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Sample records for distinct hif-alpha isoforms

  1. Distinct Functions of Endophilin Isoforms in Synaptic Vesicle Endocytosis

    PubMed Central

    Zhang, Jifeng; Tan, Minghui; Yin, Yichen; Ren, Bingyu; Jiang, Nannan; Guo, Guoqing; Chen, Yuan

    2015-01-01

    Endophilin isoforms perform distinct characteristics in their interactions with N-type Ca2+ channels and dynamin. However, precise functional differences for the endophilin isoforms on synaptic vesicle (SV) endocytosis remain unknown. By coupling RNA interference and electrophysiological recording techniques in cultured rat hippocampal neurons, we investigated the functional differences of three isoforms of endophilin in SV endocytosis. The results showed that the amplitude of normalized evoked excitatory postsynaptic currents in endophilin1 knockdown neurons decreased significantly for both single train and multiple train stimulations. Similar results were found using endophilin2 knockdown neurons, whereas endophilin3 siRNA exhibited no change compared with control neurons. Endophilin1 and endophilin2 affected SV endocytosis, but the effect of endophilin1 and endophilin2 double knockdown was not different from that of either knockdown alone. This result suggested that endophilin1 and endophilin2 functioned together but not independently during SV endocytosis. Taken together, our results indicate that SV endocytosis is sustained by endophilin1 and endophilin2 isoforms, but not by endophilin3, in primary cultured hippocampal neurons. PMID:26682072

  2. Alternative splicing results in RET isoforms with distinct trafficking properties

    PubMed Central

    Richardson, Douglas S.; Rodrigues, David M.; Hyndman, Brandy D.; Crupi, Mathieu J. F.; Nicolescu, Adrian C.; Mulligan, Lois M.

    2012-01-01

    RET encodes a receptor tyrosine kinase that is essential for spermatogenesis, development of the sensory, sympathetic, parasympathetic, and enteric nervous systems and the kidneys, as well as for maintenance of adult midbrain dopaminergic neurons. RET is alternatively spliced to encode multiple isoforms that differ in their C-terminal amino acids. The RET9 and RET51 isoforms display unique levels of autophosphorylation and have differential interactions with adaptor proteins. They induce distinct gene expression patterns, promote different levels of cell differentiation and transformation, and play unique roles in development. Here we present a comprehensive study of the subcellular localization and trafficking of RET isoforms. We show that immature RET9 accumulates intracellularly in the Golgi, whereas RET51 is efficiently matured and present in relatively higher amounts on the plasma membrane. RET51 is internalized faster after ligand binding and undergoes recycling back to the plasma membrane. This differential trafficking of RET isoforms produces a more rapid and longer duration of signaling through the extracellular-signal regulated kinase/mitogen-activated protein kinase pathway downstream of RET51 relative to RET9. Together these differences in trafficking properties contribute to some of the functional differences previously observed between RET9 and RET51 and establish the important role of intracellular trafficking in modulating and maintaining RET signaling. PMID:22875993

  3. Distinct Functional Interactions between Actin Isoforms and Nonsarcomeric Myosins

    PubMed Central

    Müller, Mirco; Diensthuber, Ralph P.; Chizhov, Igor; Claus, Peter; Heissler, Sarah M.; Preller, Matthias; Taft, Manuel H.; Manstein, Dietmar J.

    2013-01-01

    Despite their near sequence identity, actin isoforms cannot completely replace each other in vivo and show marked differences in their tissue-specific and subcellular localization. Little is known about isoform-specific differences in their interactions with myosin motors and other actin-binding proteins. Mammalian cytoplasmic β- and γ-actin interact with nonsarcomeric conventional myosins such as the members of the nonmuscle myosin-2 family and myosin-7A. These interactions support a wide range of cellular processes including cytokinesis, maintenance of cell polarity, cell adhesion, migration, and mechano-electrical transduction. To elucidate differences in the ability of isoactins to bind and stimulate the enzymatic activity of individual myosin isoforms, we characterized the interactions of human skeletal muscle α-actin, cytoplasmic β-actin, and cytoplasmic γ-actin with human myosin-7A and nonmuscle myosins-2A, -2B and -2C1. In the case of nonmuscle myosins-2A and -2B, the interaction with either cytoplasmic actin isoform results in 4-fold greater stimulation of myosin ATPase activity than was observed in the presence of α-skeletal muscle actin. Nonmuscle myosin-2C1 is most potently activated by β-actin and myosin-7A by γ-actin. Our results indicate that β- and γ-actin isoforms contribute to the modulation of nonmuscle myosin-2 and myosin-7A activity and thereby to the spatial and temporal regulation of cytoskeletal dynamics. FRET-based analyses show efficient copolymerization abilities for the actin isoforms in vitro. Experiments with hybrid actin filaments show that the extent of actomyosin coupling efficiency can be regulated by the isoform composition of actin filaments. PMID:23923011

