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PubMed1992

Phosphorylation of the budgerigar fledgling disease virus major capsid protein VP1.

The structural proteins of the budgerigar fledgling disease virus, the first known nonmammalian polyomavirus, were analyzed by isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The major capsid protein VP1 was found to be composed of at least five distinct species having isoelectric points ranging from pH 6.45 to 5.85. By analogy with the murine polyomavirus, these species apparently result from different modifications of an initial translation product. Primary chicken embryo cells were infected in the presence of 32Pi to determine whether the virus structural proteins were modified by phosphorylation. SDS-PAGE of the purified virus structural proteins demonstrated that VP1 (along with both minor capsid proteins) was phosphorylated. Two-dimensional analysis of the radiolabeled virus showed phosphorylation of only the two most acidic isoelectric species of VP1, indicating that this posttranslational modification contributes to VP1 species heterogeneity. Phosphoamino acid analysis of 32P-labeled VP1 revealed that phosphoserine is the only phosphoamino acid present in the VP1 protein.

Animals↗
PubMed2026

Feeding the epigenome: EZH2 as a metabolic integrator of cell fate in development and cancer.

Epigenetic regulation is intimately linked to cellular metabolism, enabling environmental and nutritional cues to shape gene expression programs through dynamic modifications of chromatin structure. This metabolism-epigenetics interface is mediated, in part, by the dependence of chromatin-modifying enzymes on key metabolites, including S-adenosylmethionine (SAM), acetyl-CoA, UDP-GlcNAc, and α-ketoglutarate, which serve as substrates or cofactors for DNA and histone modifications. Among these regulators, EZH2, the catalytic subunit of Polycomb Repressive Complex 2 (PRC2), has emerged as a key mediator linking metabolic state to epigenetic regulation by translating metabolic inputs into changes in chromatin architecture and gene expression. EZH2 governs developmental cell fate through H3K27me3-mediated gene repression and is frequently dysregulated in cancer, where it promotes dedifferentiation, tumor progression, and metabolic reprogramming. Importantly, EZH2 activity is itself modulated by cellular metabolic status through posttranslational modifications, including phosphorylation, acetylation, methylation, ubiquitination, and O-GlcNAcylation, which influence its stability, catalytic activity, and chromatin-binding capacity. These modifications are responsive to nutrient availability and signaling pathways involving glucose, SAM, NAD+, and other metabolic intermediates. Consequently, disruption of this finely tuned regulatory network can contribute to developmental abnormalities, metabolic dysfunction, and oncogenesis. In this review, we examine the molecular mechanisms governing EZH2 regulation and discuss how metabolic control of EZH2 shapes chromatin dynamics, cell fate decisions, and disease pathogenesis. Elucidating how metabolic signals modulate EZH2 activity will advance our understanding of development and disease while uncovering potential therapeutic opportunities to target metabolism-driven epigenetic dysregulation.

Humans↗
PubMed2005

Analysis of posttranslational regulations in the Neurospora circadian clock.

Posttranslational modification of circadian clock proteins by phosphorylation is an essential regulatory process in the control of eukaryotic circadian clocks. In the Neurospora circadian clock, the key clock protein FREQUENCY (FRQ) is progressively phosphorylated. The phosphorylation of FRQ is regulated by both kinases and phosphatases, and the phosphorylation is important for regulating FRQ stability and its function in the circadian negative feedback loop. The degradation of FRQ is mediated by the ubiquitin/proteasome pathway. This article discusses posttranslational regulations of the Neurospora clock and describes the methods used in the studies of FRQ phosphorylation, FRQ kinases and phosphatases, and FRQ degradation.

Biological Clocks↗
PubMed2001

Redox regulation of the DNA repair function of the human AP endonuclease Ape1/ref-1.

