Tissue-specific Isoforms Research Articles

Expression of a novel cardiac-specific tropomyosin isoform in humans

Tropomyosins are a family of actin binding proteins encoded by a group of highly conserved genes. Humans have four tropomyosin-encoding genes: TPM1, TPM2, TPM3, and TPM4, each of which is known to generate multiple isoforms by alternative splicing, promoters, and 3′ end processing. TPM1 is the most versatile and encodes a variety of tissue specific isoforms. The TPM1 isoform specific to striated muscle, designated TPM1α, consists of 10 exons: 1a, 2b, 3, 4, 5, 6b, 7, 8, and 9a/b. In this study, using RT-PCR with adult and fetal human RNAs, we present evidence for the expression of a novel isoform of the TPM1 gene that is specifically expressed in cardiac tissues. The new isoform is designated TPM1κ and contains exon 2a instead of 2b. Ectopic expression of human GFP.TPM1κ fusion protein can promote myofibrillogenesis in cardiac mutant axolotl hearts that are lacking in tropomyosin.

Dystroglycan in skin and cutaneous cells: beta-subunit is shed from the cell surface.

In skin, hemidesmosomal protein complexes attach the epidermis to the dermis and are critical for stable connection of the basal epithelial cell cytoskeleton with the basement membrane (BM). In muscle, a similar supramolecular aggregate, the dystrophin glycoprotein complex links the inside of muscle cells with the BM. A component of the muscle complex, dystroglycan (DG), also occurs in epithelia. In this study, we characterized the expression and biochemical properties of authentic and recombinant DG in human skin and cutaneous cells in vitro. We show that DG is present at the epidermal BM zone, and it is produced by both keratinocytes and fibroblasts in vitro. The biosynthetic precursor is efficiently processed to the alpha- and beta-DG subunits; and, in addition, a distinct extracellular segment of the transmembranous beta-subunit is shed from the cell surface by metalloproteinases. Shedding of the beta-subunit releases the alpha-subunit from the DG complex on the cell surface into the extracellular space. The shedding is enhanced by IL-1beta and phorbol esters, and inhibited by metalloproteinase inhibitors. Deficiency of perlecan, a major ligand of alpha-DG, enhanced shedding suggesting that lack of a binding partner destabilizes the epithelial DG complex and makes it accessible to proteolytic processing.

Preconditioning: evolution of basic mechanisms to potential therapeutic strategies.

Preconditioning describes the phenomenon by which a traumatic or stressful stimulus confers protection against subsequent injury. Originally recognized in dog heart subjected to ischemic challenges, preconditioning has been demonstrated in multiple species, can be induced by various stimuli, and is applicable in different organ systems. Tremendous progress has been made elucidating the signal transduction cascade of preconditioning. Preconditioning represents a potent tissue-protective condition, and mechanistic understanding may allow safe clinical application. This review recalls the history of preconditioning and how it relates to the history of the investigation of endogenous adaptation; summarizes the current mechanistic understanding of acute preconditioning; outlines the signal transduction cascade leading to the development of delayed preconditioning; discusses preconditioning in noncardiac tissue; and explores the potential of using preconditioning clinically.

Alternative splicing of mouse transcription factors affects their DNA-binding domain architecture and is tissue specific

Splice variants of 461 transcription factor loci were analyzed using a new database of splice variants in the mouse transcriptome, MouSDB3, providing quantitative evidence that alternative splicing preferentially adds or deletes domains important to the DNA-binding function of the TFs.

Transcriptional control in male germ cells: general factor TFIIA participates in CREM-dependent gene activation.

