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PubMed1986

The amino acid sequence of the NH2-terminal portion of rat and human vitamin D binding protein: evidence for a high degree of homology between rat and human vitamin D binding protein.

We determined the amino terminal sequence of rat and human vitamin D binding protein (VDBP). The sequences of the two proteins are: Rat VDBP: LeuGluArgGlyArgAspTyrGluLysAspLysValCysGlnGluLeuSerThrLeuGlyLys Human VDBP: LeuGluArgGlyArgAspTyrGluLysAsnLysValCysLysGluPheSerHisLeuGlyLys AspAspPhe GluAspPhe There are 19 matches out of a total of 24 residues sequenced giving a percent match/length of 79.2%. Differences in the composition of the two proteins at residue 10, 14, 16, and 22 can be accounted for by single base changes in the the gene for the proteins. The difference (Thr----His) at residue 18 requires a change in two bases in the respective genes. We conclude that the sequence of the amino terminus of rat and human VDBP is similar with a high degree of homology between the two proteins. Vitamin D sterols, such as 25-hydroxyvitamin D3, 24,25-dihydroxyvitamin D3, and 25,26-dihydroxyvitamin D3, are bound with high affinity by a plasma alpha-globulin - VDBP, also known as group-specific component (Gc). Other vitamin D sterols, such as 1,25-dihydroxyvitamin D3 and vitamin D3 itself, are bound to this protein with a lesser affinity. VDBP also binds actin with high affinity. Its role in vitamin D physiology is unclear, although it may play a role in the bioavailability of different D sterols. Svasti et al. have shown that human group specific component (Gc) exists as different isoforms that have rapid or slow mobility on gel electrophoresis. The different human Gc isoforms have similar NH2-terminal and COOH-terminal amino acid sequences. The difference in the mobility of the various isoforms is due to post-translational modification of the protein by various carbohydrate residues; treatment of the protein with neuraminidase results in the conversion of the different isoforms to a single isoform. The amino acid sequence of the amino terminus of rat VDBP is not known. Recently, Cooke reported preliminary data from the analysis of cDNA clones showing that rat and human VDBP are partially homologous and that rat and human VDBP exhibit homology with rat and human albumin and alpha-fetoprotein. The NH2-terminal sequence of the rat VDBP, however, has not been reported. In order to learn more about the nature of the NH2-terminal sequence of human and rat VDBP, we isolated these proteins in relatively pure form and determined the NH2-terminal amino acid sequence of both of them.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed2002

How to flip the (redox) switch.

Redox-sensitive transcription factors such as the E. coli OxyR and S. cerevisiae Yap1, are activated by conformational changes that stem from the post-translational modification of reactive protein thiols. Recent studies provide new insights on how different agents that cause redox stress mediate the activation of transcription through distinct and specific pathways.

Bacterial Proteins↗

PubMed2005

Phosphoprotein profiling of erythropoietin receptor- dependent pathways using different proteomic strategies.

Proteomic techniques provide new tools for the global analysis of protein profiles but also for the investigation of specific protein functions. The analysis of signaling cascades has traditionally been performed by the determination of enzymatic or transcription factor activities representing a certain pathway. Functional proteomics now allows more comprehensive approaches to study cellular responses induced during ligand/receptor interactions. In this study we evaluated proteomic strategies for the investigation of structure-function relationships in the erythropoietin receptor signalling complex. After expression of epidermal growth factor/erythropoietin receptor mutant molecules in an identical cellular background we characterized their potential to induce cellular activities. Using this system we focused our efforts on post-translational modifications of signalling proteins reflecting a substantial part of receptor-dependent signaling events. Although tyrosine phosphorylated proteins were enriched by immunoprecipitation the analysis using the classical approach combining two-dimensional gel electrophoresis and identification by matrix assisted laser desorption/ionization-time of flight-mass spectrometry revealed that low expressed signaling proteins cannot be detected by this technique. An alternative strategy using one-dimensional gel separation of phosphoproteins and liquid chromatography-tandem mass spectrometry, however, allowed us to identify multiple proteins involved in intracellular signalling representing already established pathways but also proteins which have not been linked to EPO-induced signaling so far. This approach offers the potential to extend functional proteomic studies to complex signaling processes.