The second enzyme in the DNA base excision repair (BER) pathway, apurinic/apyrimidinic (AP) endonuclease or Ape1, hydrolyzes the phosphodiester backbone immediately 5' to an AP site generating a normal 3'-hydroxyl group and an abasic deoxyribose-5-phosphate, which is processed by subsequent enzymes of the BER pathway. AP sites are the most common form of DNA damage, and the persistence of AP sites in DNA results in a block to DNA replication, cytotoxic mutations, and genetic instability. Interestingly, Ape1/ref-1 is a multifunctional protein that not only is a DNA repair enzyme, but also functions as a redox factor maintaining transcription factors, such as Fos, Jun, nuclear factor-kappaB, PAX (paired box-containing family of genes), hypoxia inducible factor-lalpha (HIF-1alpha), HIF-1-like factor, and p53, in an active reduced state. Apel/ref-1 has also been implicated in a number of other activities, one of which is the activation of bioreductive drugs requiring reduction for activity. In this report, we present data supporting our findings that another level of posttranslational modification of Apel/ref-1 that clearly affects the AP endonuclease activity is the reduction or oxidation of this protein. Furthermore, we show data demonstrating that at least one of the sites involved in this redox regulation is the cysteine amino acid found at position 310, immediately adjacent to the crucial histidine residue at position 309 in the DNA repair active site. These findings suggest that the Apel/ref-1 protein may be much more intimately regulated at the posttranslational level than initially imagined.

Apurinic Acid↗
PubMed2006

Production of lipid peroxidation products in osteoarthritic tissues: new evidence linking 4-hydroxynonenal to cartilage degradation.

OBJECTIVE: The lipid peroxidation product 4-hydroxynonenal (HNE) is prominently produced in osteoarthritic (OA) synovial cells, but its specific contribution to cartilage destruction is not understood. This study was designed to test whether HNE signaling and binding are involved in OA cartilage degradation through type II collagen (CII) and matrix metalloproteinase 13 (MMP-13) modulation. METHODS: HNE levels in synovial fluid and in isolated OA chondrocytes treated with free radical donors were determined by enzyme-linked immunosorbent assay. The formation of the HNE/CII adducts was measured in cartilage explants by immunoprecipitation. Levels of CII and MMP-13 messenger RNA and protein were determined by reverse transcription-polymerase chain reaction, Western blotting, and by the use of commercial kits. RESULTS: Levels of HNE/protein adducts were higher in OA synovial fluid compared with normal synovial fluid and were higher in OA chondrocytes treated with free radical donors compared with untreated cells. In cartilage explants, HNE induced CII cleavage, as established by the generation of neoepitopes. The level of HNE/CII adducts was increased in OA cartilage explants incubated with free radical donors. Modification of CII by HNE accelerated its degradation by active MMP-13. In isolated OA chondrocytes, HNE inhibited the expression of CII and tissue inhibitor of metalloproteinases 1 and induced MMP-13 mainly through activation of p38 MAPK. In vitro, HNE binding to MMP-13 activated this enzyme at a molar ratio of 1:100 (MMP-13 to HNE). CONCLUSION: The increased level of HNE in OA cartilage and the ability of HNE to induce transcriptional and posttranslational modifications of CII and MMP-13 suggest that this aldehyde could play a role in OA.

Aged↗
PubMed2003

Structural modification of the hamster sperm acrosome during posttesticular development in the epididymis.

The acrosome of the mature spermatozoon functions as a regulated secretory vesicle which performs several critical functions in mammalian fertilization. Acrosome assembly occurs throughout spermiogenesis and continues during posttesticular sperm maturation in the epididymis, resulting in a structurally polarized membrane-bounded organelle that contains an assortment of hydrolases and a stable infrastructure termed the acrosomal matrix. The role of stable acrosomal matrix assemblies in acrosomal biogenesis and function are poorly understood. This article presents ultrastructural, immunocytochemical, and biochemical data on the remodeling of the hamster acrosomal matrix during spermiogenesis and posttesticular sperm maturation in the epididymis. Specific posttranslational modifications of the major acrosomal matrix protein are evident in late, step 16, spermatids and matrix protein processing continues within specific acrosomal subdomains of caput epididymal spermatozoa. At the completion of sperm maturation, the acrosomal matrix consists of two structurally distinct domains which are adherent to the outer acrosomal membrane and exhibit a localized distribution pattern. Coincident with acrosomal matrix differentiation, a paracrystalline cytoskeletal complex is assembled onto the outer acrosomal membrane of epididymal spermatozoa. This cytoskeletal network appears to establish transmembrane structural interactions with the acrosomal matrix and may maintain attachment of the acrosomal cap to the sperm head during the early steps of the acrosome reaction.