Regulation of gene expression in haploid male germ cells follows a number of specific rules that differ from somatic cells. In this physiological context, transcriptional control mediated by the activator CREM (cAMP-responsive element modulator) represents an established paradigm. In somatic cells activation by CREM requires its phosphorylation at a unique regulatory site (Ser117) and subsequent interaction with the ubiquitous coactivator CBP (cAMP response element binding protein-binding protein). In testis, CREM transcriptional activity is controlled through interaction with a tissue-specific partner, ACT (activator of CREM in testis), which confers a powerful, phosphorylation-independent activation capacity. In addition to specialized transcription factors and coactivators, a variety of general factors of the basal transcriptional machinery, and their distinct tissue-specific isoforms, are highly expressed in testis, supporting the general notion that testis-specific gene expression requires specialized mechanisms. Here, we describe that CREM interacts with transcription factor IIA (TFIIA), a general transcription factor that stimulates RNA polymerase II-directed transcription. This association was identified by a two-hybrid screen, using a testis-derived cDNA library, and confirmed by coimmunoprecipitation. The interaction is restricted to the activator isoforms of CREM and does not require Ser117. Importantly, CREM does not interact with TFIIAtau-ALF, a testis-specific TFIIA homolog. CREM and TFIIA are expressed in a spatially and temporally coordinated fashion during the differentiation program of germ cells. The two proteins also colocalize intracellularly in spermatocyte and spermatid cells. These findings contribute to the understanding of the highly specialized rules of transcriptional regulation in haploid germ cells.

Alpha-cardiac actin (ACTC) binds to the band 3 (AE1) cardiac isoform.

The band 3 protein is the major integral protein present in the erythrocyte membrane. Two tissue-specific isoforms are also expressed in kidney alpha intercalated cells and in cardiomyocytes. It has been suggested that the cardiac isoform predominantly mediates the anion exchange in cardiomyocytes, but the role of the cytoplasmic domain of the band 3 (CDB3) protein in the cardiac tissue is unknown. In order to characterize novel associations of the CDB3 in the cardiac tissue, we performed the two-hybrid assay, using a bait comprising the region from leu 258 to leu 311 of the erythrocyte band 3, which must also be present in the cardiac isoform. The assay revealed two clones containing the C-terminal region of the alpha-cardiac actin. Immunoprecipitation of whole rat heart using an anti-actin antibody, immunoblotted with anti-human band 3, showed that actin binds to band 3 which was confirmed in the reverse assay. The confocal microscopy showed band 3 in the intercalated discs. Thus, besides the in vivo physical interaction in the Saccharomyces cerevisiae cell, we demonstrated using immunopreciptation that there is a physical association of band 3 with alpha-cardiac actin in cardiomyocyte, and we suggest that the binding occur "in situ," in the intercalated disc, a site of cell-cell contact and attachment of the sarcomere to the plasma membrane.

Distinct expression patterns of β-1,3-glucanases and chitinases during the germination of Solanaceous seeds

AbstractThe expression patterns of β-1,3-glucanases (βGlu) and chitinases (Chn) were investigated during the seed germination of members of theCestroideae(threeNicotianaspecies,Petunia hybrida) and theSolanoideae(Capsicum annuum,Physalis peruviana) subgroups of Solanaceous species. Rupture of the micropylar testa (seed coat) and rupture of the micropylar endosperm, i.e. radicle emergence, were distinct and temporally separate events during the germination ofCestroideae-type seeds. βGlu accumulation in imbibedCestroideae-type seeds, occurring after testa rupture but prior to endosperm rupture, was inhibited by abscisic acid (ABA) and promoted by gibberellins (GA) and light, in strict association with germination, and appeared to be caused by transcriptional regulation of the class I βGlu genes. The micropylar cap ofSolanoideae-type seeds does not allow a distinction between testa and endosperm rupture, but βGlu accumulation occurred prior to radicle emergence of pepper andP. peruvianaseeds. ABA inhibited endosperm rupture and βGlu accumulation in the micropylar cap of pepper seeds. In contrast to tomato, βGlu accumulation in pepper seeds was not only confined to the micropylar cap, was due to distinct, tissue-specific βGlu isoforms, and was not accompanied by Chn accumulation. In conclusion, ABA inhibition of germination and βGlu accumulation in the micropylar endosperm appears to be a widespread event during the seed germination of Solanaceous species. In contrast, accumulation of Chn and distinct βGlu isoforms in the embryo, prior to germination, appears to be a species-specific phenomenon within theSolanaceae. In addition, a post-germination co-induction of βGlu and Chn in the root of the emerged seedling was found in endospermic and non-endospermic species and could represent an evolutionarily conserved event during dicot seedling development.