  4. GAGA Factor Isoforms Have Distinct but Overlapping Functions In Vivo

    PubMed Central

    Greenberg, Anthony J.; Schedl, Paul

    2001-01-01

    The Drosophila melanogaster GAGA factor (encoded by the Trithorax-like [Trl] gene) is required for correct chromatin architecture at diverse chromosomal sites. The Trl gene encodes two alternatively spliced isoforms of the GAGA factor (GAGA-519 and GAGA-581) that are identical except for the length and sequence of the C-terminal glutamine-rich (Q) domain. In vitro and tissue culture experiments failed to find any functional difference between the two isoforms. We made a set of transgenes that constitutively express cDNAs coding for either of the isoforms with the goal of elucidating their roles in vivo. Phenotypic analysis of the transgenes in Trl mutant background led us to the conclusion that GAGA-519 and GAGA-581 perform different, albeit largely overlapping, functions. We also expressed a fusion protein with LacZ disrupting the Q domain of GAGA-519. This LacZ fusion protein compensated for the loss of wild-type GAGA factor to a surprisingly large extent. This suggests that the Q domain either is not required for the essential functions performed by the GAGA protein or is exclusively used for tetramer formation. These results are inconsistent with a major role of the Q domain in chromatin remodeling or transcriptional activation. We also found that GAGA-LacZ was able to associate with sites not normally occupied by the GAGA factor, pointing to a role of the Q domain in binding site choice in vivo. PMID:11713290

  5. Distinct roles for protein kinase C isoforms in regulating platelet purinergic receptor function.

    PubMed

    Mundell, Stuart J; Jones, Matthew L; Hardy, Adam R; Barton, Johanna F; Beaucourt, Stephanie M; Conley, Pamela B; Poole, Alastair W

    2006-09-01

    ADP is a critical regulator of platelet activation, mediating its actions through two G protein-coupled receptors (GPCRs), P2Y1 and P2Y12. We have shown previously that the receptors are functionally desensitized, in a homologous manner, by distinct kinase-dependent mechanisms in which P2Y1 is regulated by protein kinase C (PKC) and P2Y12 by G protein-coupled receptor kinases. In this study, we addressed whether different PKC isoforms play different roles in regulating the trafficking and activity of these two GPCRs. Expression of PKCalpha and PKCdelta dominant-negative mutants in 1321N1 cells revealed that both isoforms regulated P2Y1 receptor signaling and trafficking, although only PKCdelta was capable of regulating P2Y12, in experiments in which PKC was directly activated by the phorbol ester phorbol 12-myristate 13-acetate (PMA). These results were paralleled in human platelets, in which PMA reduced subsequent ADP-induced P2Y1 and P2Y12 receptor signaling. PKC isoform-selective inhibitors revealed that novel, but not conventional, isoforms of PKC regulate P2Y12 function, whereas both novel and classic isoforms regulate P2Y1 activity. It is also noteworthy that we studied receptor internalization in platelets by a radioligand binding approach showing that both receptors internalize rapidly in these cells. ADP-induced P2Y1 receptor internalization is attenuated by PKC inhibitors, whereas that of the P2Y12 receptor is unaffected. Both P2Y1 and P2Y12 receptors can also undergo PMA-stimulated internalization, and here again, novel but not classic PKCs regulate P2Y12, whereas both novel and classic isoforms regulate P2Y1 internalization. This study therefore is the first to reveal distinct roles for PKC isoforms in the regulation of platelet P2Y receptor function and trafficking. PMID:16804093

  6. Distinct roles of class IA PI3K isoforms in primary and immortalised macrophages.

    PubMed

    Papakonstanti, Evangelia A; Zwaenepoel, Olivier; Bilancio, Antonio; Burns, Emily; Nock, Gemma E; Houseman, Benjamin; Shokat, Kevan; Ridley, Anne J; Vanhaesebroeck, Bart