Animals↗

PubMed1983

mRNA from mutant Y1 adrenal cells directs the synthesis of altered regulatory subunits of type 1 cAMP-dependent protein kinase.

The molecular basis for altered cyclic AMP-dependent protein kinase activity was examined in three different mutant clones (Kin-1, Kin-7, and Kin-8) derived from the Y1 mouse adrenocortical cell line. Parental Y1 cells and the Kin mutants were labeled with L-[35S] methionine and the regulatory subunit of the type 1 cAMP-dependent protein kinase isozyme (RI) was immunoprecipitated from each clone with a specific guinea pig antiserum. When analyzed by electrophoresis on isoelectric focusing gels, the immunoprecipitates from mutant clones exhibited parental forms of RI plus an additional acidic variant form which likely accounted for altered cAMP-dependent protein kinase activity. Poly(A+) RNA was isolated from Y1 and Kin mutant cells and was translated in a cell-free, reticulocyte lysate system in the presence of L-[35S]methionine. The RI synthesized from poly(A+) RNA was immunoprecipitated from the translation mixture and analyzed on isoelectric focusing gels. The poly(A+) RNA from the Kin mutant clones directed the synthesis of parental and acidic variant forms of RI. These results suggest that the altered electrophoretic forms of RI arise from mutations in one of two RI genes rather than from post-translational modifications of the protein. The coexistence of parental and variant forms of RI in the Kin mutants indicate that the mutations are codominant.

Adrenal Cortex↗

PubMed1998

Aldose reductase: a window to the treatment of diabetic complications?

Kinetic studies on the aldose reductase protein (AR2) have shown that it does not behave as a classical enzyme in relation to ring aldose sugars. These results have been confirmed by X-ray crystallography studies, which have pinpointed binding sites for pharmacological "aklose reductase inhibitors" (ARIs). As with non-enzymic glycation reactions, there is probably a free-radical element involved derived from monosaccharide autoxidation. In the case of AR2, there is free radical oxidation of NADPH by autoxidising monosaccharides, enhanced in the presence of the NADPH-binding protein. Whatever the behaviour of AR2, many studies have showed that sorbitol production is not an initiating aetiological factor in the development of diabetic complications in humans. Vitamin E (alpha-tocopherol), other antioxidants and high fat diets can delay or prevent cataract in diabetic animals even though sorbitol and fructose levels are not modified; vitamin C acts as an AR1 in humans. Protein post-translational modification by glyc-oxidation or other events is probably the key factor in the aetiology of diabetic complications. There is now no need to invoke AR2 in xylitol biosynthesis. Xylitol can be produced in the lens from glucose, via a pathway involving the enzymes myo-inositol-oxygen oxidoreductase, D-glucuronate reductase. L-gulonate NAD(+)-3-oxidoreductase and L-iditol-NAD(+)-5-oxidoreductase, all of which have recently been found in bovine and rat lens. This chapter investigates the molecular events underlying AR2 and its binding and kinetics. Induction of the protein by osmotic response elements is discussed, with detailed analysis of recent in vitro and in vivo experiments on numerous ARIs. These have a number of actions in the cell which are not specific, and which do not involve them binding to AR2. These include peroxy-radical scavenging and recently discovered effects of metal ion chelation. In controlled experiments, it has been found that incubation of rat lens homogenate with glucose and the copper chelator o-phenanthroline abolishes production of sorbitol. Taken together, these results suggest AR2 is a vestigial NADPH-binding protein, perhaps similar in function to a number of non-mammalian crystallins which have been recruited into the lens. There is mounting evidence for the binding of reactive aldehyde moieties to the protein, and the involvement of AR2 either as a 'housekeeping' protein, or in a free-radial-mediated 'catalytic' role. Interfering with the NADPH binding and flux levels--possibly involving free radicals and metal ions--has a deleterious effect. We have yet to determine whether aldose reductase is the black sheep of the aldehyde reductase family, or whether it is a skeleton in the cupboard, waiting to be clothed in the flesh of new revelations in the interactions between proteins, metal ions and redox metabolites.

Aldehyde Reductase↗

PubMed2006

Nuclear mRNA retention and aberrant doppel protein expression in human astrocytic tumor cells.