Study of structure–function relationships in human glutamate dehydrogenases reveals novel molecular mechanisms for the regulation of the nerve tissue-specific (GLUD2) isoenzyme

In mammalian brain, glutamate dehydrogenase (GDH) is located predominantly in astrocytes, where is thought to play a role in transmitter glutamate’s metabolism. Human GDH exists in GLUD1 (housekeeping) and GLUD2 (nerve tissue-specific) isoforms, which share all but 15 out of their 505 amino acids. The GLUD1 GDH is potently inhibited by GTP, whereas the GLUD2 enzyme is resistant to this compound. On the other hand, the GLUD2 isoform assumes in the absence of GTP a conformational state associated with little catalytic activity, but it remains amenable to full activation by ADP and/or l-leucine. Site-directed mutagenesis of the GLUD1 gene at sites that differ from the corresponding residues of the GLUD2 gene showed that replacement of Gly456 by Ala made the enzyme resistant to GTP (IC50=2.8±0.15μM) compared to the wild-type GDH (IC50=0.19±0.01μM). In addition, substitution of Ser for Arg443 virtually abolished basal activity and rendered the enzyme dependent on ADP for its function. These properties may permit the neural enzyme to be recruited under conditions of low energy charge (high ADP:ATP ratio), similar to those that prevail in synaptic astrocytes during intense glutamatergic transmission. Hence, substitution of Ser for Arg443 and Ala for Gly456 are the main evolutionary changes that led to the adaptation of the GLUD2 GDH to the unique metabolic needs of the nerve tissue.

Spliced oct-1 mRNA isoforms with untranslated exons and a partly deleted region coding for the POU-specific domain

Transcription factor Oct-1 is involved in expression regulation of housekeeping genes, in lymphocyte differentiation, and in the immune response. Tissue-specific oct-1 mRNA isoforms are known to be expressed in lymphoid cells. Four new mouse isoforms were identified. Of these, two were tissue-specific (oct-1Rα and oct-1Rβ) and contained exon 1L. The oct-1Rα was shown to contain an additional fragment, which corresponds to an exon located in the 3"-region of mouse otf-1. No homolog was found in human OTF-1. The oct-1Rβ isoform proved to lack an exon coding for a fragment of the POU domain. This deletion results in a loss of the first helix of the domain, and the mutant protein is devoid of affinity for octamer ATGCAAAT. Two other mRNA isoforms, oct-1d and oct-1e, were shown to contain untranslated regions between exons 1U and 2. The regions correspond to exons 1i and 2i located between exons 1U and 1L in the 5"-region of the mouse oct-1 gene. Human OTF-1 was not found to contain exon 1i. On evidence of these and published data, it was assumed that a set of oct-1 isoforms is present in the cell, reflecting the complexity of expression regulation of оct-l and the multiplicity of its functions.

Glycogen phosphorylase as a molecular target for type 2 diabetes therapy.

The regulation of the hepatic glucose output through glycogenolysis is an important target for type 2 diabetes therapy. Glycogenolysis is catalyzed in liver, muscle and brain by tissue specific isoforms of glycogen phosphorylase (GP). Because of its central role in glycogen metabolism, GP has been exploited as a model for structure-assisted design of potent inhibitors, which may be relevant to the control of blood glucose concentrations in type 2 diabetes. Several regulatory binding sites have been identified in GP, such as the catalytic, the allosteric, and the inhibitor binding sites. Protein crystallography has contributed significant structural information on the specificity and interactions that distinguish the binding sites, and also revealed a new unexpected binding site (new allosteric site). In this review, the kinetic, crystallographic binding, and physiological studies of a number of compounds, inhibitors of GP, are described, and the essential inhibitory and binding properties of specific compounds are analyzed in an effort to provide rationalizations for the affinities of these compounds and to exploit the molecular interactions that might give rise to a better inhibitor. These studies have given new insights into fundamental structural aspects of the enzyme enhancing our understanding of how the enzyme recognizes and specifically binds ligands, that could be of potential therapeutic value in the treatment of type 2 diabetes.