    2008-12-15

    The class IA isoforms of phosphoinositide 3-kinase (p110alpha, p110beta and p110delta) often have non-redundant functions in a given cell type. However, for reasons that are unclear, the role of a specific PI3K isoform can vary between cell types. Here, we compare the relative contributions of PI3K isoforms in primary and immortalised macrophages. In primary macrophages stimulated with the tyrosine kinase ligand colony-stimulating factor 1 (CSF1), all class IA PI3K isoforms participate in the regulation of Rac1, whereas p110delta selectively controls the activities of Akt, RhoA and PTEN, in addition to controlling proliferation and chemotaxis. The prominent role of p110delta in these cells correlates with it being the main PI3K isoform that is recruited to the activated CSF1 receptor (CSF1R). In immortalised BAC1.2F5 macrophages, however, the CSF1R also engages p110alpha, which takes up a more prominent role in CSF1R signalling, in processes including Akt phosphorylation and regulation of DNA synthesis. Cell migration, however, remains dependent mainly on p110delta. In other immortalised macrophage cell lines, such as IC-21 and J774.2, p110alpha also becomes more prominently involved in CSF1-induced Akt phosphorylation, at the expense of p110delta.These data show that PI3K isoforms can be differentially regulated in distinct cellular contexts, with the dominant role of the p110delta isoform in Akt phosphorylation and proliferation being lost upon cell immortalisation. These findings suggest that p110delta-selective PI3K inhibitors may be more effective in inflammation than in cancer. PMID:19033389

  7. Distinct Temporal Regulation of RET Isoform Internalization: Roles of Clathrin and AP2.

    PubMed

    Crupi, Mathieu J F; Yoganathan, Piriya; Bone, Leslie N; Lian, Eric; Fetz, Andrew; Antonescu, Costin N; Mulligan, Lois M

    2015-11-01

    The RET receptor tyrosine kinase (RTK) contributes to kidney and nervous system development, and is implicated in a number of human cancers. RET is expressed as two protein isoforms, RET9 and RET51, with distinct interactions and signaling properties that contribute to these processes. RET isoforms are internalized from the cell surface into endosomal compartments in response to glial cell line-derived neurotropic factor (GDNF) ligand stimulation but the specific mechanisms of RET trafficking remain to be elucidated. Here, we used total internal reflection fluorescence (TIRF) microscopy to demonstrate that RET internalization occurs primarily through clathrin coated pits (CCPs). Activated RET receptors colocalize with clathrin, but not caveolin. The RET51 isoform is rapidly and robustly recruited to CCPs upon GDNF stimulation, while RET9 recruitment occurs more slowly and is less pronounced. We showed that the clathrin-associated adaptor protein complex 2 (AP2) interacts directly with each RET isoform through its AP2 μ subunit, and is important for RET internalization. Our data establish that interactions with the AP2 complex promote RET receptor internalization via clathrin-mediated endocytosis but that RET9 and RET51 have distinct internalization kinetics that may contribute to differences in their biological functions. PMID:26304132

  8. The Splice Isoforms of the Drosophila Ecdysis Triggering Hormone Receptor Have Developmentally Distinct Roles.

    PubMed

    Diao, Feici; Mena, Wilson; Shi, Jonathan; Park, Dongkook; Diao, Fengqiu; Taghert, Paul; Ewer, John; White, Benjamin H

    2016-01-01

    To grow, insects must periodically shed their exoskeletons. This process, called ecdysis, is initiated by the endocrine release of Ecdysis Trigger Hormone (ETH) and has been extensively studied as a model for understanding the hormonal control of behavior. Understanding how ETH regulates ecdysis behavior, however, has been impeded by limited knowledge of the hormone's neuronal targets. An alternatively spliced gene encoding a G-protein-coupled receptor (ETHR) that is activated by ETH has been identified, and several lines of evidence support a role in ecdysis for its A-isoform. The function of a second ETHR isoform (ETHRB) remains unknown. Here we use the recently introduced "Trojan exon" technique to simultaneously mutate the ETHR gene and gain genetic access to the neurons that express its two isoforms. We show that ETHRA and ETHRB are expressed in largely distinct subsets of neurons and that ETHRA- but not ETHRB-expressing neurons are required for ecdysis at all developmental stages. However, both genetic and neuronal manipulations indicate an essential role for ETHRB at pupal and adult, but not larval, ecdysis. We also identify several functionally important subsets of ETHR-expressing neurons including one that coexpresses the peptide Leucokinin and regulates fluid balance to facilitate ecdysis at the pupal stage. The general strategy presented here of using a receptor gene as an entry point for genetic and neuronal manipulations should be useful in establishing patterns of functional connectivity in other hormonally regulated networks. PMID:26534952

  9. RAS and RHO families of GTPases directly regulate distinct phosphoinositide 3-kinase isoforms.

    PubMed

    Fritsch, Ralph; de Krijger, Inge; Fritsch, Kornelia; George, Roger; Reason, Beth; Kumar, Madhu S; Diefenbacher, Markus; Stamp, Gordon; Downward, Julian