Doppel (Dpl) is a paralogue of the mammalian Prion (PrP) protein. It is abundant in testis and, unlike PrP, it is expressed at low levels in the adult central nervous system (CNS). Besides, Dpl overexpression correlates with some prion-disease pathological features, such as ataxia and death of cerebellar neurons. Recently, ectopic expression of doppel was found in two different tumor types, specifically in glial and haematological cancers. In this study the doppel gene (PRND) mRNA and protein expression in PRT-HU2 and IPDDC-A2 astrocytoma-derived cell lines was investigated. Northern blot analysis revealed two equally abundant PRND mRNA isoforms, while real-time PCR, on nuclear and cytoplasmic RNA fractions, and cRNA in situ hybridization, on astrocytoma cells and bioptical specimens, showed a nuclear retention of PRND transcripts. Western blot analysis showed that the amount of protein expressed is low compared to the level of mRNA. Moreover deglycosylation studies indicated that Dpl undergoes unusual glycosylation processes. Immunohistochemistry experiments demonstrated that Dpl was mainly localised in the cytoplasm of the astrocytic tumor cells, and that it failed to be GPI-anchored to the cell membrane. This unusual cellular localization was also confirmed through EGFP-Dpl expression in astrocytomas; on the contrary, HeLa cells exhibited the expected Dpl membrane localization. Our findings suggest an aberrant doppel gene expression pattern, characterized by a substantial nuclear retention of the transcript, an altered post-translational modification of the protein and an unusual cytoplasmic localization.

Astrocytoma↗

PubMed1993

One-step affinity isolation of recombinant protein using the baculovirus/insect cell expression system.

We have developed two baculovirus transfer vectors which allow single-step affinity isolation of recombinant proteins after expression in insect cells. Using these vectors, recombinant proteins are synthesized as fusions with glutathione-S-transferase and are amenable to enrichment from a crude insect cell lysate using glutathione affinity agarose. After affinity isolation, glutathione-S-transferase can be cleaved from the recombinant polypeptides of interest at an engineered thrombin cleavage site. We used this approach to successfully isolate glutathione-S-transferase, the human low density lipoprotein receptor, two large polypeptides containing cytoplasmic domains of the cystic fibrosis transmembrane conductance regulator (CFTR), and the full-length CFTR. The approach has potential advantages over prokaryotic overexpression of foreign polypeptides, including: (i) eukaryotic post-translational modification of expressed protein, (ii) increased solubility of recombinant fusion proteins synthesized in insect cells leading to increased affinity yield under mild conditions, and (iii) production of large and/or complex polypeptides which might be difficult to purify from prokaryotic cells. The method also allows enrichment of recombinant protein representing a small fraction (less than 5%) of total insect cell protein produced and provides a general method for eukaryotic protein synthesis and isolation which is independent of the particular protein being expressed.

Animals↗

PubMed2001

Nuclear apoptotic changes: an overview.

Apoptosis is a form of active cell death essential for morphogenesis, development, differentiation, and homeostasis of multicellular organisms. The activation of genetically controlled specific pathways that are highly conserved during evolution results in the characteristic morphological features of apoptosis that are mainly evident in the nucleus. These include chromatin condensation, nuclear shrinkage, and the formation of apoptotic bodies. The morphological changes are the result of molecular alterations, such as DNA and RNA cleavage, post-translational modifications of nuclear proteins, and proteolysis of several polypeptides residing in the nucleus. During the last five years our understanding of the process of apoptosis has dramatically increased. However, the mechanisms that lead to apoptotic changes in the nucleus have been only partially clarified. Here, we shall review the most recent findings that may explain why the nucleus displays these striking modifications. Moreover, we shall take into consideration the emerging evidence about apoptotic events as a trigger for the generation of autoantibodies to nuclear components.

Apoptosis↗

PubMed1985

The association of human c-Ha-ras sequences with chromatin and nuclear proteins.

