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PubMed2006

Posttranslational phosphorylation of mutant p53 protein in tumor development.

p53 has been called the "cellular gatekeeper" and the "genome guard," because in response to exposure to DNA-damaging agents, it induces cell-cycle arrest in G1 or apoptosis and also directly affects DNA replication. Multiple mechanisms regulate p53 activity and posttranslational modification, including multisite phosphorylation of wild-type p53, in particular. Normal functions of wild-type p53 are abrogated by mutation of this gene, and oncogenic studies have revealed that p53 mutation is among the most common genetic alteration in human cancers. It is generally accepted that mutant p53 protein may not only lose the tumor suppressor functions of wild-type p53 but also acquire additional tumorigenetic roles, including dominant-negative effects and gain of function. Although many studies have revealed such aberrant functions of mutant p53, less is known about the posttranslational phosphorylation status of mutant p53 and novel biological functions of phosphorylation in carcinogenesis.

Humans↗

PubMed1988

Regulation of glucocorticoid receptor expression: evidence for transcriptional and posttranslational mechanisms.

The mechanism of ligand-induced (homologous) down-regulation of the glucocorticoid receptor (GR) has been studied. Dexamethasone caused a down-regulation of the levels of GR mRNA and protein both in hepatoma tissue culture cells and rat liver in vivo. The decrease in the level of rat liver GR mRNA was due to a reduced transcription rate of the GR gene, as assessed by nuclear run-on transcription experiments. The half-life of GR mRNA in hepatoma tissue culture cells was determined to be approximately 4.5 h and was unaffected by dexamethasone. In addition to the transcriptional regulation of GR gene expression, a dexamethasone-dependent posttranslational modification in the rate of GR protein turnover was observed. In the absence of dexamethasone, GR protein half life was approximately 25 h whereas it decreased to approximately 11 h in the presence of hormone. Down-regulation of GR protein occurred with a 6- to 24-h delay as compared to the decline in GR mRNA. This is most likely due to the differences in half-lives of GR mRNA and protein, respectively. Our results suggest that auto-regulation of GR by its cognate ligand is complex and occurs at both transcriptional and posttranslational levels.

Animals↗

PubMed2003

Molecular modifications of MC31/CE9, a sperm surface molecule, during sperm capacitation and the acrosome reaction in the rat: is MC31/CE9 required for fertilization?

We examined the modification of the MC31 molecule during capacitation, the acrosome reaction, and studied its role in fertilization. These studies revealed that the molecular mass of MC31 in cauda spermatozoa was approximately 28,000-26,000 Dalton (28-26 kDa). A limited change in molecular mass was seen in capacitated spermatozoa. Treatment of sperm extracts with peptide-N-glycosidase (PN glycosidase) reduced the molecular mass of MC31 in both cauda and capacitated spermatozoa from 28-26 kDa to 23-20 kDa, suggesting that MC31 from both cauda and capacitated spermatozoa is glycosylated, and indicating that capacitation induces minor posttranslational modifications in the structure of the MC31 antigen. The MC31 antigen was redistributed from the midpiece of cauda epididymal spermatozoa to the head and equatorial segment after capacitation and acrosome reaction, respectively, when traced by indirect immunofluorescence under in vitro fertilization (IVF) conditions. Some spermatozoa did not stain for the MC31 antigen and might represent spermatozoa that have shed the antigen. IVF experiments conducted to assess the effect of an anti-MC31 monoclonal antibody (mMC31) revealed that this antibody significantly (P < 0.01) inhibited fertilization of cumulus-invested zona pellucida-intact and the zona pellucida-free oocytes in a dose-dependent manner. However, sperm-oolemma binding was not affected. These findings suggest the MC31 antigen facilitates sperm-oocyte interactions.

Acrosome Reaction↗

PubMed2003

Enhancement of chaperone function of alpha-crystallin by methylglyoxal modification.

