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Results for “Protein Isoforms”

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PubMed2007

Expression of embryonic tau protein isoforms persist during adult neurogenesis in the hippocampus.

Tau is a microtubule-associated protein with a developmentally regulated expression of multiple isoforms. The neonatal isoform is devoid of two amino terminal inserts and contains only three instead of four microtubule-binding repeats (0N/3R-tau). We investigated the temporal expression pattern of 0N-tau and 3R-tau in the rat hippocampus. After the decline of 0N- and 3R-tau immunoreactivity during the postnatal development both isoforms remain highly expressed in a few cells residing beneath the granule cell layer. Coexpression of the polysialylated neuronal cell adhesion molecule, doublecortin, and incorporated bromodeoxyuridine showed that these cells are proliferating progenitor cells. In contrast mature granule cells express the adult tau protein isoform containing one aminoterminal insert domain (1N-tau). Therefore a shift in tau isoform expression takes place during adult neurogenesis, which might be related to migration, differentiation, and integration in the granule cell layer. A model for studying shifts in tau isoform expression in a defined subset of neurons might help to understand the etiology of tauopathies, when isoform composition is crucial for neurodegeneration, as in Pick's disease or FTDP-17.

Animals↗

PubMed2002

Alternate exon insertion controls selective ubiquitination and degradation of different AUF1 protein isoforms.

The A+U-rich element (ARE) in the 3' non-coding region (3' NCR) of short-lived cytokine mRNAs binds several regulatory proteins, including hnRNP D/AUF1, which comprises four isoforms of 37, 40, 42 and 45 kDa. ARE-mRNA degradation involves ubiquitin-proteasome activity, and one or more AUF1 proteins are thought to be ubiquitinated. Here we have characterized the mechanism for differential ubiquitination and degradation of the different AUF1 protein isoforms. We demonstrate in an in vitro ubiquitination system that the p37, followed by the p40 protein, are strongly ubiquitinated, whereas the p42 and p45 forms are not. Over expression in cells of enzymes that control the ubiquitin cycle were found to control p37 and p40 AUF1 protein levels through ubiquitination and proteasome activity, but not p42 and p45 forms. The p42 and p45 AUF1 proteins share a C-terminal exon 7 that is not found in the p37/p40 isoforms. Our studies show that exon 7 blocks ubiquitination and rapid degradation of AUF1 proteins, whereas its deletion permits ubiquitination to occur and promotes rapid turnover of AUF1 proteins. Thus, the stabilities of AUF1 isoforms are differentially controlled by insertion of an alternate exon that regulates ubiquitin targeting activity.

Exons↗

PubMed1997

Allelic origin of the abnormal prion protein isoform in familial prion diseases.

The hallmark of prion diseases is the presence of an aberrant isoform of the prion protein (PrP(res)) that is insoluble in nondenaturing detergents and resistant to proteases. We investigated the allelic origin of PrP(res) in brains of subjects heterozygous for the D178N mutation linked to fatal familial insomnia (FFI) and a subtype of Creutzfeldt-Jakob disease (CJD178), as well as for insertional mutations associated with another CJD subtype. We found that in FFI and CJD178 subjects, only mutant PrP was detergent-insoluble and protease-resistant. Therefore, PrP(res) derives exclusively from the mutant allele carrying the D178N mutation. In contrast, in the CJD subtype harboring insertional mutations, wild-type PrP was also detergent-insoluble and likely to be protease-resistant. Our findings indicate that the participation of the wild-type PrP in the formation of PrP(res) depends on the type of mutations, providing an insight into the molecular mechanisms underlying the phenotypic heterogeneity in familial prion diseases.

Alleles↗

PubMed1994

Isolation and characterization of an olive allergen-like protein from lilac pollen. Sequence analysis of three cDNA encoding protein isoforms.

An olive allergen-like protein has been isolated from lilac (Syringa vulgaris) pollen extract. The protein can be considered as an allergen since is recognized by IgE from olive hypersensitive human sera, and has been called Syr v I (IUIS nomenclature). This protein consists of a glycosylated polypeptide of 20 kDa, which has an amino acid composition, spectroscopic properties, and an N-terminal sequence similar to the major allergen from olive pollen, Ole e I. The lilac allergen is recognized by rabbit polyclonal antisera raised against olive allergen as well as by an Ole e I-specific monoclonal antibody. Using a polymerase chain reaction strategy, based on the similarities observed between these olive and lilac proteins, three cDNA clones encoding Syr v I have been isolated and sequenced. These clones code for a polymorphic protein of 145 residues with a derived molecular mass of about 16,400Da, which contains a potential N-glycosylation site. Comparison of the deduced amino acid sequences of these Syr v I isoforms to each other revealed identities of 90-97%. Moreover, these sequences showed a high degree of similarity (85.5-89.6% identity) with Ole e I. The structural and immunological characterization of Syr v I justify the cross-reactions observed between olive and lilac pollen extracts. The molecular cloning of Syr v I is relevant for the epitope mapping in Oleaceae allergens, and may contribute to an improvement in the design of reagents for diagnosis and therapy of IgE-dependent allergic reactions.