Acrosome↗
PubMed2002

Lens proteomics: the accumulation of crystallin modifications in the mouse lens with age.

PURPOSE: To identify modified crystallins associated with aging of lens and produce two-dimensional electrophoresis (2-DE) proteome maps of crystallins in mouse lens. METHODS: Lens proteins from mice of increasing age or different strains were separated by either chromatography or 2-DE. Masses of whole proteins or tryptic peptides were analyzed by mass spectrometry. Changes in the abundance of individual crystallins were determined by image analysis of 2-DE gels. RESULTS: The measured masses of all known mouse crystallins, with the exception of gammaD and gammaF, matched the masses calculated from their reported sequences. Analysis by 2-DE indicated that most posttranslational modifications took place in mice after 6 weeks of age. Partially degraded crystallins, including betaB1, betaB2, betaB3, betaA3, alphaA, and alphaB, were found in greater proportion in the insoluble fractions. gamma-Crystallins A through F also became insoluble during aging. However, insolubilization of gamma-crystallins was associated with a decrease in isoelectric point (pI). Aging was also associated with increased phosphorylation of soluble alphaA- and alphaB-crystallins, confirmed by mass measurements of these proteins eluted from 2-DE gels. Comparison of protein profiles between several strains of mice used to produce transgenic or knockout models of cataract indicated few differences, except for an additional acidic form of a gamma-crystallin, possibly due to a polymorphism. CONCLUSIONS: These results suggest that partial degradation of alpha- and beta-crystallins and increased acidity of gamma-crystallins may cause insolubilization during aging. The 2-DE proteome maps of mouse lens proteins created in this study, using immobilized pH gradients, will be useful for comparison with maps of lens proteins of mice with cataracts so that cataract-specific modifications may be identified.

Aging↗
PubMed2004

Intranuclear membrane structure formations by CaaX-containing nuclear proteins.

The nuclear lamina is a protein meshwork lining the nucleoplasmic face of the nuclear envelope. Association of lamins with the inner nuclear membrane is mediated by specific modifications in the CaaX motif at their C-termini. B-type lamins are permanently isoprenylated whereas lamin A loses its modification by a lamin A-specific processing step after incorporation into the lamina. Lamins are differentially expressed during development and tissue differentiation. Here we show that an increased synthesis of lamins B1 and B2 in amphibian oocytes induces the formation of intranuclear membrane structures that form extensive arrays of stacked cisternae. These 'lamin membrane arrays' are attached to the inner nuclear membrane but are not continuous with it. Induction of this membrane proliferation depends on CaaX-specific posttranslational modification. Moreover, in transfected HeLa cells, chimeric GFP containing a nuclear localization signal and a C-terminal CaaX motif of N-Ras induces intranuclear membrane stacks that resemble those induced by lamins and ER-like cisternae that are induced in the cytoplasm upon increased synthesis of integral ER membrane proteins. Implications for the synthesis of CaaX-containing proteins are discussed and the difference from intranuclear fibrous lamina annulate lamellae formations is emphasized.

Amino Acid Motifs↗
PubMed2005

The evolution of meiosis: recruitment and modification of somatic DNA-repair proteins.