    2013-05-23

    RAS proteins are important direct activators of p110α, p110γ, and p110δ type I phosphoinositide 3-kinases (PI3Ks), interacting via an amino-terminal RAS-binding domain (RBD). Here, we investigate the regulation of the ubiquitous p110β isoform of PI3K, implicated in G-protein-coupled receptor (GPCR) signaling, PTEN-loss-driven cancers, and thrombocyte function. Unexpectedly, RAS is unable to interact with p110β, but instead RAC1 and CDC42 from the RHO subfamily of small GTPases bind and activate p110β via its RBD. In fibroblasts, GPCRs couple to PI3K through Dock180/Elmo1-mediated RAC activation and subsequent interaction with p110β. Cells from mice carrying mutations in the p110β RBD show reduced PI3K activity and defective chemotaxis, and these mice are resistant to experimental lung fibrosis. These findings revise our understanding of the regulation of type I PI3K by showing that both RAS and RHO family GTPases directly regulate distinct ubiquitous PI3K isoforms and that RAC activates p110β downstream of GPCRs. PMID:23706742

  10. Two alternatively spliced isoforms of the Arabidopsis SR45 protein have distinct roles during normal plant development.

    PubMed

    Zhang, Xiao-Ning; Mount, Stephen M

    2009-07-01

    The serine-arginine-rich (SR) proteins constitute a conserved family of pre-mRNA splicing factors. In Arabidopsis (Arabidopsis thaliana), they are encoded by 19 genes, most of which are themselves alternatively spliced. In the case of SR45, the use of alternative 3' splice sites 21 nucleotides apart generates two alternatively spliced isoforms. Isoform 1 (SR45.1) has an insertion relative to isoform 2 (SR45.2) that replaces a single arginine with eight amino acids (TSPQRKTG). The biological implications of SR45 alternative splicing have been unclear. A previously described loss-of-function mutant affecting both isoforms, sr45-1, shows several developmental defects, including defects in petal development and root growth. We found that the SR45 promoter is highly active in regions with actively growing and dividing cells. We also tested the ability of each SR45 isoform to complement the sr45-1 mutant by overexpression of isoform-specific green fluorescent protein (GFP) fusion proteins. As expected, transgenic plants overexpressing either isoform displayed both nuclear speckles and GFP fluorescence throughout the nucleoplasm. We found that SR45.1-GFP complements the flower petal phenotype, but not the root growth phenotype. Conversely, SR45.2-GFP complements root growth but not floral morphology. Mutation of a predicted phosphorylation site within the alternatively spliced segment, SR45.1-S219A-GFP, does not affect complementation. However, a double mutation affecting both serine-219 and the adjacent threonine-218 (SR45.1-T218A + S219A-GFP) behaves like isoform 2, complementing the root but not the floral phenotype. In conclusion, our study provides evidence that the two alternatively spliced isoforms of SR45 have distinct biological functions. PMID:19403727

  11. Distinct or shared actions of peptide family isoforms: II. Multiple pyrokinins exert similar effects in the lobster stomatogastric nervous system.

    PubMed

    Dickinson, Patsy S; Kurland, Sienna C; Qu, Xuan; Parker, Brett O; Sreekrishnan, Anirudh; Kwiatkowski, Molly A; Williams, Alex H; Ysasi, Alexandra B; Christie, Andrew E

    2015-09-01

    Many neuropeptides are members of peptide families, with multiple structurally similar isoforms frequently found even within a single species. This raises the question of whether the individual peptides serve common or distinct functions. In the accompanying paper, we found high isoform specificity in the responses of the lobster (Homarus americanus) cardiac neuromuscular system to members of the pyrokinin peptide family: only one of five crustacean isoforms showed any bioactivity in the cardiac system. Because previous studies in other species had found little isoform specificity in pyrokinin actions, we examined the effects of the same five crustacean pyrokinins on the lobster stomatogastric nervous system (STNS). In contrast to our findings in the cardiac system, the effects of the five pyrokinin isoforms on the STNS were indistinguishable: they all activated or enhanced the gastric mill motor pattern, but did not alter the pyloric pattern. These results, in combination with those from the cardiac ganglion, suggest that members of a peptide family in the same species can be both isoform specific and highly promiscuous in their modulatory capacity. The mechanisms that underlie these differences in specificity have not yet been elucidated; one possible explanation, which has yet to be tested, is the presence and differential distribution of multiple receptors for members of this peptide family. PMID:26206359

  12. Distinct or shared actions of peptide family isoforms: II. Multiple pyrokinins exert similar effects in the lobster stomatogastric nervous system

    PubMed Central

    Dickinson, Patsy S.; Kurland, Sienna C.; Qu, Xuan; Parker, Brett O.; Sreekrishnan, Anirudh; Kwiatkowski, Molly A.; Williams, Alex H.; Ysasi, Alexandra B.; Christie, Andrew E.

