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Results for “post translational modification”

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PubMed1997

Tubulin post-translational modifications--enzymes and their mechanisms of action.

This review describes the enzymes responsible for the post-translational modifications of tubulin, including detyrosination/tyrosination, acetylation/deacetylation, phosphorylation, polyglutamylation, polyglycylation and the generation of non-tyrosinatable alpha-tubulin. Tubulin tyrosine-ligase, which reattaches tyrosine to detyrosinated tubulin, has been extensively characterized and its gene sequenced. Enzymes such as tubulin-specific carboxypeptidase and alpha-tubulin acetyltransferase, required, respectively, for detyrosination and acetylation of tubulin, have yet to be purified to homogeneity and examined in defined systems. This has produced some conflicting results, especially for the carboxypeptidase. The phosphorylation of tubulin by several different types of kinases has been studied in detail but drawing conclusions is difficult because many of these enzymes modify proteins other than their actual substrates, an especially pertinent consideration for in vitro experiments. Tubulin phosphorylation in cultured neuronal cells has proven to be the best model for evaluation of kinase effects on tubulin/microtubule function. There is little information on the enzymes required for polyglutamylation, polyglycylation, and production of non-tyrosinatable tubulin, but the available data permit interesting speculation of a mechanistic nature. Clearly, to achieve a full appreciation of tubulin post-translational changes the responsible enzymes must be characterized. Knowing when the enzymes are active in cells, if soluble or polymerized tubulin is the preferred substrate and the amino acid residues modified by each enzyme are all important. Moreover, acquisition of purified enzymes will lead to cloning and sequencing of their genes. With this information, one can manipulate cell genomes in order to either modify key enzymes or change their relative amounts, and perhaps reveal the physiological significance of tubulin post-translational modifications.

Acetyltransferases↗

PubMed2006

Characterization of histones and their post-translational modifications by mass spectrometry.

Histone proteins and their accompanying post-translational modifications have received much attention for their ability to affect chromatin structure and, hence, regulate gene expression. Recently, mass spectrometry has become an important complementary tool for the analysis of histone variants and modification sites, for determining the degree of occupancy of these modifications and for quantifying differential expression of these modifications from various samples. Additionally, as advancements in mass spectrometry technologies continue, the ability to read entire 'histone codes' across large regions of histone polypeptides or intact protein is possible. As chromatin biology demands, mass spectrometry has adapted and continues as a key technology for the analysis of gene regulation networks involving histone modifications.

Amino Acid Sequence↗

PubMed2004

Application of mass spectrometry to the identification and quantification of histone post-translational modifications.

The core histones are the primary protein component of chromatin, which is responsible for the packaging of eukaryotic DNA. The NH(2)-terminal tail domains of the core histones are the sites of numerous post-translational modifications that have been shown to play an important role in the regulation of chromatin structure. In this study, we discuss the recent application of modern analytical techniques to the study of histone modifications. Through the use of mass spectrometry, a large number of new sites of histone modification have been identified, many of which reside outside of the NH(2)-terminal tail domains. In addition, techniques have been developed that allow mass spectrometry to be effective for the quantitation of histone post-translational modifications. Hence, the use of mass spectrometry promises to dramatically alter our view of histone post-translational modifications.

Animals↗

PubMed2005

Quantitative analysis of both protein expression and serine / threonine post-translational modifications through stable isotope labeling with dithiothreitol.

While phosphorylation and O-GlcNAc (cytoplasmic and nuclear glycosylation) are linked to normal and pathological changes in cell states, these post-translational modifications have been difficult to analyze in proteomic studies. We describe advances in beta-elimination / Michael addition-based approaches which allow for mass spectrometry-based identification and comparative quantification of O-phosphate or O-GlcNAc-modified peptides, as well as cysteine-containing peptides for expression analysis. The method (BEMAD) involves differential isotopic labeling through Michael addition with normal dithiothreitol (DTT) (d0) or deuterated DTT (d6), and enrichment of these peptides by thiol chromatography. BEMAD was comparable to isotope-coded affinity tags (ICAT; a commercially available differential isotopic quantification technique) in protein expression analysis, but also provided the identity and relative amounts of both O-phosphorylation and O-GlcNAc modification sites. Specificity of O-phosphate vs. O-GlcNAc mapping is achieved through coupling enzymatic dephosphorylation or O-GlcNAc hydrolysis with differential isotopic labeling. Blocking of cysteine labeling by prior oxidation of a cytosolic lysate from mouse brain allowed specific targeting of serine / threonine post-translational modifications as demonstrated through identification of 21 phosphorylation sites (5 previously reported) in a single mass spectrometry analysis. These results demonstate BEMAD is suitable for large-scale quantitative analysis of both protein expression and serine / threonine post-translational modifications.