Several DNA-damage detection and repair mechanisms have evolved to repair double-strand breaks induced by mutagens. Later in evolutionary history, DNA single- and double-strand cuts made possible immune diversity by V(D)J recombination and recombination at meiosis. Such cuts are induced endogenously and are highly regulated and controlled. In meiosis, DNA cuts are essential for the initiation of homologous recombination, and for the formation of joint molecule and crossovers. Many proteins that function during somatic DNA-damage detection and repair are also active during homologous recombination. However, their meiotic functions may be altered from their somatic roles through localization, posttranslational modifications and/or interactions with meiosis-specific proteins. Presumably, somatic repair functions and meiotic recombination diverged during evolution, resulting in adaptations specific to sexual reproduction. (c) 2005 Wiley Periodicals, Inc.

Animals↗
PubMed1996

Regulation and physiological role of insulin-like growth factor binding proteins.

The insulin-like growth factors (IGFs) are potent anabolic agents, structurally related to insulin, that can influence growth processes in virtually every system of the body. Unlike insulin, these peptides associate with distinct binding proteins present in serum and other biological fluids as well as in medium conditioned by cultured cells. The IGFs are pleiotropic in activity and near ubiquitous in distribution, making the IGF binding proteins (IGFBPs) integral components of IGF physiology that define and coordinate IGF and insulin action at the level of the target cell. Thus, to fully understand and, ultimately, manipulate IGF-directed growth, it is critical to take into account the IGFBPs. Six distinct IGFBPs have been cloned and characterized, and they are postulated to serve specific, but as yet poorly defined, functional roles. Expression of these multiple IGFBP species is under developmental, hormonal, and nutritional control. In addition, IGFBPs can undergo a variety of posttranslational modifications that can have profound effects on IGFBP structure/function, and, hence, IGF action. The significance of IGFBP glycosylation, phosphorylation, proteolysis, and cell-membrane and extracellular matrix association as components of IGF physiology has only begun to be appreciated. In each case identified so far the modified IGFBP acts dramatically different from native or recombinant IGFBP in solution. In this presentation, I will briefly review the IGF/IGFBP system and use data from in vitro models to illustrate the potential power of posttranslational regulation of IGFBP in cellular response to IGFs.

Fibroblasts↗
PubMed2002

Gas-phase ion unimolecular dissociation for rapid phosphopeptide mapping by IRMPD in a Penning ion trap: an energetically favored process.

This communication discusses the efficient detection of the most important and common protein modification, phosphorylation, using ESI-FTICR-MS and IRMPD. Preliminary studies have demonstrated that within a complex protein digest, all phosphopeptides can be identified by a single IR laser irradiation event due to preferential dissociation of the modified peptides. This research demonstrates that the energy of activation for dissociation of the phosphopeptides is lower than that of the unmodified analogues providing the basis for the success of this technique. However, the P-O stretch (9.6-11 mum or 1042-909 cm-1) of this posttranslational modification is in direct resonance with the CO2 IR laser (10.6 mum or 943 cm-1) used for IRMPD. Therefore, the vibrational frequency of the phosphate moiety may be an additional factor in the rapid first-order decay of phosphopeptides. Based upon the energetics of dissociation discussed in this manuscript, IRMPD of ions in a Penning ion trap is an ideal platform for rapid phosphopeptide mapping.

Amino Acid Sequence↗
PubMed2006

Tip60-dependent acetylation of p53 modulates the decision between cell-cycle arrest and apoptosis.

Upon DNA damage and other types of stress, p53 induces either cell-cycle arrest or apoptosis depending on the cellular context. However, the molecular mechanisms that govern the choice between cell-cycle arrest and apoptosis are not well understood. Here, we show that Tip60 is required for both cell growth arrest and apoptosis mediated by p53 and also induces its acetylation specifically at lysine 120 (K120) within the DNA-binding domain. Interestingly, this modification is crucial for p53-dependent apoptosis but is dispensable for its mediated growth arrest. K120 is a recurrent site for p53 mutation in human cancer, and the corresponding acetylation-defective tumor mutant (K120R) abrogates p53-mediated apoptosis, but not growth arrest. Thus, our study demonstrates that Tip60-dependent acetylation of p53 at K120 modulates the decision between cell-cycle arrest and apoptosis, and it reveals that the DNA-binding core domain is an important target for p53 regulation by posttranslational modifications.