Molecular cloning and characterization of novel tissue-specific isoforms of the human vacuolar H +-ATPase C, G and d subunits, and their evaluation in autosomal recessive distal renal tubular acidosis

Several of the 13 subunits comprising mammalian H +-ATPases have multiple isoforms, encoded by separate genes and with differing tissue expression patterns, which may play an important role in the intracellular localization and activity of H +-ATPases. Here we report the cloning of three previously uncharacterized human genes, ATP6V1C2, ATP6V1G3 and ATP6V0D2, encoding novel H +-ATPase subunit isoforms C2, G3 and d2, respectively. We demonstrate that these novel genes are expressed in kidney and few other tissues, and confirm previous reports that the C1, G1 and d1 isoforms are ubiquitously expressed, while G2 is brain-specific. Previously we have shown that mutations in two kidney-specific genes, ATP6V1B1 and ATP6V0A4, encoding the H +-ATPase B1 and a4 subunit isoforms, cause recessive distal renal tubular acidosis (dRTA). As the genes reported here are expressed mainly in kidney, we assessed their candidacy as causative genes for recessive dRTA in eight kindreds unlinked to either known disease locus. Although no potential disease-causing mutations were seen in this cohort, this does not rule out a role for these genes in other families. The identification of these three novel tissue-specific isoforms supports the hypothesis that subunit differences may play a key role in the structure, site and function of H +-ATPases within the cell.

Alternative Splicing of Brain-Specific PTB Defines a Tissue-Specific Isoform Pattern That Predicts Distinct Functional Roles

Splicing of neural-specific exons is differentially regulated in neuronal and non-neuronal cells. The polypyrimidine tract binding protein (PTB) has been implicated as a negative regulator for exon splicing, whereas the brain-specific homolog of PTB, termed nPTB, promotes exon splicing exclusively in neurons. We have now isolated a novel mRNA splice variant of nPTB from non-neuronal cells. In contrast to the neural nPTB transcript, the expression of this novel isoform was absent from brain tissue and was generated in non-neuronal cells by alternative splicing to include five additional amino acid residues encoded by exon 9. In addition, we identified a brain-specific transcript containing a novel, alternatively spliced, internal exon 10. The exclusion of this 34-nucleotide exon 10 in non-neuronal tissues generates a premature termination codon and results in the truncation of the open reading frame. Our findings suggest that alternative splicing of nPTB has an important role in regulation of tissue-specific gene expression and thus in the functional activity of nPTB in neuronal and non-neuronal cells.

A tissue-specific exon of myosin Va is responsible for selective cargo binding in melanocytes.

Class V myosins are molecular motors used for intracellular transportation and organelle tethering. The mouse Myosin Va (MyoVa) is encoded by the dilute locus, which is alternatively spliced to generate several tissue specific isoforms. The tail of MyoVa is the putative cargo-binding domain. To determine the functions of different isoforms of MyoVa and the minimal cargo-binding region, we tagged various isoforms and different portions of the mouse MyoVa tail with a green fluorescent protein and examined their intracellular localizations in the mouse melan-a cells. We found that the amino acid sequence encoded by an alternatively spliced exon, exon F, is necessary for the selective binding of MyoVa to melanosome. The MyoVa isoforms lacking this amino acid sequence are not targeted to the melanosomes, but localized to the perinuclear region instead. These findings suggested that MyoVa is able to bind to more than one types of cargos, with the selectivities determined by alternative spliced sequences.

Proteomic survey of metabolic pathways in rice.