Animals↗

PubMed1995

Isolation, identification and functional significance of [Hyp2]Met-callatostatin and des Gly-Pro Met-callatostatin, two further post-translational modifications of the blowfly neuropeptide Met-callatostatin.

Two post-translationally modified neuropeptides of the Met-callatostatin (Gly-Pro-Pro-Tyr-Asp-Phe-Gly-Met-NH2) family have been identified from head extracts of the blowfly Calliphora vomitoria. They are the octapeptide, [Hyp2]Met-callatostatin, (Gly-Hyp-Pro-Tyr-Asp-Phe-Gly-Met-NH2) and the truncated hexapeptide, des Gly-Pro Met-callatostatin (Pro-Tyr-Asp-Phe-Gly-Met-NH2). The existence of the [Hyp2]Met-callatostatin variant, in addition to the previously identified [Hyp3]Met-callatostatin peptide, suggests that the motif for prolyl hydroxylation in C. vomitoria is more variable than those known from mammalian and other invertebrate studies where, in those regulatory peptides containing a pair of adjacent prolyl residues so far studied, e.g., bradykinin, and the mosquito peptide Aea HP-I, only one of the pair (the second) is known to undergo hydroxylation. The truncated hexapeptide, des Gly-Pro Met-callatostatin could be produced as a result of the action of a dipeptidyl peptidase II type of enzyme which is known from mammalian studies to be unique in its ability to cleave between the two prolyl residues of an Xaa-Pro-Pro- sequence, where Xaa is any unprotected NH2-terminal amino acid. This enzyme is, however, considered unlikely to be able to cleave the Gly-Hyp-Pro-sequence, which would suggest a functional significance for such a post-translational modification. For this reason, it is of interest that [Hyp2]Met-callatostatin (and earlier, [Hyp3]Met-callatostatin) have been shown to be potent inhibitors of the spontaneous contractions of the hindgut of C. vomitoria (biphasic dose-response curve with IC50 values of 10(-14) M and 10(-7) M).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed2004

Post-translational modifications of proteins: implications for aging, antigen recognition, and autoimmunity.

Proteins are complex organic molecules susceptible to numerous post-translational modifications occurring spontaneously during aging or as a consequence of physiologic or pathologic processes. Antigenicity and interactions of proteins with components of the immune system may be profoundly affected by post-translational modifications. Thus, modified self-antigens may be absent (not-tolerated) during early T-cell selection and trigger reactions by the immune system as they arise later in life. In turn, this may play a role in the initiation and pathogenesis of autoimmune diseases. This Review article presents an overview of protein modifications that have been shown to affect antigenicity and presentation of protein antigens in autoimmune diseases. The relevance of these observations is discussed, and the implications for future prophylactic and therapeutic interventions are outlined.

Aging↗

PubMed2000

Differences in the post-translational modifications of human papillomavirus type 6b major capsid protein expressed from a baculovirus system compared with a vaccinia virus system.

Virus-like particles (VLPs) are being currently investigated in vaccines against viral infections in humans. There are different recombinant-protein-expression systems available for obtaining the necessary VLP preparation for vaccination. However, the differences in post-translational modifications of the recombinant proteins obtained and their differences in efficacy in eliciting an anti-viral response in vaccines are not well established. In this study we have compared the post-translational modifications of human papillomavirus type-6b major capsid protein L1 (HPV 6bL1) expressed using recombinant baculovirus (rBV) in Sf9 (Spodoptera frugiperda) insect cells, with the protein expressed using recombinant vaccinia virus (rVV) in CV-1 kidney epithelial cells. Two-dimensional gel electrophoresis of biosynthetically labelled rBV-expressed HPV 6bL1 showed several post-translationally modified variants of the protein, whereas rVV-expressed HPV 6bL1 showed only a few variants. Phosphorylations were detected at threonine and serine residues for the L1 expressed from rBV compared with phosphorylation at serine residues only for the L1 expressed from rVV. HPV 6bL1 expressed using rBV incorporated [(3)H]mannose and [(3)H]galactose, whereas HPV 6bL1 expressed using rVV incorporated only [(3)H]galactose. We conclude that post-translational modification of recombinant HPV 6bL1 can differ according to the system used for its expression. Since recombinant L1 protein is a potential human-vaccine candidate, the implication of the observed differences in post-translational modifications on immunogenicity of L1 VLPs warrants investigation.