Acetylation↗
PubMed2004

Parallel identification of O-GlcNAc-modified proteins from cell lysates.

We report a new strategy for the parallel identification of O-GlcNAc-glycosylated proteins from cell lysates. The approach permits specific proteins of interest to be rapidly interrogated for the modification in any tissue or cell type and can be extended to peptides to facilitate the mapping of glycosylation sites. As an illustration of the approach, we identified four new O-GlcNAc-glycosylated proteins of low cellular abundance (c-Fos, c-Jun, ATF-1, and CBP) and two short regions of glycosylation in the enzyme O-GlcNAc transferase (OGT). The ability to target specific proteins across various tissue or cell types complements emerging proteomic technologies and should advance our understanding of this important posttranslational modification.

Acetylglucosamine↗
PubMed1999

Tryptophan hydroxylase regulation. Drug-induced modifications that alter serotonin neuronal function.

Tryptophan hydroxylase is the initial and rate-limiting enzyme in the biosynthesis of the neurotransmitter serotonin. A variety of drugs are known to diminish the function of this enzyme, and possibly cause damage to serotonin neurons. These include the substituted amphetamines methamphetamine and 3,4-methylenedioxy-methamphetamine, as well as L-DOPA, the most common therapy for Parkinsons Disease. In view of the important role for dopamine in the effects of these drugs on tryptophan hydroxylase and on serotonin neurons, we tested whether dopamine could alter the activity of this important enzyme. We found that dopamine-derived quinones, but not dopamine, inactivate tryptophan hydroxylase and convert the protein to a redox-cycling quinoprotein. This posttranslational modification of tryptophan hydroxylase could play a role in the drug-induced reduction in serotonin synthesis.

Amphetamines↗
PubMed1997

Developmental expression and regulation of the gap junction protein and transcript in rat ovaries.

The extensively developed network of cell-to-cell communication, generated by gap junctions, mediates transmission of small molecules between the cells of the ovarian follicle. Our study aimed at the analysis of the ontogeny and regulation of connexin43 (Cx43) the ovarian gap junction protein and its gene expression throughout folliculogenesis. Developmental analysis was performed using ovaries of immature rats at different ages and selected ovarian follicles of sexually mature female rats at different phases of their estrous cycle. In order to establish the effect of hormones involved in regulation of folliculogenesis on Cx43 modulation, the experimental animal model of sexually immature female rats administered with exogenous gonadotropins was employed. Developmental and hormonal modulations of Cx43 protein and its mRNA expression were studied by Western and Northern blot analysis, respectively. We found that Cx43 was undetectable in ovaries of rats on the first postnatal day, with a low level of this protein observed in 11-day-old rats ovaries. Some increase in the amount of Cx43 was observed at the age of 25 days with a dramatic elevation accompanied by phosphorylation of this protein that was specific to large antral follicles of sexually mature proestrous rats. Elimination of the protein was observed at estrus and could be prevented by cancellation of the preovulatory surge of luteinizing hormone (LH). This pattern of Cx43 modifications was mimicked by exogenous administration of hormones as follows: Pregnant mare's serum gonadotropin (PMSG) increased the Cx43 protein expression with a concurrent induction of its phosphorylation while a further human chorionic gonadotropin (hCG) injection resulted in a decrease of the signal. Analysis of the Cx43 mRNA showed a direct correlation between the Cx43 protein level and its gene expression. We conclude that: 1) At early folliculogenesis the ovarian gap junction protein Cx43 and its gene are developmentally regulated; and 2) After antrum formation, transcription, translation, and posttranslational modifications of Cx43 are regulated by gonadotropins.

Animals↗
PubMed1990

Secretion in yeast of mutant human lysozymes with and without glutathione bound to cysteine 95.