A systematic proteomic analysis of rice (Oryza sativa) leaf, root, and seed tissue using two independent technologies, two-dimensional gel electrophoresis followed by tandem mass spectrometry and multidimensional protein identification technology, allowed the detection and identification of 2,528 unique proteins, which represents the most comprehensive proteome exploration to date. A comparative display of the expression patterns indicated that enzymes involved in central metabolic pathways are present in all tissues, whereas metabolic specialization is reflected in the occurrence of a tissue-specific enzyme complement. For example, tissue-specific and subcellular compartment-specific isoforms of ADP-glucose pyrophosphorylase were detected, thus providing proteomic confirmation of the presence of distinct regulatory mechanisms involved in the biosynthesis and breakdown of separate starch pools in different tissues. In addition, several previously characterized allergenic proteins were identified in the seed sample, indicating the potential of proteomic approaches to survey food samples with regard to the occurrence of allergens.

Generation of a cell line with smooth muscle phenotype from hypertrophied urinary bladder.

We have established a cell line from hypertrophied rabbit urinary bladder smooth muscle (SM) that stably expresses SM myosin (SMM). These cells, termed BSM, are spindle shaped and form swirls, similar to the "hills and valleys" described for cultured aortic SM cells. Western blotting revealed that BSM expresses the amino-terminal SMM heavy chain isoform SM-B, the carboxy-terminal SM1 and SM2 isoforms, and SM alpha-actin. In addition, they express cGMP-dependent protein kinase G, made by contractile SM cells in vitro but not by noncontractile cells synthesizing extracellular matrix. Immunofluorescence studies indicate a homogeneous population of cells expressing alpha-actin and SMM, including the SM-B isoform, and karyotyping demonstrates a stable 4N chromosomal pattern. These cells also express calcium-dependent myosin light chain kinase and phosphatase activity and contract in response to the muscarinic agonist bethanechol. To our knowledge, BSM is the first visceral SM cell line that expresses the SM-B isoform and might serve as a useful model to study the transcriptional regulation of tissue-specific SMM isoforms in differentiation and pathological SM.

Mapping the human translation elongation factor eEF1H complex using the yeast two-hybrid system.

In eukaryotes, the eukaryotic translation elongation factor eEF1A responsible for transporting amino-acylated tRNA to the ribosome forms a higher-order complex, eEF1H, with its guanine-nucleotide-exchange factor eEF1B. In metazoans, eEF1B consists of three subunits: eEF1B alpha, eEF1B eta and eEF1B gamma. The first two subunits possess the nucleotide-exchange activity, whereas the role of the last remains poorly defined. In mammals, two active tissue-specific isoforms of eEF1A have been identified. The reason for this pattern of differential expression is unknown. Several models on the basis of in vitro experiments have been proposed for the macromolecular organization of the eEF1H complex. However, these models differ in various aspects. This might be due to the difficulties of handling, particularly the eEF1B beta and eEF1B gamma subunits in vitro. Here, the human eEF1H complex is for the first time mapped using the yeast two-hybrid system, which is a powerful in vivo technique for analysing protein-protein interactions. The following complexes were observed: eEF1A1:eEF1B alpha, eEF1A1:eEF1B beta, eEF1B beta:eEF1B beta, eEF1B alpha:eEF1B gamma, eEF1B beta:eEF1B gamma and eEF1B alpha:eEF1B gamma:eEF1B beta, where the last was observed using a three-hybrid approach. Surprisingly, eEF1A2 showed no or only little affinity for the guanine-nucleotide-exchange factors. Truncated versions of the subunits of eEF1B were used to orientate these subunits within the resulting model. The model unit is a pentamer composed of two molecules of eEF1A, each interacting with either eEF1B alpha or eEF1B beta held together by eEF1B gamma. These units can dimerize via eEF1B beta. Our model is compared with other models, and structural as well as functional aspects of the model are discussed.

Glycogen and its metabolism.