Animals↗

PubMed1998

Post-translational modifications of beta subunits of voltage-dependent calcium channels.

Different post-translational modifications of Ca channel beta subunits have been identified. Recent studies have characterized the palmitoylation of the Ca channel beta2a subunit, as well as one effect of this modification on channel function. The potential importance of palmitoylation on other channel properties is discussed. Other studies have addressed the role of phosphorylation of beta subunits in the regulation of voltage-dependent Ca channels. Phosphorylation of beta subunits by second messenger-activated protein kinases, as well as by unidentified protein kinases, may affect interactions between channel subunits and other aspects of channel function. The differential modification of Ca channel beta subunit isoforms by post-translational events likely results in diversely regulated channels with unique properties.

Animals↗

PubMed2003

The characterization of protein post-translational modifications by mass spectrometry.

Most biological processes are regulated by post-translational modifications of proteins, and conditions that disrupt the regulation of such events can lead to disease. In the past decade, the identification and characterization of covalent modifications have been driven by advances in mass spectrometry. Here, we discuss current mass spectrometric and proteomic approaches for the identification of proteins and their covalent modifications, and we highlight high-throughput strategies for comprehensive analysis of cell proteomes.

Amino Acid Sequence↗

PubMed1983

Mechanism for the regulation of post-translational modifications of procollagens synthesized by matrix-free cells from chick embryos.

Three possible mechanisms are considered to account for the variations of post-translational modifications in different collagen types. 1) The cells have different amounts of post-translational modifying enzymes, 2) the rate of prolylhydroxylation of different procollagen types is varied, and 3) the rate of chain association of pro-alpha chains of different collagen types is modulated. In an attempt to examine the three possibilities, we have determined the activities of prolyl hydroxylase and lysyl hydroxylase, and we have examined the kinetics of the secretion of procollagens and the kinetics of pro-gamma chain formation of different procollagen types in matrix-free cells isolated from tissues of 17-day-old chick embryos. Type II collagen synthesized by cartilage cells contains more hydroxylysine than type I collagen synthesized by tendon and cornea cells. It was found, however, that cartilage cells contain significantly less lysyl hydroxylase than tendon and cornea cells. In contrast, we found only a small difference in the amount of prolyl hydroxylase in tendon, cornea, and cartilage cells. The secretion of type I procollagen by tendon and cornea cells can be described by two first order processes. In contrast, the secretion of type II procollagen by cartilage cells, type IV procollagen by lens cells, and type V procollagen by cornea cells can be described by single first order processes. Examination of the formation of pro-gamma components of procollagen types I and II revealed that it occurs via intermediate dimers of two pro-alpha chains. The formation or pro-gamma(I) chains in tendon and cornea cells is about three times faster than the formation of pro-gamma(II) chains in cartilage cells. These results are consistent with the hypothesis that the rate of association of pro-alpha chains regulates the synthesis of procollagens with different degrees of post-translational modifications.

Animals↗

PubMed1996

Tubulin post-translational modifications in the primitive protist Trichomonas vaginalis.

Using several specific monoclonal antibodies, we investigated the occurrence and distribution of different post-translationally modified tubulin during interphase and division of the primitive flagellated protist Trichomonas vaginalis. Immunoblotting and immunofluorescence experiments revealed that interphasic microtubular structures of T. vaginalis contained acetylated and glutamylated but non-tyrosinated and non-glycylated [Brugerolle and Adoutte, 1988: Bio Systems 21: 255-268] tubulin. Immunofluorescence studies performed on dividing cells showed that the extranuclear mitotic spindle (or paradesmosis) was acetylated and glutamylated, which contrast with the ephemeral nature of this structure. Newly formed short axostyles also contained acetylated and glutamylated tubulin suggesting that both post-translational modifications might take place very early after assembly of microtubular structures. Our results indicate that acetylation and glutamylation of tubulin appeared early in the history of eukaryotes and could reflect the occurrence of post-translational modifications of tubulin in the primitive eukaryotic cells. These cells probably had a highly ordered cross-linked microtubular cytoskeleton in which microtubules showed a low level of subunit exchange dynamics.

Acetylation↗

PubMed2001

Post-translational modifications of proteins: acetylcholinesterase as a model system.