Allergens↗

PubMed1993

Application of an ectopic expression system for the selection of protein-isoform-specific antibodies. The monoclonal antibody K1C3 is specific for the RCK1 potassium channel.

Monoclonal antibodies were raised against a fusion protein consisting of a fragment of 141 amino acids of the C-terminal region of the rat brain voltage-gated K(+)-channel protein (RCK1) and the lambda N protein (fusion protein I). Selection of K(+)-channel-specific hybridoma cell lines was performed by means of an ELISA employing a fusion protein consisting of the K(+)-channel-specific peptide sequence and glutathione S-transferase (fusion protein II). For final selection of RCK1 isoform-specific antibodies, a panel of Xenopus oocytes was employed, each injected with cRNA coding for a specific RCK isoform (RCK 1, 2, 4 or 5). Several days after injection, cryosections of embedded oocytes were obtained and were employed in immunohistochemical analysis of antibody binding. Of five hybridoma supernatants from stable growing hybridoma cell lines, selected by the fusion-protein ELISA, one monoclonal antibody (denoted K1C3) recognized exclusively the RCK1-protein isoform, with the other four exhibiting different levels of cross-reactivity with other K(+)-channel isoforms, or with unknown protein(s) of non-injected oocytes. The expression of the RCK1 protein in the postnatal brain was studied using, as far as we are aware, the first example of the application of such isoform-specific antibodies.

Amino Acid Sequence↗

PubMed2004

Studying protein isoforms of the adaptor SETA/CIN85/Ruk with monoclonal antibodies.

SETA/CIN85/Ruk is a multifunctional adaptor protein involved in signal transduction and attenuation downstream of receptor tyrosine kinases. It has a modular structure, and various isoforms that combine different protein-protein interaction domains have been proposed based on cDNA analysis. As a first step towards understanding SETA/CIN85/Ruk isoforms at the protein level, we have characterized 5 monoclonal antibodies against this protein. Three of these were used to study lysates fractionated on a pH gradient, leading to the identification of various SETA/CIN85/Ruk proteins on the basis of pI and apparent molecular weight. While good correspondence with proteins predicted from cDNA analysis was found for two isoforms, in most cases it was not possible to make an unequivocal assignment. We conclude that additional splice variants remain to be described, and that a deeper understanding of SETA/CIN85/Ruk post-translational processing and modification is necessary to gain further understanding of this complex gene product.

Amino Acid Sequence↗

PubMed2004

Astrocytes and oligodendrocytes express different STOP protein isoforms.

Many cell types contain subpopulations of microtubules that resist depolymerizing conditions, such as exposure to cold or to the drug nocodazole. This stabilization is due mainly to polymer association with STOP proteins. In mouse, neurons express two major variants of these proteins, N-STOP and E-STOP (120 kDa and 79 kDa, respectively), whereas fibroblasts express F-STOP (42 kDa) and two minor variants of 48 and 89 kDa. N- and E-STOP induce microtubule resistance to both cold and nocodazole exposure, whereas F-STOP confers microtubule stability only to the cold. Here, we investigated the expression of STOP proteins in oligodendrocytes and astrocytes in culture. We found that STOP proteins were expressed in precursor cells, in immature and mature oligodendrocytes, and in astrocytes. We found that oligodendrocytes express a major STOP variant of 89 kDa, which we called O-STOP, and two minor variants of 42 and 48 kDa. The STOP variants expressed by oligodendrocytes induce microtubule resistance to the cold and to nocodazole. For astrocytes, we found the expression of two STOP variants of 42 and 48 kDa and a new STOP isoform of 60 kDa, which we called A-STOP. The STOP variants expressed by astrocytes induce microtubule resistance to the cold but not to nocodazole, as fibroblast variants. In conclusion, astrocytes and oligodendrocytes express different isoforms of STOP protein, which show different microtubule-stabilizing capacities.

Animals↗

PubMed2003

Differential expression of equine myosin heavy-chain mRNA and protein isoforms in a limb muscle.