    2015-01-01

    ABSTRACT Many neuropeptides are members of peptide families, with multiple structurally similar isoforms frequently found even within a single species. This raises the question of whether the individual peptides serve common or distinct functions. In the accompanying paper, we found high isoform specificity in the responses of the lobster (Homarus americanus) cardiac neuromuscular system to members of the pyrokinin peptide family: only one of five crustacean isoforms showed any bioactivity in the cardiac system. Because previous studies in other species had found little isoform specificity in pyrokinin actions, we examined the effects of the same five crustacean pyrokinins on the lobster stomatogastric nervous system (STNS). In contrast to our findings in the cardiac system, the effects of the five pyrokinin isoforms on the STNS were indistinguishable: they all activated or enhanced the gastric mill motor pattern, but did not alter the pyloric pattern. These results, in combination with those from the cardiac ganglion, suggest that members of a peptide family in the same species can be both isoform specific and highly promiscuous in their modulatory capacity. The mechanisms that underlie these differences in specificity have not yet been elucidated; one possible explanation, which has yet to be tested, is the presence and differential distribution of multiple receptors for members of this peptide family. PMID:26206359

  13. NFAT isoforms play distinct roles in TNFα-induced retinal leukostasis

    PubMed Central

    Bretz, Colin A.; Savage, Sara R.; Capozzi, Megan E.; Suarez, Sandra; Penn, John S.

    2015-01-01

    The objective of this study was to determine the role of individual NFAT isoforms in TNFα-induced retinal leukostasis. To this end, human retinal microvascular endothelial cells (HRMEC) transfected with siRNA targeting individual NFAT isoforms were treated with TNFα, and qRT-PCR was used to examine the contribution of each isoform to the TNFα-induced upregulation of leukocyte adhesion proteins. This showed that NFATc1 siRNA increased ICAM1 expression, NFATc2 siRNA reduced CX3CL1, VCAM1, SELE, and ICAM1 expression, NFATc3 siRNA increased CX3CL1 and SELE expression, and NFATc4 siRNA reduced SELE expression. Transfected HRMEC monolayers were also treated with TNFα and assayed using a parallel plate flow chamber, and both NFATc2 and NFATc4 knockdown reduced TNFα-induced cell adhesion. The effect of isoform-specific knockdown on TNFα-induced cytokine production was also measured using protein ELISAs and conditioned cell culture medium, and showed that NFATc4 siRNA reduced CXCL10, CXCL11, and MCP-1 protein levels. Lastly, the CN/NFAT-signaling inhibitor INCA-6 was shown to reduce TNFα-induced retinal leukostasis in vivo. Together, these studies show a clear role for NFAT-signaling in TNFα-induced retinal leukostasis, and identify NFATc2 and NFATc4 as potentially valuable therapeutic targets for treating retinopathies in which TNFα plays a pathogenic role. PMID:26527057

  14. Distinct Regulation of Transmitter Release at the Drosophila NMJ by Different Isoforms of nemy

    PubMed Central

    Knight, David; Iliadi, Konstantin G.; Iliadi, Natalia; Wilk, Ronit; Hu, Jack; Krause, Henry M.; Taylor, Paul; Moran, Michael F.; Boulianne, Gabrielle L.

    2015-01-01

    Synaptic transmission is highly plastic and subject to regulation by a wide variety of neuromodulators and neuropeptides. In the present study, we have examined the role of isoforms of the cytochrome b561 homologue called no extended memory (nemy) in regulation of synaptic strength and plasticity at the neuromuscular junction (NMJ) of third instar larvae in Drosophila. Specifically, we generated two independent excisions of nemy that differentially affect the expression of nemy isoforms. We show that the nemy45 excision, which specifically reduces the expression of the longest splice form of nemy, leads to an increase in stimulus evoked transmitter release and altered synaptic plasticity at the NMJ. Conversely, the nemy26.2 excision, which appears to reduce the expression of all splice forms except the longest splice isoform, shows a reduction in stimulus evoked transmitter release, and enhanced synaptic plasticity. We further show that nemy45 mutants have reduced levels of amidated peptides similar to that observed in peptidyl-glycine hydryoxylating mono-oxygenase (PHM) mutants. In contrast, nemy26.2 mutants show no defects in peptide amidation but rather display a decrease in Tyramine β hydroxylase activity (TβH). Taken together, these results show non-redundant roles for the different nemy isoforms and shed light on the complex regulation of neuromodulators. PMID:26237434