The molecular chaperone function of alpha-crystallin in the lens prevents the aggregation and insolubilization of lens proteins that occur during the process of aging. We found that chemical modification of alpha-crystallin by a physiological alpha-dicarbonyl compound, methylglyoxal (MG), enhances its chaperone function. Protein-modifying sugars and ascorbate have no such effect and actually reduce chaperone function. Chaperone assay after immunoprecipitation or with immunoaffinity-purified argpyrimidine-alpha-crystallin indicates that 50-60% of the increased chaperone function is due to argpyrimidine-modified protein. Incubation of alpha-crystallin with DL-glyceraldehyde and arginine-modifying agents also enhances chaperone function, and we believe that the increased chaperone activity depends on the extent of arginine modification. Far- and near-UV circular dichroism spectra indicate modest changes in secondary and tertiary structure of MG-modified alpha-crystallin. LC MS/MS analysis of MG-modified alpha-crystallin following chymotryptic digestion revealed that R21, R49, and R103 in alphaA-crystallin were converted to argpyrimidine. 1,1'-Bis(4-anilino)naphthalene-5,5'-disulfonic acid binding, an indicator of hydrophobicity of proteins, increased in alpha-crystallin modified by low concentrations of MG (2-100 microM). MG similarly enhances chaperone function of another small heat shock protein, Hsp27. Our results show that posttranslational modification by a metabolic product can enhance the chaperone function of alpha-crystallin and Hsp27 and suggest that such modification may be a protective mechanism against environmental and metabolic stresses. Augmentation of the chaperone function of alpha-crystallin might have evolved to protect the lens from deleterious protein modifications associated with aging.

Alcohol Dehydrogenase↗

PubMed2003

A chemical approach for identifying O-GlcNAc-modified proteins in cells.

The glycosylation of serine and threonine residues with a single GlcNAc moiety is a dynamic posttranslational modification of many nuclear and cytoplasmic proteins. We describe a chemical strategy directed toward identifying O-GlcNAc-modified proteins from living cells or proteins modified in vitro. We demonstrate, in vitro, that each enzyme in the hexosamine salvage pathway, and the enzymes that affect this dynamic modification (UDP-GlcNAc:polypeptidtyltransferase and O-GlcNAcase), tolerate analogues of their natural substrates in which the N-acyl side chain has been modified to bear a bio-orthogonal azide moiety. Accordingly, treatment of cells with N-azidoacetylglucosamine results in the metabolic incorporation of the azido sugar into nuclear and cytoplasmic proteins. These O-azidoacetylglucosamine-modified proteins can be covalently derivatized with various biochemical probes at the site of protein glycosylation by using the Staudinger ligation. The approach was validated by metabolic labeling of nuclear pore protein p62, which is known to be posttranslationally modified with O-GlcNAc. This strategy will prove useful for both the identification of O-GlcNAc-modified proteins and the elucidation of the specific residues that bear this saccharide.

Acetylglucosamine↗

PubMed1990

Peroxisome proliferator-induced alterations in the expression and modification of rat hepatocyte plasma membrane proteins.

Rats were fed the peroxisome proliferator ciprofibrate (0.025%), and the effects on the expression, modification, and localization of seven domain-specific integral proteins of the rat hepatocyte plasma membrane were assessed using a combination of immunoblotting, -precipitation, and -fluorescence. Ciprofibrate caused the down-regulation of five of the plasma membrane proteins (the epidermal growth factor receptor, the asialoglycoprotein receptor, HA 321, HA 4, and dipeptidylpeptidase IV) and induced the expression of a more basic, lower-Mr isoform of the basolateral plasma membrane protein CE 9. Pulse labeling, chemical deglycosylation, and 125I-wheat germ lectin blotting suggested that the ciprofibrate-induced isoform of CE 9 differed in the posttranslational modification of its oligosaccharides and contained more sialic acid. These changes in hepatocyte surface differentiation were first observed between Days 1 and 5 on the ciprofibrate-containing diet, coincident with other aspects of the pleiotropic response of the hepatocyte to peroxisome proliferators, e.g., the induction of the Mr 78,000 peroxisome proliferation-associated protein. The effects were reversed within 2-3 weeks upon removal of ciprofibrate. The three other peroxisome proliferators tested, di(2-ethylhexyl)phthalate, clofibrate, and Wy-14,643, were found to exert most of these same effects on the expression and modification of the hepatocyte plasma membrane proteins, but the compounds differed in relative potency. The ciprofibrate-induced decreases in the concentrations of the epidermal growth factor receptor, the asialoglycoprotein receptor, HA 321, and HA 4 were similar to the selective down-regulation of these proteins observed transiently during the period of hepatocyte proliferation following two-thirds hepatectomy. Other compounds frequently used in studies of liver enzyme induction and carcinogenesis, the antioxidants ethoxyquin and butylated hydroxyanisole and the liver tumor promoter phenobarbital, were not as effective as ciprofibrate or two-thirds hepatectomy at causing the down-regulation of these proteins. The induction of the lower-Mr isoform of the basolateral plasma membrane protein CE 9 was not observed following two-thirds hepatectomy or upon the feeding of the antioxidants or phenobarbital but was specific to the feeding of the peroxisome proliferators.