The horse is one of the few animals kept and bred for its athletic performance and is therefore an interesting model for human sports performance. The regulation of the development of equine locomotion in the first year of life, and the influence of early training on later performance, are largely unknown. The major structural protein in skeletal muscle, myosin heavy-chain (MyHC), is believed to be primarily transcriptionally controlled. To investigate the expression of the MyHC genes at the transcriptional level, we isolated cDNAs encoding the equine MyHC isoforms type 1 (slow), type 2a (fast oxidative), and type 2d/x (fast glycolytic). cDNAs encoding the 2b gene were not identified. The mRNA expression was compared to the protein expression on a fiber-to-fiber basis using in situ hybridization (non-radioactive) and immunohistochemistry. Marked differences were detected between the expression of MyHC transcripts and MyHC protein isoforms in adult equine gluteus medius muscle. Mismatches were primarily due to the presence of hybrid fibers expressing two fast (2ad) MyHC protein isoforms, but only one fast (mainly 2a) MyHC RNA isoform. This discrepancy was most likely not due to differential mRNA expression of myonuclei.

Amino Acid Sequence↗

PubMed2006

A distinct p53 protein isoform signature reflects the onset of induction chemotherapy for acute myeloid leukemia.

PURPOSE: The antioncogene protein product p53 has not been studied previously in cancer patients during in vivo chemotherapy. This study examined the early p53 protein and gene expression during induction chemotherapy in acute myeloid leukemia (AML). EXPERIMENTAL DESIGN: Leukemic cells were collected from five AML patients during their first 18 hours of induction chemotherapy and examined for p53 protein and gene expression by one- and two-dimensional gel immunoblot and high-density gene expression arrays. RESULTS: Up-regulation of p53 protein expression was detected in AML patients posttreatment in vivo. One- and two-dimensional gel immunoblots showed two main forms of p53, denominated alpha p53 and delta p53, both recognized by various NH2-terminal directed antibodies. As a response to treatment, we detected rapid accumulation of alpha p53, with significantly altered protein expression levels already after 2 hours. The accumulation of alpha p53 was accompanied by increased transcription of putative p53 target genes and subsequent cytopenia in the patients. CONCLUSION: Up-regulation of the p53 protein and target genes seems to be a prominent feature in induction chemotherapy of AML. The rapid shift from a shorter p53 protein form (delta) toward the full-length protein (alpha) underscores the complexity of p53 protein modulation in patients undergoing chemotherapy.

Acute Disease↗

PubMed1994

Primary structure and distribution of ryanodine-binding protein isoforms of the bullfrog skeletal muscle.

We have cloned two groups of cDNAs which encode isoforms of ryanodine-binding protein/Ca2+ release channel of the bullfrog skeletal muscle sarcoplasmic reticulum. One of the cDNA groups encodes the protein of 5,037 (or 5,031 with a deletion) amino acids with a molecular weight of 571,262 (or 570,607), which is identified as the alpha-isoform of the ryanodine-binding protein based on the amino acid sequence of three tryptic fragments of the purified protein. The other group of cDNAs encodes the protein of 4,868 amino acids with molecular weight of 553,029, which contains the sequences of three proteolytic fragments derived from the beta-isoform protein. About 70% of the amino acid sequence identity is present between alpha- and beta-isoforms of the bullfrog. The primary structure of the alpha-isoform is highly (80%) homologous to the ryanodine-binding protein cloned from rabbit skeletal muscle (type 1). The beta-isoform, on the other hand, is more than 85% identical with that from the rabbit brain (type 3), while it has only 67% overall identity with type 1. Analyses of RNA from various tissues of the bullfrog demonstrate that the beta-isoform is widely expressed, while the alpha-isoform is expressed mainly in skeletal muscle. A phylogenetic analysis of the ryanodine-binding protein/Ca2+ release channel family suggests that the various types of Ca2+ release channels have evolved from an ancestor gene. Possible differential roles of alpha- and beta-isoforms of ryanodine-binding protein in Ca2+ release mechanisms including skeletal muscle excitation-contraction coupling were discussed.

Animals↗

PubMed2005

Cholinesterase inhibitor affects the amyloid precursor protein isoforms in patients with Alzheimer's disease.

An altered platelet ratio of amyloid precursor protein (APP) isoforms might be a diagnostic, predictive, or therapeutic marker for Alzheimer's disease (AD). Our purpose was to test the hypothesis that this ratio might serve as a therapeutic marker for AD patients treated with the cholinesterase inhibitor, galantamine. Thirty-nine patients (mean age 76.6 +/- 9.4 years) with AD were treated with galantamine for 12 weeks. Patients were evaluated at baseline, 4 and 12 weeks by cognitive testing along with a determination of their platelet APP isoform ratio. Western blotting was performed to calculate the APP isoform ratio. At the end of the treatment, cognitive scores significantly improved, and the ratio of the high-molecular-weight (130 kDa) isoform to the low-molecular-weight (110-106 kDa) isoforms increased. These results suggest that cholinesterase inhibition might be involved in APP processing.