A mutant human lysozyme C77A, in which Cys-77 is replaced with Ala, was secreted by Saccharomyces cerevisiae as two proteins (C77A-a and C77A-b) with different specific activities. A peptide fragment from Val93 to Ala108 was obtained from C77A-a by pepsin digestion, and examined by fast atom bombardment mass spectrometry and amino acid analysis. The results showed that glutathione was attached to the thiol group of Cys95 of the fragment through a disulfide linkage. This observation was confirmed by quantitative formation of free glutathionesulfonic acid from C77A-a by performic acid treatment. In contrast, there was no modification in the case of C77A-b. These results indicate that C77A-a contained a mixed disulfide with glutathione attached to cysteine residue 95. In C77A-b, there appears to be a free thiol of Cys95 surrounded by many side chains, which was not modified by iodoacetic acid under native conditions, suggesting that the attachment of glutathione occurs during folding. These findings further suggest that in the oxidation step of disulfide bond formation in human lysozyme secreted by yeast, mixed disulfides are formed with glutathione and that posttranslational modification with glutathione can occur even in a protein secreted by yeast.

Amino Acid Sequence↗
PubMed1996

Experimental modification of rat pituitary prolactin cell function during and after spaceflight.

This study was done to evaluate the effects of microgravity on prolactin (PRL) cells of the male rat pituitary gland. We used the identical passive closed-vial cell culture system that was described for the culture of growth hormone cells (W. C. Hymen, R. E. Grindeland, T. Salada, P. Nye, E. Grossman, and P. Lane. J. Appl. Physiol. 80:955-970, 1996). After an 8-day spaceflight, all flight media (containing released PRL), as well as extracts (containing intracellular PRL), contained significantly lower amounts of immunoreactive PRL than their corresponding ground control samples. On the other hand, these same samples, when assessed for their biological activities by two different in vitro lymphocyte assays, yielded disparate results that may reflect posttranslational modifications to the hormone molecule. Other data showed that 1) the apparent molecular weights of released PRL molecules were not altered by microgravity, but 2) the region from which the PRL cells came (dorsal or ventral) made a significant difference in the amount and activity of PRL released from the flight cells. Because there is much current interest in the role that PRL may play in the regulation of the immune system and because changes in both cellular and humoral immunity accompany spaceflight, this study could help define future microgravity research in this area.

Animals↗
PubMed1990

Vesicle-mediated transport of membrane and proteins in malaria-infected erythrocytes.

Malaria parasites during intraerythrocytic development change the ultrastructure, biophysics, and the antigens of the host red blood cell membrane. Parasite-encoded proteins are associated with, inserted into, or secreted across the infected erythrocyte membrane. Since parasites of the genus Plasmodium are eukaryotic cells, it must be assumed that they possess essentially eukaryotic modes of vesicle-mediated transport and translocation of proteins and membranes. Numerous studies have demonstrated vesicular structures in the cytoplasm of malaria-infected red blood cells and an assortment of parasite proteins associated with the different vesicles, membranes, and membrane-defined compartments. Some parasite polypeptides remain trapped between the parasite and the parasitophorous vacuole membranes PVM, whereas others are associated with morphologically distinct membrane-limited vesicles and vacuoles. Some of these same parasite protein antigens also associate with the erythrocyte membrane or with parasite-induced ultrastructural modifications in the membrane of the parasitized red blood cells. This implies that intracellular transport occurs in malaria-infected erythrocytes, a capacity that uninfected red blood cells normally lose upon enucleation. The specific locations of parasite antigens within the infected cell also implys the existence of targeting signals in the translocated parasite polypeptides and perhaps transport-mediating proteins. The genes corresponding to some of these translocated proteins have been sequenced. Typical (and in some cases atypical) signal peptide sequences occur, as well as a number of sequences that may result in posttranslational modifications. How or if these features figure in to the translocation across, and targeting to a particular membrane compartment of the intraerythrocytic parasite remains unknown.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