Antibodies, Monoclonal↗

PubMed1999

Incorporation of mouse zona pellucida proteins into the envelope of Xenopus laevis oocytes.

All vertebrate eggs have extracellular matrices, referred to as the zona pellucida in Mus musculus and the vitelline envelope in Xenopus laevis. The mouse zona, composed of three sulfated glycoproteins (ZP1, ZP2, ZP3), is critical for fertilization and early development, and mice lacking a zona pellucida produce no live offspring. The primary structures of mouse ZP1 (623 amino acids), ZP2 (713 amino acids) and ZP3 (424 amino acids) have been deduced from full-length cDNAs, but posttranslational modifications result in mature zona proteins with molecular masses of 200-180 kDa, 140-120 kDa, and 83 kDa, respectively. The vitelline envelope forms a similar structure around Xenopus eggs and contains three glycoproteins that are structurally related (39-48% amino acid similarity) to the three mouse zona proteins. To investigate whether the structural semblances are sufficient to allow incorporation of the mouse zona proteins into the Xenopus vitelline envelope, capped synthetic mRNAs encoding ZP1, ZP2, and ZP3 proteins were injected into the cytoplasm of stage VI Xenopus oocytes. After 20 h of incubation the oocytes were harvested, and posttranslationally modified zona proteins were detected with monoclonal antibodies specific to mouse ZP1, ZP2, and ZP3. The oocytes were imaged with confocal microscopy to detect individual zona proteins in the extracellular matrix of the oocytes, and this localization was confirmed biochemically. Thus the mouse zona proteins appear to have been sufficiently conserved through 350 million years of evolution to be incorporated into the extracellular envelope surrounding Xenopus eggs.

Animals↗

PubMed2001

Role of glycopeptide-specific T cells in collagen-induced arthritis: an example how post-translational modification of proteins may be involved in autoimmune disease.

Immunization of mice with type II collagen (CII), a cartilage-restricted protein, leads to collagen-induced arthritis (CIA), a model for rheumatoid arthritis (RA). CIA symptoms consist of an erosive joint inflammation caused by an autoimmune attack, mediated by both T and B lymphocytes. CD4+ alphabeta T cells play a central role in CIA, both by helping B cells to produce anti-CII antibodies, and by interacting with other cells in the joints, eg macrophages. In H-2q mice, most CII-specific CD4+ T cells recognize the CII(256-270) peptide presented on the major histocompatibility complex (MHC) class II Aq molecule. Post-translational modifications (hydroxylation and variable glycosylation) of the lysine residue at position 264 of CII generate at least four different T-cell determinants that are specifically recognized by distinct T-cell subsets. Most T cells recognize CII(256-270) glycosylated with the monosaccharide galactose, which is consequently immunodominant in CIA. Recent studies indicate that the arthritogenic T cells in CIA are glycopeptide-specific, suggesting that induction of self-tolerance may be rendered more difficult by glycosylation of CII. These data open the possibility that outoimmune disease may be caused by the creation of new epitopes by posttranslational modification of proteins under circumstances such as trauma, inflammation or ageing.

Animals↗

PubMed1997

Dynamic O-linked glycosylation of nuclear and cytoskeletal proteins.