Glycogen is a branched polymer of glucose which serves as a reservoir of glucose units. The two largest deposits in mammals are in the liver and skeletal muscle but many cells are capable synthesizing glycogen. Its accumulation and utilization are under elaborate controls involving primarily covalent phosphorylation and allosteric ligand binding. Both muscle and liver glycogen reserves are important for whole body glucose metabolism and their replenishment is linked hormonally to nutritional status. Control differs between muscle and liver in part due to the existence of different tissue-specific isoforms at key steps. Control of synthesis is shared between transport into the muscle and the step catalyzed by glycogen synthase. Breakdown of liver glycogen, as part of blood glucose homeostasis, is also in response to nutritional cues. Muscle glycogen serves only to fuel muscular activity and its utilization is controlled by muscle contraction and by catecholamines. Though the number of enzymes directly involved in the metabolism of glycogen is quite small, many more proteins act indirectly in a regulatory capacity. Defects in the basic metabolizing enzymes lead to severe consequences whereas, with some exceptions, mutations in the regulatory proteins appear to cause a more subtle phenotypic change.

Calpain

The calcium-dependent thiol proteases, calpains, are widely expressed with ubiquitous and tissue specific isoforms. Calpains have been implicated in basic cellular processes including cell proliferation, apoptosis and differentiation. The focus of the current review is to summarize recent findings implicating calpains in cytoskeletal rearrangements and cell migration. Calpain cleaves many cytosolic proteins and therefore to be effective and limited in its scope, calpain activity has to be tightly regulated both temporally and spatially. Some mechanisms of regulation include calcium, growth factor-mediated phosphorylation and membrane targeting. Calpain inhibition reduces migration rates and inhibits cell invasiveness. Two putative mechanisms of calpain action during migration include its role as a signaling intermediate, acting upstream of Rho, and its effects on focal adhesion structure and disassembly. Therefore, calpains and downstream signaling molecules may be future targets for therapeutic interventions to treat cancer or chronic inflammation.

Isoform-specific expression of hypoxia-inducible factor-1alpha during the late stages of mouse spermiogenesis.

The heterodimeric hypoxia-inducible factor (HIF)-1 is a transcriptional master regulator of several genes involved in mammalian oxygen homeostasis, including erythropoietin, vascular endothelial growth factor, and factors involved in glucose transport and metabolism. The mouse Hif1a gene is expressed from two distinct promoter/first exon combinations resulting in tissue-specific (mHIF-1alphaI.1) and ubiquitous (mHIF-1alphaI.2) mRNA isoforms. By in situ hybridization, we detected mHIF-1alphaI.1 mRNA exclusively in the elongated spermatids of the testis. In vitro studies indicated that the switch from mHIF-1alphaI.2 to mHIF-1alphaI.1 mRNA expression does not occur at the premeiotic stages of mouse spermatogenesis. Exposure of mice to hypoxic conditions induced mHIF-1alphaI.2 protein in spermatocytes and probably in Sertoli cells but not in spermatogonia. In contrast, expression of the putative mHIF-1alphaI.1 protein in spermatozoa of the testis and epididymis was oxygen independent and located to the midpiece of the spermatozoal flagellum. Both the switch in transcript expression during spermiogenesis and the unexpected protein localization in mature sperm cells suggest a so far unrecognized function of HIF-1alpha.

Membrane cofactor protein (MCP; CD46) expression in transgenic mice.

Human membrane cofactor protein (MCP; CD46) is a widely distributed complement regulator. In the mouse, expression of MCP is largely restricted to the testis while a related, widely expressed protein (Crry) appears to perform MCP's (CD46) regulatory activity. We have developed two mouse strains transgenic for human MCP (CD46) utilizing an approximately 400 kb YAC clone carrying the complete gene. A third mouse strain was generated using an overlapping YAC clone isolated from a second library. The expression of human MCP (CD46) in these mouse strains was characterized by immunohistochemistry, FACS, Western blotting and RT-PCR. No differences were detected in the isoform pattern or distribution among the three strains, although the expression level varied according to how many copies of the gene were integrated. The expression profile closely mimicked that observed in humans, including the same pattern of isoform expression as the donor. In addition, tissue-specific isoform expression in the kidney, salivary gland and brain paralleled that observed in man. The transgenic mice expressed low levels of MCP (CD46) on their E, in contrast to humans but in line with most other primates. These mice should be a useful tool to analyse tissue-specific expression, to establish animal models of infections and to characterize the role of MCP (CD46) in reproduction.