Analysis of the expressed protein complement of cells requires knowledge of the diversity of post-translational modifications that can occur and which can be transient or permanent. The modifications range from amino acid changes through to the addition of macromolecules: lipid, carbohydrate or protein. Many variants of the common amino acids can occur, which can affect the structure or function of the protein. The major class of modification, however, is represented by glycosylation, N-linked, O-linked, or glycosylphosphatidylinositol(GPI)-linked. Such modifications have roles in protein stability and folding, targeting and recognition. Glycosylated proteins can be found in all cellular compartments and, intracellularly, O-GlcNAc modification is commonplace. Lipid modification of proteins (acylation, prenylation, GPI-anchoring) is also common, resulting in membrane association, and can play an important role in cell signalling. Targeting and turnover of proteins can also be mediated via covalent protein addition, for example by members of the ubiquitin family. Limited proteolysis as a post-translational modification will be discussed, focusing on the family of membrane protein secretases, in particular in relation to the Alzheimer's amyloid precursor protein. Finally, acetylcholinesterase will be used as a model example to illustrate the diversity of modifications occurring on a single protein.

Acetylcholinesterase↗

PubMed2006

dbPTM: an information repository of protein post-translational modification.

dbPTM is a database that compiles information on protein post-translational modifications (PTMs), such as the catalytic sites, solvent accessibility of amino acid residues, protein secondary and tertiary structures, protein domains and protein variations. The database includes all of the experimentally validated PTM sites from Swiss-Prot, PhosphoELM and O-GLYCBASE. Only a small fraction of Swiss-Prot proteins are annotated with experimentally verified PTM. Although the Swiss-Prot provides rich information about the PTM, other structural properties and functional information of proteins are also essential for elucidating protein mechanisms. The dbPTM systematically identifies three major types of protein PTM (phosphorylation, glycosylation and sulfation) sites against Swiss-Prot proteins by refining our previously developed prediction tool, KinasePhos (http://kinasephos.mbc.nctu.edu.tw/). Solvent accessibility and secondary structure of residues are also computationally predicted and are mapped to the PTM sites. The resource is now freely available at http://dbPTM.mbc.nctu.edu.tw/.

Amino Acids↗

PubMedDate not supplied

Post-translational modifications of immunoglobulin G: a mouse IgG variant that lacks the entire CH1 domain.

In the present study, we characterized the post-translational modifications of a short-chain variant of mouse IgG2a that lacks the entire CH 1 domain. The short-chain IgG2a and its proteolytic fragments were subjected to electrospray ionization- and fast atom bombardment-mass spectrometric analyses. It has been demonstrated that approximately 14% of the heavy chain of the short-chain IgG2a is O-glycosylated with a disaccharide of Ga1-GalNAc- at Thr220A in the hinge region. while the Oglycosylation does not occur in its parent IgG2a molecule. Two additional modifications have been detected at the C-termini of both the heavy and light chains of the short-chain IgG2a. Biological significance of the post-translational modifications of the short-chain IgG2a variant is briefly discussed.

Alkylation↗

PubMed1996

Studies on [3H]palmitate-binding proteins of rat spermatozoa: a post-translational modification of membrane proteins by fatty acid acylation.

The purpose of the present study was to demonstrate the post-translational modifications of sperm plasma membrane proteins by fatty acid acylation during sperm maturation in the epididymis. Rat epididymal spermatozoa were incubated at 37 degrees C with various concentrations (100 microCi and 1 mCi) of [9-10(n)3H]palmitic acid in a medium containing Tyrode's solution supplemented with sodium bicarbonate, sodium pyruvate and sodium lactate. The incorporation of [3H]palmitate in vitro was determined in epididymal spermatozoa and an attempt was made to identify the lipid-linked proteins of purified plasma membranes of maturing epididymal spermatozoa by autoradiography. The studies demonstrated that [3H]palmitate was covalently linked to a subset of membrane cytoskeleton proteins of maturing rat spermatozoa. The pattern of incorporation of lipid was a maturation-associated phenomenon as caput spermatozoa incorporated more radioactivity than did caudal spermatozoa. The labelled proteins appeared to be membrane-bound since 82% of radioactivity was associated with membrane fractions. Autoradiograms of SDS-PAGE gels of labelled caput sperm extract showed three prominent palmitate-incorporating protein bands of about 70, 56 and 36 kDa and few minor bands. Most of these proteins were present in the membrane fraction of caput spermatozoa. Labelled gels of both the sperm extracts and of purified membranes showed resistance to hydroxylamine treatment, suggesting that there are amide bonds between lipid and proteins. The higher incorporation of labelled palmitate by immature spermatozoa of the caput epididymis compared with mature spermatozoa from the cauda epididymis and the addition of palmitate to plasma membrane proteins of caput epididymal spermatozoa suggest that fatty acylation is a post-translational modification of sperm membrane proteins.