Modification of Ser and Thr residues by attachment of O-linked N-acetylglucos-amine [Ser(Thr)-O-GlcNAcylation] to eukaryotic nuclear and cytosolic proteins is as dynamic and possibly as abundant as Ser(Thr) phosphorylation. Known O-GlcNAcylated proteins include cytoskeletal proteins and their regulatory proteins; viral proteins; nuclear-pore, heat-shock, tumor-suppressor, and nuclearoncogene proteins; RNA polymerase II catalytic subunit; and a multitude of transcription factors. Although functionally diverse, all of these proteins are also phosphoproteins. Most O-GlcNAcylated proteins form highly regulated multimeric associations that are dependent upon their posttranslational modifications. Evidence is mounting that O-GlcNAcylation is an important regulatory modification that may have a reciprocal relationship with O-phosphorylation and may modulate many biological processes in eukaryotes.

Animals↗

PubMed2002

Maturation of the lantibiotic subtilin: matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to monitor precursors and their proteolytic processing in crude bacterial cultures.

Bacillus subtilis synthesizes the lanthionine containing 32-amino-acid peptide antibiotic (lanti-biotic) subtilin from a ribosomally generated 56-amino-acid precursor pre-propeptide by extensive posttranslational modifications. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) was used to monitor the production of matured subtilin within crude samples taken from B. subtilis culture media without prior fractionation. The processing reaction of subtilin was blocked with the serine protease inhibitor phenylmethylsulfonyl fluoride and different subtilin precursor peptides in the molecular mass range up to 6220 were observed. Two of these species were isolated by reversed-phase high-performance liquid chromatography (HPLC) and structurally analyzed by post-source decay MALDI-TOFMS. We provide evidence that the precursor species comprise the posttranslational modified C-terminal part of subtilin to which leader peptide moieties with different chain lengths are attached. These antimicrobial-inactive species could be processed to antibiotic-active subtilin by incubation with culture media of different subtilin-nonproducing B. subtilis strains as indicated by a combination of antimicrobial growth assays and MALDI-TOFMS analyses. These achievements are strong evidence for the sensitivity of MALDI-TOFMS methodology that allows straightforward investigations of analytes even in complex mixtures without time-consuming sample preparations.

Amino Acid Sequence↗

PubMed1995

Rapid mass spectrometric peptide sequencing and mass matching for characterization of human melanoma proteins isolated by two-dimensional PAGE.

We report a general mass spectrometric approach for the rapid identification and characterization of proteins isolated by preparative two-dimensional polyacrylamide gel electrophoresis. This method possesses the inherent power to detect and structurally characterize covalent modifications. Absolute sensitivities of matrix-assisted laser desorption ionization and high-energy collision-induced dissociation tandem mass spectrometry are exploited to determine the mass and sequence of subpicomole sample quantities of tryptic peptides. These data permit mass matching and sequence homology searching of computerized peptide mass and protein sequence data bases for known proteins and design of oligonucleotide probes for cloning unknown proteins. We have identified 11 proteins in lysates of human A375 melanoma cells, including: alpha-enolase, cytokeratin, stathmin, protein disulfide isomerase, tropomyosin, Cu/Zn superoxide dismutase, nucleoside diphosphate kinase A, galaptin, and triosephosphate isomerase. We have characterized several posttranslational modifications and chemical modifications that may result from electrophoresis or subsequent sample processing steps. Detection of comigrating and covalently modified proteins illustrates the necessity of peptide sequencing and the advantages of tandem mass spectrometry to reliably and unambiguously establish the identity of each protein. This technology paves the way for studies of cell-type dependent gene expression and studies of large suites of cellular proteins with unprecedented speed and rigor to provide information complementary to the ongoing Human Genome Project.

Amino Acid Sequence↗

PubMed2006

Assays of protein palmitoylation.

