cells; however, there was no detectable synthesis in vitro of alpha-3 in either. These results suggest that alpha -3 tubulin is formed in vivo by posttranslational modification of a form co- migrating with, and possibly identical to, cytoskeletal alpha-tubulin. In the related alga chlamydomonas reinhardii deflagellation greatly stimulates synthesis of tubulin and tubulin mRNA. As in polytomella, the principal alpha-tubulin synthesized both in vivo and in vitro following deflagellation in chlamydomonas is more basic than the major alpha-tubulin and appears to correspond to alpha-1 tubulin in polytomella. The conversion of alpha-1 to alpha-3 receives additional support from in vivo labeling and pulse-chase experiments. In addition, in both polytomella and chlamydomonas some conversion of alpha-1 to alpha-3 appears to occur even when protein synthesis is inhibited.

Chlamydomonas↗
PubMed1998

SPIN, a substrate in the MAP kinase pathway in mouse oocytes.

The newly cloned gene Spin encodes a 30-kDa protein, a well-defined abundant molecule found in mouse oocytes and early embryos. This protein SPIN undergoes metaphase-specific phosphorylation and binds to the spindle. To understand the role of SPIN in oocyte meiosis, oocytes were treated with drugs that affect the cell cycle by activating or inactivating specific kinases. The posttranslational modification of SPIN in the treated oocytes was then investigated by one- and two-dimensional gel electrophoresis. Modification of SPIN is inhibited by treatment with 6-dimethylaminopurine (DMAP), suggesting that SPIN is phosphorylated by a serine-threonine kinase. Furthermore, SPIN from cycloheximide-treated oocytes that lack detectable MAP kinase activity is only partially phosphorylated, indicating that SPIN may be phosphorylated by the MOS/MAP kinase pathway. To confirm this observation, SPIN was analyzed in Mos-null mutant mice lacking MAP kinase activity. Normal posttranslational modification of SPIN did not occur in Mos-null mutant oocytes. In addition, there is reduced association of SPIN with the metaphase I spindle in Mos-null mutant oocytes, as determined by immunohistochemical analysis. These findings suggest that SPIN is a substrate in the MOS/ MAP kinase pathway and further that this phosphorylation of SPIN may be essential for its interaction with the spindle.

Adenine↗
PubMed2002

Identification of modified proteins by mass spectrometry.

Because it is obvious that high-throughput genomics do not lead to a molecular description or even a prediction of protein function, modern techniques for protein analysis become increasingly more important. Sequence analysis of proteins and peptides is not limited to the elucidation of the primary structure of a protein. The analysis of posttranslational modifications is an important task of protein chemistry in proteome research. Increased sensitivity in mass spectrometry as a result of more efficient ionization techniques and better detection systems has allowed the stepwise reduction of protein quantity for analysis. Protein spots of 2D-PAGE separated samples are now sufficient for an unequivocal identification of a protein by mass spectrometry. In addition to protein identification, a closer look at posttranslational modifications is now also possible. It is assumed that modifications such as phosphorylation or glycosylation exist on every second protein and that they are important for the protein function.

Algorithms↗
PubMed2002

Altered post-translational modification of redox factor 1 protein in human uterine smooth muscle tumors.

Uterine leiomyomas are the most common benign smooth muscle tumors in the myometrium. The expression of redox factor 1 (Ref-1), a DNA repair enzyme and redox-modifying factor, was studied in the myometrium and uterine smooth muscle tumors to investigate the relevance of Ref-1 in the growth regulation of the tumors. Two forms of Ref-1 protein were detected, using three antibodies against different epitopes of Ref-1. The abundance of the large form of Ref-1 was increased in leiomyoma extracts relative to myometrial tissue extracts, and the large form was dominant in cell lines derived from leiomyosarcomas. A single mRNA transcript was detected in the same samples, leading us to hypothesize that the differentially migrating forms are the result of posttranslational modification(s). In vitro incubation of leiomyoma tissue extract lead to a shift from the large form to the small form, and this conversion was inhibited by either protease or phosphatase inhibitors. Finally, the relative abundance of the large form of Ref-1 was found to correlate with proliferating cell nuclear antigen levels, suggesting a correlation with increased proliferation. These results indicate that altered posttranslational modification of Ref-1 is involved in uterine smooth muscle tumorigenesis.

Adult↗