Aged↗

PubMed1998

A comparative study of abnormal prion protein isoforms between Gerstmann-Sträussler-Scheinker syndrome and Creutzfeldt-Jakob disease.

Proteinase K (PK)-resistant prion protein (PrPres) isoforms were examined in three patients with Gerstmann-Sträussler-Scheinker syndrome (GSS) carrying proline-to-leucine mutation at codon 102 in prion protein gene (PRNP), and in nine patients with sporadic Creutzfeldt-Jakob disease (CJD). PrPres isoform termed 'type A', which showed a more prominent band of highly glycosylated form than both a lower glycosylated band and an unglycosylated band in immunoblotting, was exclusively found in the GSS patients examined. In eight of nine CJD patients, electrophoretic mobilities of three PrPres glycoforms were similar to type A, but the ratio of these glycoforms termed 'type B' was distinct from that of type A. On the other hand, one sporadic CJD case with wild-type PRNP had a different PrPres isoform termed type C, which showed higher molecular shift of each of the PrPres glycoforms. There was no significant relationships among genotypes, clinical features and PrPres isoforms in sporadic CJD cases. Our finding suggests that type A PrPres isoform is specifically found in the patients with GSS carrying codon 102 mutation, and there are at least two different PrPres isoforms in the patients with sporadic CJD.

Amyloid↗

PubMed2006

Strategies for examination of Alzheimer's disease amyloid precursor protein isoforms.

We describe a proteomics procedure using bioinformatics, immunoprecipitation, two-dimensional gel electrophoresis, Western blotting, in-gel digestion, LC-MS, MALDI-MS, and MS-MS for isolation and identification of amyloid precursor protein (APP) isoforms APP695, APP751, and APP770. Retinoic acid-induced Ntera 2 cell line, derived from a human teratocarcinoma cells, was the in-vitro source of APP. Initial isolation of whole APP was performed by immunoprecipitation, using AB10, a monoclonal antibody raised to amino acids 1-17 of the beta-amyloid peptide sequence, which is present in all three alpha secretase-cleaved isoforms of interest. The next stage was separation of whole APP into its isoform components by two-dimensional gel electrophoresis. Because of low APP concentrations, detection by the usual staining methods, for example Sypro Ruby, able to detect low picomole concentrations, did not enable visualisation of the isoforms. Western analysis, however, enabled primary detection of APP, because of the inherent sensitivity of antibodies raised to specific isoform regions. This initial visualization acted as a template for excision of isoforms from 2D gels, which were then subjected to peptide mass mapping. Initial theoretical digestion of each isoform revealed the presence of specific peptides, which were then used as "tags" for isoform detection.

Alzheimer Disease↗

PubMed2000

Biotin carboxyl carrier protein isoforms in Brassicaceae oilseeds.

De novo fatty acid biosynthesis occurs predominantly in plastids. The committed step for this pathway is the production of malonyl-CoA catalysed by acetyl-CoA carboxylase (ACCase). In most plants, plastidial ACCase is a multisubunit complex minimally comprised of four polypeptides, which catalyse two reactions. In the simple oilseed plant, Arabidopsis thaliana, two cDNAs encoding biotin carboxyl carrier protein (BCCP) isoforms have been identified. The remaining three subunits of ACCase appear to be single gene members in A. thaliana [Mekhedov, Martinez de Ilarduya and Ohlrogge (2000) Plant Physiol. 122, 389-401]. Transcript and protein analyses indicate that BCCP isoform 1 is constitutively expressed while isoform 2 is predominantly expressed in developing seeds. The apparent masses of constitutive and seed-enriched BCCP isoforms agree with the apparent masses of recombinantly expressed isoforms 1 and 2, respectively. In a related oilseed, Brassica napus, multiple putative BCCP polypeptides were also observed in developing seeds. The presence of a divergent class of BCCP genes in A. thaliana and B. napus, coincident with appropriately sized biotin-containing proteins expressed specifically in developing seeds, suggests that these BCCPs play an evolutionarily conserved role in oil deposition.

Acetyl-CoA Carboxylase↗

PubMed2004

Selective association of sterol regulatory element-binding protein isoforms with target promoters in vivo.