  15. Modeling autosomal recessive cutis laxa type 1C in mice reveals distinct functions for Ltbp-4 isoforms

    PubMed Central

    Bultmann-Mellin, Insa; Conradi, Anne; Maul, Alexandra C.; Dinger, Katharina; Wempe, Frank; Wohl, Alexander P.; Imhof, Thomas; Wunderlich, F. Thomas; Bunck, Alexander C.; Nakamura, Tomoyuki; Koli, Katri; Bloch, Wilhelm; Ghanem, Alexander; Heinz, Andrea; von Melchner, Harald; Sengle, Gerhard; Sterner-Kock, Anja

    2015-01-01

    Recent studies have revealed an important role for LTBP-4 in elastogenesis. Its mutational inactivation in humans causes autosomal recessive cutis laxa type 1C (ARCL1C), which is a severe disorder caused by defects of the elastic fiber network. Although the human gene involved in ARCL1C has been discovered based on similar elastic fiber abnormalities exhibited by mice lacking the short Ltbp-4 isoform (Ltbp4S−/−), the murine phenotype does not replicate ARCL1C. We therefore inactivated both Ltbp-4 isoforms in the mouse germline to model ARCL1C. Comparative analysis of Ltbp4S−/− and Ltbp4-null (Ltbp4−/−) mice identified Ltbp-4L as an important factor for elastogenesis and postnatal survival, and showed that it has distinct tissue expression patterns and specific molecular functions. We identified fibulin-4 as a previously unknown interaction partner of both Ltbp-4 isoforms and demonstrated that at least Ltbp-4L expression is essential for incorporation of fibulin-4 into the extracellular matrix (ECM). Overall, our results contribute to the current understanding of elastogenesis and provide an animal model of ARCL1C. PMID:25713297

  16. Distinct freshwater and seawater isoforms of Na+/K+-ATPase in gill chloride cells of Atlantic salmon

    USGS Publications Warehouse

    McCormick, Stephen D.; Regish, A.M.; Christensen, A.K.

    2009-01-01

    Gill Na(+)/K(+)-ATPase (NKA) in teleost fishes is involved in ion regulation in both freshwater and seawater. We have developed and validated rabbit polyclonal antibodies specific to the NKA alpha1a and alpha1b protein isoforms of Atlantic salmon (Salmo salar Linnaeus), and used western blots and immunohistochemistry to characterize their size, abundance and localization. The relative molecular mass of NKA alpha1a is slightly less than that for NKA beta1b. The abundance of gill NKA alpha1a was high in freshwater and became nearly undetectable after seawater acclimation. NKA alpha1b was present in small amounts in freshwater and increased 13-fold after seawater acclimation. Both NKA isoforms were detected only in chloride cells. NKA alpha1a was located in both filamental and lamellar chloride cells in freshwater, whereas in seawater it was present only as a faint background in filamental chloride cells. In freshwater, NKA alpha1b was found in a small number of filamental chloride cells, and after seawater acclimation it was found in all chloride cells on the filament and lamellae. Double simultaneous immunofluorescence indicated that NKA alpha1a and alpha1b are located in different chloride cells in freshwater. In many chloride cells in seawater, NKA alpha1b was present in greater amounts in the subapical region than elsewhere in the cell. The combined patterns in abundance and immunolocalization of these two isoforms can explain the salinity-related changes in total NKA and chloride cell abundance. The results indicate that there is a freshwater and a seawater isoform of NKA alpha-subunit in the gills of Atlantic salmon and that they are present in distinct chloride cells.

  17. Distinct Transcript Isoforms of the Atypical Chemokine Receptor 1 (ACKR1) / Duffy Antigen Receptor for Chemokines (DARC) Gene Are Expressed in Lymphoblasts and Altered Isoform Levels Are Associated with Genetic Ancestry and the Duffy-Null Allele

    PubMed Central

    Davis, Melissa B.; Walens, Andrea; Hire, Rupali; Mumin, Kauthar; Brown, Andrea M.; Ford, DeJuana; Howerth, Elizabeth W.; Monteil, Michele