Acylation↗

PubMed2004

Prediction of sequence signals for lipid post-translational modifications: insights from case studies.

In silico annotation techniques for post-translational modifications (PTMs) are important to generate biologically meaningful descriptions for sequences of experimentally uncharacterized proteins. Having previously contributed with predictors for lipid PTMs, we summarize our methodological experience. Rather than only looking for the sequence pattern in substrate sequences, a strategy aimed at creating a generalized model of substrate protein/enzyme interaction appears more appropriate since the number of known substrate sequences is small, and some of them are not sufficiently verified experimentally. Such a physical approach (in contrast to a mere textual analysis of substrate sequences) can also take into account other, heterogeneous biological data (mutations of substrate sequences, kinetic data, enzyme sequences/structures) with simple analytical expressions in the score function. Several lipid PTMs are encoded in the form of a small sequence region (with pronounced amino acid type preferences) that is connected to the substrate protein by a linker region with many conformationally flexible, hydrophilic residues. A score function composed of terms penalizing sequence properties known to be incompatible with productive substrate protein/enzyme complexes essentially unselects inappropriate queries. Also, we estimate the number of nonredundant sequences necessary for robust profile computation with statistical criteria, a number that is not reached in most cases of PTM prediction. Finally, we discuss the usage of evolutionary information in evaluating the functional importance of predicted PTMs in cases of motif conservation within sequence families.

Acetyltransferases↗

PubMed1988

Biosynthesis of placental alkaline phosphatase and its post-translational modification by glycophospholipid for membrane-anchoring.

The biosynthesis and post-translational modification of placental alkaline phosphatase were studied in human choriocarcinoma cells, JEG-3. Pulse-chase experiments with [35S]methionine demonstrated that placental alkaline phosphatase was synthesized as a major precursor form with Mr 63,000, which was then converted to a mature form with Mr 66,000, by processing of its N-linked oligosaccharides from the high-mannose type to the complex type. In addition, the two forms of the protein were found to be modified by a glycophospholipid, components of which were characterized by metabolic incorporation into placental alkaline phosphatase of 3H-labeled compounds such as myo-inositol, palmitic acid, stearic acid, mannose, glucosamine, and ethanolamine. When placental alkaline phosphatase labeled with these compounds was treated with phosphatidylinositol-specific phospholipase C or papain, the phospholipase C removed only the 3H-labeled fatty acids, whereas papain, that is known to cleave the C-terminal region, released all the radioactive glycolipid components including [3H]ethanolamine. More detailed analysis with shorter pulse-chase experiments demonstrated that placental alkaline phosphatase was primarily synthesized as a form with Mr 64,500 which was not yet labeled with [3H]palmitic acid. This form was converted by papain digestion to the above-mentioned major precursor with Mr 63,000. Taken together, these results suggest that placental alkaline phosphatase is initially synthesized as the precursor with Mr 64,500, which is immediately converted to the intermediate form with Mr 63,000 by simultaneously occurring proteolysis of the C terminus and replacement by the glycophospholipid, and finally to the mature form with Mr 66,000 by terminal glycosylation of its N-linked oligosaccharides. The glycophospholipid thus attached is considered to function as the membrane-anchoring domain of placental alkaline phosphatase.

Alkaline Phosphatase↗

PubMed2004

Antibodies immobilized as arrays to profile protein post-translational modifications in mammalian cells.

Previously, we demonstrated that antibodies printed on a solid support were able to detect protein-protein interaction in mammalian cells. Here we further developed the antibody array system for detecting proteins with various post-translational modifications in mammalian cells. In this novel approach, immunoprecipitated proteins were labeled with fluorescent dye followed by incubation over antibody arrays. Targeted proteins, captured by the antibodies immobilized on PVDF membrane or glass slide, were detected by means of near infrared fluorescent scanner or fluorescent microscopy. To demonstrate the application of the antibody arrays in protein post-translational modifications, we profiled protein tyrosine phosphorylation, ubiquitination, and acetylation in mammalian cells under different conditions. Our results indicate that antibody array technology can provide a powerful means of profiling a large number of proteins with different post-translational modifications in cells.

3T3 Cells↗