Protein palmitoylation plays an important role in the structure and function of a wide array of proteins. Unlike other lipid modifications, protein palmitoylation is highly dynamic and cycles of palmitoylation and depalmitoylation can regulate protein function and localization. The dynamic nature of palmitoylation is poorly resolved because of limitations in assay methods. Here, we discuss various methods that can be used to measure protein palmitoylation and identify sites of palmitoylation. We describe new methodology based on "fatty acyl exchange labeling" in which palmitate is removed via hydroxylamine-mediated cleavage of the palmitoyl-thioester bond and then exchanged with a sulfhydryl-specific labeling compound. The techniques are highly sensitive and allow for quantitative estimates of palmitoylation. Unlike other techniques used to assay posttranslational modifications, the techniques we have developed can label all sites of modification with a variety of probes, radiolabeled or non-radioactive, and can be used to assay the palmitoylation of proteins from tissue samples.

Alkylation↗

PubMed2002

Sulfonation and molecular action.

The sulfonation of endogenous molecules is a pervasive biological phenomenon that is not always easily understood, and although it is increasingly recognized as a function of fundamental importance, there remain areas in which significant cognizance is still lacking or at most minimal. This is particularly true in the field of endocrinology, in which the sulfoconjugation of hormones is a widespread occurrence that is only partially, if at all, appreciated. In the realm of steroid/sterol sulfoconjugation, the discovery of a novel gene that utilizes an alternative exon 1 to encode for two sulfotransferase isoforms, one of which sulfonates cholesterol and the other pregnenolone, has been an important advance. This is significant because cholesterol sulfate plays a crucial role in physiological systems such as keratinocyte differentiation and development of the skin barrier, and pregnenolone sulfate is now acknowledged as an important neurosteroid. The sulfonation of thyroglobulin and thyroid hormones has been extensively investigated and, although this transformation is better understood, there remain areas of incomplete comprehension. The sulfonation of catecholamines is a prevalent modification that has been extensively studied but, unfortunately, remains poorly understood. The sulfonation of pituitary glycoprotein hormones, especially LH and TSH, does not affect binding to their cognate receptors; however, sulfonation does play an important role in their plasma clearance, which indirectly has a significant effect on biological activity. On the other hand, the sulfonation of distinct neuroendocrine peptides does have a profound influence on receptor binding and, thus, a direct effect on biological activity. The sulfonation of specific extracellular structures plays an essential role in the binding and signaling of a large family of extracellular growth factors. In summary, sulfonation is a ubiquitous posttranslational modification of hormones and extracellular components that can lead to dramatic structural changes in affected molecules, the biological significance of which is now beginning to be appreciated.

Amino Acid Sequence↗

PubMed1998

Alpha-galactosidase transgenic mouse: heterogeneous gene expression and posttranslational glycosylation in tissues.

We produced six transgenic mouse lines expressing human alpha-galactosidase (alpha-Gal) in order to evaluate its posttranslational modification. Among them, serum alpha-Gal activity increased 3000-fold in two transgenic mouse lines (TgN2 and TgN51), as compared to that in non-transgenic lines. The heart and liver of the TgN2 mouse expressed a high amount of transcript as well as high alpha-Gal activity. Its gene products in the heart and kidney were sensitive to endoglycosidase H digestion, but those in the spleen and liver were largely resistant. Glycopeptidase F treatment confirmed an identical molecular mass for the peptide moiety of the enzyme. We concluded that heterogeneous molecular mass of the gene products was caused by different degrees of posttranslational glycosylation in murine tissues.

Animals↗

PubMed2005

Insights into the role of histone H3 and histone H4 core modifiable residues in Saccharomyces cerevisiae.

The biological significance of recently described modifiable residues in the globular core of the bovine nucleosome remains elusive. We have mapped these modification sites onto the Saccharomyces cerevisiae histones and used a genetic approach to probe their potential roles both in heterochromatic regions of the genome and in the DNA repair response. By mutating these residues to mimic their modified and unmodified states, we have generated a total of 39 alleles affecting 14 residues in histones H3 and H4. Remarkably, despite the apparent evolutionary pressure to conserve these near-invariant histone amino acid sequences, the vast majority of mutant alleles are viable. However, a subset of these variant proteins elicit an effect on transcriptional silencing both at the ribosomal DNA locus and at telomeres, suggesting that posttranslational modification(s) at these sites regulates formation and/or maintenance of heterochromatin. Furthermore, we provide direct mass spectrometry evidence for the existence of histone H3 K56 acetylation in yeast. We also show that substitutions at histone H4 K91, K59, S47, and R92 and histone H3 K56 and K115 lead to hypersensitivity to DNA-damaging agents, linking the significance of the chemical identity of these modifiable residues to DNA metabolism. Finally, we allude to the possible molecular mechanisms underlying the effects of these modifications.