    2015-01-01

    The Atypical ChemoKine Receptor 1 (ACKR1) gene, better known as Duffy Antigen Receptor for Chemokines (DARC or Duffy), is responsible for the Duffy Blood Group and plays a major role in regulating the circulating homeostatic levels of pro-inflammatory chemokines. Previous studies have shown that one common variant, the Duffy Null (Fy-) allele that is specific to African Ancestry groups, completely removes expression of the gene on erythrocytes; however, these individuals retain endothelial expression. Additional alleles are associated with a myriad of clinical outcomes related to immune responses and inflammation. In addition to allele variants, there are two distinct transcript isoforms of DARC which are expressed from separate promoters, and very little is known about the distinct transcriptional regulation or the distinct functionality of these protein isoforms. Our objective was to determine if the African specific Fy- allele alters the expression pattern of DARC isoforms and therefore could potentially result in a unique signature of the gene products, commonly referred to as antigens. Our work is the first to establish that there is expression of DARC on lymphoblasts. Our data indicates that people of African ancestry have distinct relative levels of DARC isoforms expressed in these cells. We conclude that the expression of both isoforms in combination with alternate alleles yields multiple Duffy antigens in ancestry groups, depending upon the haplotypes across the gene. Importantly, we hypothesize that DARC isoform expression patterns will translate into ancestry-specific inflammatory responses that are correlated with the axis of pro-inflammatory chemokine levels and distinct isoform-specific interactions with these chemokines. Ultimately, this work will increase knowledge of biological mechanisms underlying disparate clinical outcomes of inflammatory-related diseases among ethnic and geographic ancestry groups. PMID:26473357

  18. Alternative 5' exons and differential splicing regulate expression of protein 4.1R isoforms with distinct N-termini.

    PubMed

    Parra, Marilyn K; Gee, Sherry L; Koury, Mark J; Mohandas, Narla; Conboy, John G

    2003-05-15

    Among the alternative pre-mRNA splicing events that characterize protein 4.1R gene expression, one involving exon 2' plays a critical role in regulating translation initiation and N-terminal protein structure. Exon 2' encompasses translation initiation site AUG1 and is located between alternative splice acceptor sites at the 5' end of exon 2; its inclusion or exclusion from mature 4.1R mRNA regulates expression of longer or shorter isoforms of 4.1R protein, respectively. The current study reports unexpected complexity in the 5' region of the 4.1R gene that directly affects alternative splicing of exon 2'. Identified far upstream of exon 2 in both mouse and human genomes were 3 mutually exclusive alternative 5' exons, designated 1A, 1B, and 1C; all 3 are associated with strong transcriptional promoters in the flanking genomic sequence. Importantly, exons 1A and 1B splice differentially with respect to exon 2', generating transcripts with different 5' ends and distinct N-terminal protein coding capacity. Exon 1A-type transcripts splice so as to exclude exon 2' and therefore utilize the downstream AUG2 for translation of 80-kDa 4.1R protein, whereas exon 1B transcripts include exon 2' and initiate at AUG1 to synthesize 135-kDa isoforms. RNA blot analyses revealed that 1A transcripts increase in abundance in late erythroblasts, consistent with the previously demonstrated up-regulation of 80-kDa 4.1R during terminal erythroid differentiation. Together, these results suggest that synthesis of structurally distinct 4.1R protein isoforms in various cell types is regulated by a novel mechanism requiring coordination between upstream transcription initiation events and downstream alternative splicing events. PMID:12522012

  19. Alternative 5' exons and differential splicing regulate expression of protein 4.1R isoforms with distinct n-termini

    SciTech Connect

    Parra, Marilyn K.; Gee, Sherry L.; Koury, Mark J.; Mohandas, Narla; Conboy, John G.

    2003-03-25

    Among the alternative pre-mRNA splicing events that characterize protein 4.1R gene expression, one involving exon 2' plays a critical role in regulating translation initiation and N-terminal protein structure. Exon 2' encompasses translation initiation site AUG1 and is located between alternative splice acceptor sites at the 5' end of exon 2; its inclusion or exclusion from mature 4.1R mRNA regulates expression of longer or shorter isoforms of 4.1R protein, respectively. The current study reports unexpected complexity in the 5' region of the 4.1R gene that directly affects alternative splicing of exon 2'. Three mutually exclusive alternative 5' exons, designated 1A, 1B, and 1C, were identified far upstream of exon 2 in both mouse and human genomes; all three are associated with strong transcriptional promoters in the flanking genomic sequence. Importantly, exons 1A and 1B splice differentially with respect to exon 2', generating transcripts with different 5' ends and distinct N-terminal protein coding capacity. Exon 1A-type transcripts splice so as to exclude exon 2' and therefore utilize the downstream AUG2 for translation of 80kD 4.1R protein, whereas exon 1B transcripts include exon 2' and initiate at AUG1 to synthesize 135kD isoforms. RNA blot analyses revealed that 1A transcripts increase in abundance in late erythroblasts, consistent with the previously demonstrated upregulation of 80kD 4.1R during terminal erythroid differentiation. Together these results suggest that synthesis of structurally distinct 4.1R protein isoforms in various cell types is regulated by a novel mechanism requiring coordination between upstream transcription initiation events and downstream alternative splicing events.