As a step towards the understanding of possible relationship between chromatin organization and regulation of the oncogene expression, we have investigated the chromatin structure of one of the more frequently activated oncogenes, c-Ha-ras, in HeLa-S3 cells. This was accomplished by isolation of the chromatin fractions (soluble and insoluble) after micrococcal nuclease digestion of purified nuclei and probing for the distribution of ras sequences. The polynucleosomal fraction was further resolved by sucrose gradient sedimentation. Southern-blot hybridization of the DNA isolated from various fractions yielded following results: (1) c-Ha-ras sequences segregated predominantly in the lysate fraction. (2) Unlike the B-globin (transcriptionally inactive) sequences, ras-H associated chromatin lacked typical nucleosomal packaging. Furthermore, since post-translational modifications of nuclear proteins have been suggested to modulate the nucleosome structure during DNA transcription and replication, ras sequences, in polynucleosomes immunofractionated on anti-poly (ADP-Ribose) Sepharose were also examined. The data suggested that the major class of this oncogene sequence exists in chromatin more distal to the sites of this particular chromatin modification.

Base Sequence↗

PubMed1995

Characterization of a recombinant antihaemophilia-A factor (factor VIII-delta II) by matrix-assisted laser desorption/ionization mass spectrometry.

Factor VIII-delta II is a genetically engineered deletion variant of factor VIII, expressed by recombinant Chinese hamster ovary cells. This 1436-residues-long protein has a molecular mass, calculated from its sequence, of 164,954 Da and exhibits seven potential glycosylation sites. The glycoprotein, secreted as a single polypeptide chain, can be cleaved after Arg740 to generate a heavy-light chain complex of 90-80 kDa as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Due to its high mass range and excellent sensitivity, matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) has been chosen to play a key role in the precise determination of the molecular masses of recombinant factor VIII and the localization of the post-translational modifications within the protein. Native factor VIII-delta II displays a molecular mass of 178 kDa. The masses measured by MALDI for the heavy and light chains are respectively 89,900 Da and 87,100 Da. These mass values, found reproducible from batch to batch, are used to characterize factor VIII-delta II during the course of preclinical studies. The difference from the theoretical molecular molecular masses and the observation of broad molecular peaks suggest that recombinant FVIII-delta II has been effectively glycosylated by the host cell on both heavy and light chains. Similarly to plasma-derived factor VIII, the recombinant protein is proteolyzed by thrombin to generate the A1/A2/A3-C1-C2 trimer that is the active form of factor VIII in the coagulation pathway. MALDI-MS analysis of activated factor VIII-delta II suggested the presence of N-linked oligosaccharides in the proteolyzed light chain (A3-C1-C2 of 77,750 Da) and in the A1 domain (46,400 Da) of the heavy chain. By contrast, the similarity between the experimental and theoretical masses of the A2 domain indicated that its single potential glycosylation site has not been utilized.

Enzyme Activation↗

PubMed2006

Histone acetylation in gene regulation.

Genetic information is packaged in the highly dynamic nucleoprotein structure called chromatin. Many biological processes are regulated via post-translational modifications of key proteins. Acetylation of lysine residues at the N-terminal histone tails is one of the most studied covalent modifications influencing gene regulation in eukaryotic cells. This review focuses on the role of enzymes involved in controlling both histone and non-histone proteins acetylation levels in the cell, with particular emphasis on their effects on cancer.

Acetylation↗

PubMed2002

Primary skin fibroblasts as human model system for proteome analysis.

Elucidation of cellular processes and their changes at the level of protein expression and post-translational modification patterns may allow identification of novel proteins and thereby mechanisms involved in the pathogenesis of multigenic diseases. The aim of this study was to test cultured, nontransformed primary fibroblasts derived from human skin biopsies as a suitable model system for proteome analysis. Therefore soluble protein fractions were separated on several overlapping ultrazoom gels covering the pH range from 3.5-9. Correlation analysis of gel-pairs revealed a highly reproducible protein expression pattern within (intra-assay) and between (inter-assay) independent experiments of a single fibroblast cell line (intra-cell line comparison). Spot intensity variations were less than a factor of two for more than 80% of identical spots. In addition, inter-cell line comparison exhibits no significant variations in spot intensities. To achieve further improvements in reproducibility we generated master gels for each pH range by combining averaged spot information derived from two different cell lines each analysed by two independent experiments using the raw master gel algorithm of the Z3 image analysis software. The resulting reference images of primary human fibroblasts provided a basis for investigating regulation by extracellular stimuli and drugs as well as their alterations in patients with different diseases.