Alleles↗

PubMed2006

Protein processing and other modifications analyzed by diagonal peptide chromatography.

Diagonal peptide chromatography consists of two consecutive, identical peptide separations with in between an enzymatic or chemical alteration of the side-chain structure of selected peptides. Such selected and altered peptides acquire different chromatographic properties thereby segregating from non-altered peptides in a series of secondary peptide separations. Originally described by Brown and Hartley in 1966, we have modified the technique such that it can be used for higher throughput gel-free proteomics. Our technique is termed COmbined FRActional DIagonal Chromatography (COFRADIC) and exploits evoked differences of the hydrophobicity of peptides in reverse-phase liquid chromatography. One important advantage of COFRADIC is its versatility: by changing the alteration reaction, different classes of peptides are sorted and finally analyzed. We previously published protocols and applications for separating methionyl, cysteinyl, amino terminal and phosphorylated peptides. In this review, we assess the potential of COFRADIC for the analysis of several posttranslational modifications emphasizing on in vivo protein processing events. Additional modifications that can be analyzed include phosphorylation and N-glycosylation. The potential of COFRADIC for isolating peptides holding such modified amino acids are discussed here.

Carbon Isotopes↗

PubMed1999

Biochemical studies of the structure and function of the N-methyl-D-aspartate subtype of glutamate receptors.

The N-methyl-D-aspartate (NMDA) subtype of glutamate receptors plays a key role in synaptic transmission, synaptic plasticity, synaptogenesis, and excitotoxicity in the mammalian central nervous system. The NMDA receptor channel is formed from two gene products from two glutamate receptor subunit families, termed NR1 and NR2. Although the subunit composition of native NMDA receptors is incompletely understood, electrophysiological studies using recombinant receptors suggest that functional NMDA receptors consist of heteromers containing combinations of NR1, which is essential for channel activity, and NR2, which modulates the properties of the channels. The lack of agonists or antagonists selective for a given subunit of NMDA receptors has made it difficult to understand the subunit expression, subunit composition, and posttranslational modification mechanisms of native NMDA receptors. Therefore, most studies on NMDA receptors that examine regional expression and ontogeny have been focused at the level of the mRNAs encoding the different subunits using northern blotting, ribonuclease protection, and in situ hybridization techniques. However, the data from these studies do not provide clear information about the resultant subunit protein. To directly examine the protein product of the NMDA receptor subunit genes, the development of subunit-specific antibodies using peptides and fusion proteins has provided a good approach for localizing, quantifying, and characterizing the receptor subunits in tissues and transfected cell lines, and to study the subunit composition and the functional effects of posttranslational processing of the NMDA subunits, particularly the phosphorylation profiles of NMDA glutamate receptors.

Animals↗

PubMed2005

Palmitoylation at Cys574 is essential for MT1-MMP to promote cell migration.

MT1-MMP is a type I transmembrane proteinase that promotes cell migration and invasion. Here, we report that MT1-MMP is palmitoylated at Cys574 in the cytoplasmic domain, and this lipid modification is critical for its promotion of cell migration and clathrin-mediated internalization. The palmitoylation-defective mutant (C574A) failed to promote cell migration and was not internalized through clathrin pathway like wild-type, but it was internalized through the caveolae pathway. Reintroducing a cysteine at different positions in the cytoplasmic tail of the C574A mutant revealed that the position of the palmitoylated cysteine relative to LLY573, a motif that interacts with mu2 subunit of adaptor protein 2, is critical for the cell motility-promoting activity of MT1-MMP and its clathrin-mediated internalization. Taken together, palmitoylation of MT1-MMP is one of the key posttranslational modifications that determines MT1-MMP-dependent cell migration.

Amino Acid Motifs↗