The mRNAs for all three members of the sterol regulatory element-binding protein (SREBP) family are widely expressed, and the proteins are highly similar. They have potential to both hetero- and homodimerize through their bHLHLZ domains, so it has been difficult to definitively study the role of each one apart from the other two. In the current study, we have utilized cell lines that express only one functional SREBP and the chromatin immunoprecipitation technique to analyze individual SREBP binding to three specific target genes: hydroxymethylglutaryl-CoA reductase (Red), fatty acid synthase (FAS), and squalene synthase (SQS). Our studies show that SREBP-2 binds to promoters for all three genes, and in agreement with the original report using these cells, all three mRNAs are also induced. In the line expressing only SREBP-1a, mRNAs for Red and FAS are induced, but SQS is not. Chromatin immunoprecipitation also shows that SREBP-1a is recruited efficiently to Red and FAS promoters but not to SQS. This observation indicates SREBP-2 selectively binds the SQS promoter and is sufficient to explain the lack of SQS mRNA induction in the SREBP-1a-expressing cells. SREBP-1c protein was not stably recruited to any SREBP target promoter despite being fully active in DNA binding when purified from extracts of the corresponding cells. This is also sufficient to explain the lack of SREBP target gene induction by the singular expression of SREBP-1c. We also show that whereas SREBP-1a and -2 proteins interact efficiently with transcriptional co-activators that modify cellular chromatin, SREBP-1c does not. Taken together, our data support a model suggesting that chromatin modification is required during the initial stage of specific site recognition by SREBPs in native chromatin in vivo.

Base Sequence↗

PubMed1986

Activity of acyl carrier protein isoforms in reactions of plant Fatty Acid metabolism.

Two forms of spinach acyl carrier protein (ACP-I and ACP-II) have recently been characterized and found to be expressed in a tissue-specific manner (JB Ohlrogge, TM Kuo, 1985 J Biol Chem 260: 8032). To examine possible different functions for these ACP isoforms, we have tested purified preparations of spinach leaf ACP-I and ACP-II and Escherichia coli ACP in several in vitro reactions of fatty acid metabolism. Total de novo fatty acid synthesis and malonyl-CoA:ACP transacylase do not appear to discriminate between acyl carrier protein isoforms. In contrast, the K(m) of oleoyl-ACP thioesterase for oleoyl-ACP-II is 10-fold higher than for oleoyl-ACP-I, whereas the K(m) of acyl-ACP glycerol-3-phosphate acyl transferase is 5-fold higher for oleoyl-ACP-I than for oleoyl-ACP-II. A characterization of these reactions and a possible role for ACP isoforms in regulation of fatty acid metabolism in plants are described.

Journal Article↗

PubMed1999

Myelin basic protein isoforms in myelinating and remyelinating rat brain aggregate cultures.

Recent evidence suggests that myelin basic protein (MBP) exon-2-containing isoforms play a significant role in the onset of myelination because they are more abundant during early development. The pattern of expression of MBP exon-2-containing isoforms was studied in rat brain aggregate cultures during myelination to draw comparisons with the developing brain and at remyelination after demyelinative treatment. The pattern of MBP isoform expression in the aggregate cultures was found to be similar to that of the brain and was recapitulated after demyelination with antimyelin antibodies. Macrophage enrichment, resulting in increased accumulation of total MBP in the cultures, did not alter the isoform distribution. Both control and enriched cultures expressed a 16-kDa protein (26+/-9.8% of total MBP for control samples) that reacted with MBP antisera at the onset of myelination (day in vitro 14) but was barely detectable by day in vitro 21. The expression of this protein, also present in postnatal day 6 rat brain but no longer by day 11, has been predicted by reverse transcription polymerase chain reaction in embryonic mouse brain. The results of the present study reinforce the value of the aggregate culture system as a versatile yet accurate model of myelination and remyelination.

Animals↗

PubMed1999

Reduced levels of a specific myelin-associated oligodendrocytic basic protein isoform in shiverer myelin.

Myelin-associated oligodendrocytic basic protein (MOBP) and myelin basic protein (MBP) share many structural similarities. MOBP is synthesised by mature oligodendrocytes and localised at the major dense line (MDL), suggesting a role in the myelin compaction process. The shiverer mouse, a deletion mutant of the myelin basic protein (Mbp) gene, has poorly compacted myelin with essentially no MDL. In this study we compare the developmental expression of the Mobp gene in wild-type and shiverer mice. The significant finding is that one of the two abundant MOBP isoforms, the approximately 20-kD species, is poorly incorporated into shiverer myelin. The absence is specific to shiverer and is not a feature of dysmyelinating mutants with an abnormal intraperiod line. Our data suggest that incorporation of this MOBP isoform into shiverer myelin may be influenced by the presence of MBP or be a consequence of a disrupted MDL.

Aging↗