Cells, Cultured↗

PubMedDate not supplied

Phosphoproteomics by mass spectrometry and classical protein chemistry approaches.

The general fields of biological sciences have seen phenomenal transformations in the past two decades at the level of data acquisition, understanding biological processes, and technological developments. Those advances have been made partly because of the advent of molecular biology techniques (which led to genomics) coupled to the advances made in mass spectrometry (MS) to provide the current capabilities and developments in proteomics. However, our current knowledge that approximately 30,000 human genes may code for up to 1 million or more proteins disengage the interface between the genome sequence database algorithms and MS to generate a major interest in independent de novo MS/MS sequence determination. Significant progress has been made in this area through procedures to covalently modify peptide N- and C-terminal amino-acids by sulfonation and guanidination to permit rapid de novo sequence determination by MS/MS analysis. A number of strategies that have been developed to perform qualitative and quantitative proteomics range from 2D-gel electrophoresis, affinity tag reagents, and stable-isotope labeling. Those procedures, combined with MS/MS peptide sequence analysis at the subpicomole level, permit the rapid and effective identification and quantification of a large number of proteins within a given biological sample. The identification of proteins per se, however, is not always sufficient to interpret biological function because many of the naturally occurring proteins are post-translationally modified. One such modification is protein phosphorylation, which regulates a large array of cellular biochemical pathways of the biological system. Traditionally, the study of phosphoprotein structure-function relationships involved classical protein chemistry approaches that required protein purification, peptide mapping, and the identification of the phosphorylated peptide regions and sites by N-terminal sequence analysis. Recent advances made in mass spectrometry have clearly revolutionized the studies of phosphoprotein biochemistry, and include the development of specific strategies to preferentially enrich phosphoproteins by covalent-modifications that incorporate affinity tags that use the physicochemical properties of phosphoaminoacids. The phosphoserine/phosphothreonine-containing proteins/peptides are derivatized under base-catalyzed conditions by thiol agents; mono- and di-thiol reagents both have been used in such studies. The thiol agent may have: (i) an affinity tag for protein enrichment; (ii) stable-isotopic variants for relative quantitation; or (iii) a combination of the moieties in (i) and (ii). These strategies and techniques, together with others, are reviewed, including their practical application to the study of phosphoprotein biochemistry and structure-function. The consensus of how classical protein chemistry and current MS technology overlap into special case of proteomics, namely "phosphoproteomics," will be discussed.

Amino Acid Sequence↗

PubMedDate not supplied

Proteolytic 18O-labeling strategies for quantitative proteomics.

A number of proteomic techniques have been developed to quantify proteins in biological systems. This review focuses on the quantitative proteomic technique known as "proteolytic 18O-labeling." This technique utilizes a protease and H(2)18O to produce labeled peptides, with subsequent chromatographic and mass spectrometric analysis to identify and quantify (relative) the proteins from which the peptides originated. The technique determines the ratio of individual protein's expression level between two samples relative to each other, and can be used to quantitatively examine protein expression (comparative proteomics) and post-translational modifications, and to study protein-protein interactions. The present review discusses various aspects of the 18O-labeling technique, including: its history, the advantages and disadvantages of the proteolytic 18O-labeling technique compared to other techniques, enzymatic considerations, the problem of variable incorporation of 18O atoms into peptides with a discussion on recent advancements of the technique to overcome it, computational tools to interpret the data, and a review of the biological applications.

Gene Expression Profiling↗

PubMed2005

Interplay of isoprenoid and peptide substrate specificity in protein farnesyltransferase.

Protein farnesyltransferase (FTase) catalyzes the post-translational modification of many important cellular proteins, and is a potential anticancer drug target. Crystal structures of the FTase ternary complex illustrate an unusual feature of this enzyme, the fact that the isoprenoid substrate farnesyl diphosphate (FPP) forms part of the binding site for the peptide substrate. This implies that changing the structure of FPP could alter the specificity of the FPP-FTase complex for peptide substrates. We have found that this is the case; a newly synthesized FPP analogue, 3-MeBFPP, is a substrate with three peptide cosubstrates, but is not an effective substrate with a fourth peptide (dansyl-GCKVL). Addition of this analogue also inhibits farnesylation of dansyl-GCKVL by FPP. Surprisingly, the differential substrate abilities of these four peptides with FPP-FTase and 3-MeBFPP-FTase complexes do not correlate with their binding affinities for these isoprenoid-enzyme complexes. The possible mechanistic rationales for this observation, along with its potential utility for the study of protein prenylation, are discussed.

Alkyl and Aryl Transferases↗

PubMed1988

The development of hemopoietic cells in response to stromal cells or growth factors is modified by agents that influence ADP-ribosylation.

The in vitro differentiation of multipotent stem cells in long-term marrow cultures can be blocked by treatment with agents that modify cholera toxin induced ADP-ribosylation of proteins. The latter agents also inhibit the growth and development of progenitor cells in soft gels in response to interleukin-3 but have little effect upon the development of progenitor cells that respond to the macrophage colony stimulating factor (CSF-1). Cholera toxin, in the same system, inhibits the development of CSF-1 responsive progenitor cells but has little effect on the development of cells that respond to IL-3. Similarly, progenitor cells that respond to IL-3 are relatively more resistant to pertussis toxin than cells that respond to CSF-1. These data indicate that ADP-ribosylation may be an important post-translational modification of regulatory proteins concerned with hemopoietic cell differentiation and growth in response to stromal cells or growth factors.

Adenosine Diphosphate↗

PubMed1994

Adenovirus vectored vaccines.

Human recombinant adenoviruses (Ad) have been employed to develop experimental vaccines against a number of infectious agents. Ad-vectored vaccines express recombinant proteins, including any post-translational modifications, into functioning replicas of the native proteins capable of eliciting neutralizing antibodies in both abortive and permissive animal models. Human Ad types 4, 5, and 7 were used to construct recombinant viruses that express the respiratory syncytial virus F or G glycoproteins, the hepatitis B surface antigen, and the HIV env or gag genes. The recombinant Ad-HIV viruses are of particular interest and have been examined for their immunogenicity in dogs and chimpanzees. Dogs were immunized intratracheally with Ad-env recombinants (10(9) pfu/dog). Excellent humoral anti-HIV responses, including neutralizing antibodies, were detected in the sera following booster immunization (12-18 weeks after primary immunization) with a second Ad-env recombinant made in a different Ad serotype (heterotypic booster). Chimpanzees were immunized in two ways, orally with lyophilized virus (10(9) to 10(10) pfu/virus) in enteric-coated capsules or intranasally (10(7) pfu/virus). Intranasal immunization was superior to oral immunization with respect to replication of recombinant viruses as well as induction of anti-Ad and anti-HIV antibodies. Administration by both routes resulted in stimulation of cellular immune responses, as measured by antigen proliferation assays. Anti-HIV antibodies were detected in chimpanzee secretions (salivary, nasal, rectal, vaginal) taken from animals following intranasal immunization with a heterotypic recombinant. Intranasal administration effectively primed chimpanzees to produce high-titred (320-640) serum neutralizing antibodies to HIV following boosting with a baculovirus-derived env (gp160) subunit vaccine.(ABSTRACT TRUNCATED AT 250 WORDS)

AIDS Vaccines↗

PubMed2002

PKC regulation of the human equilibrative nucleoside transporter, hENT1.

Regulation of nucleoside transporters is poorly understood. We show that acute stimulation of protein kinase C (PKC) causes a rapid increase in S-(4-nitrobenzyl)-6-thioinosine-sensitive (human equilibrative nucleoside transporter 1, hENT1) nucleoside uptake, in human cultured cells, which is not due to increased metabolism and which can be blocked by PKC inhibitors. Use of isoform-specific inhibitors indicates that PKC delta and/or epsilon (but not alpha, beta or gamma) are responsible for the acute effects. Down-regulation of PKC decreases hENT1-dependent uridine uptake. These are the first data to show rapid PKC delta/epsilon-dependent stimulation of hENT1 transport by a mechanism that may involve activation of transporters at the membrane possibly by post-translational modification of the protein.

Amphetamine↗