  20. Distinct Functions of Different scl Isoforms in Zebrafish Definitive Hematopoietic Stem Cell Initiation and Maintenance

    NASA Astrophysics Data System (ADS)

    Lan, Yahui

    2011-07-01

    The establishment of entire blood system relies on the multi-potent hematopoietic stem cells (HSCs), thus identifying the molecular mechanism in HSC generation is of importance for not only complementing the fundamental knowledge in stem cell biology, but also providing insights to the regenerative therapies. Recent researches have documented the formation of nascent HSCs through a direct transition from ventral aortic endothelium, named as endothelial hematopoietic transition (EHT) process. However, the precise genetic program engaged in this process remains largely elusive. The transcription factor scl plays pivotal and conserved roles in embryonic and adult hematopoiesis from teleosts to mammals. Our lab have previously identified a new truncated scl isoform, scl-beta, which is indispensible for the specification of HSCs in the ventral wall of dorsal aorta (VDA), the zebrafish equivalent of mammalian fetal hematopoietic organ. Here we observe that, by combining time-lapse confocal imaging of transgenic zebrafish and genetic epistasis analysis, scl-beta is expressed in a subset of ventral aortic endothelial cells and critical for their forthcoming transformation to hemogenic endothelium; in contrast, runx1 is required downstream to govern the successful egress of the hemogenic endothelial cells to become naive HSCs. In addition, the traditional known full-length scl-alpha isoform is firstly evidenced to be required for the maintenance or survival of newly formed HSCs in VDA. Collectively our data has established the genetic hierarchy controlling discrete steps in the consecutive process of HSC formation from endothelial cells and further development in VDA.

  1. Novel alternative PBX3 isoforms in leukemia cells with distinct interaction specificities.

    PubMed

    Milech, N; Kees, U R; Watt, P M

    2001-11-01

    PBX3 is a member of the PBX family of TALE homeobox genes. The prototypic member, PBX1, was first identified in chromosomal translocations in B-lineage leukemia and is required for normal hematopoiesis. PBX2 and PBX3 were later identified as members of this highly conserved family by their strong homology to PBX1. While the expression pattern of PBX1 is restricted, PBX2 and PBX3 are ubiquitously expressed. Little is known about the functional role of PBX3. Our studies identified two PBX3 transcripts alternative to the canonical forms, PBX3A and PBX3B, resulting from a novel splice in PBX3. These new isoforms, named PBX3C and PBX3D, have been detected in all tissues and cell lines tested. Intriguingly, expression of PBX3D is favored in normal cells, whereas PBX3C expression is favored in leukemia cells. Functional studies showed that PBX3C and PBX3D proteins were unable to interact with the PBX-interacting factor PREP1 and weakly interacted with MEIS proteins. We propose that PBX3C and PBX3D may affect PBX3-mediated transcriptional regulation by acting in opposition to the known PBX proteins through alternative PBX3 complex formation. The identification and characterization of these novel PBX3 isoforms provide a foundation for a better understanding of the biological role of PBX3. PMID:11579467

  2. Four Isoforms of Arabidopsis 4-Coumarate:CoA Ligase Have Overlapping yet Distinct Roles in Phenylpropanoid Metabolism1[OPEN

    PubMed Central

    Kim, Jeong Im

    2015-01-01

    The biosynthesis of lignin, flavonoids, and hydroxycinnamoyl esters share the first three enzymatic steps of the phenylpropanoid pathway. The last shared step is catalyzed by 4-coumarate:CoA ligase (4CL), which generates p-coumaroyl CoA and caffeoyl CoA from their respective acids. Four isoforms of 4CL have been identified in Arabidopsis (Arabidopsis thaliana). Phylogenetic analysis reveals that 4CL1, 4CL2, and 4CL4 are more closely related to each other than to 4CL3, suggesting that the two groups may serve different biological functions. Promoter-GUS analysis shows that 4CL1 and 4CL2 are expressed in lignifying cells. In contrast, 4CL3 is expressed in a broad range of cell types, and 4CL3 has acquired a distinct role in flavonoid metabolism. Sinapoylmalate, the major hydroxycinnamoyl ester found in Arabidopsis, is greatly reduced in the 4cl1 4cl3 mutant, showing that 4CL1 and 4CL3 function redundantly in its biosynthesis. 4CL1 accounts for the majority of the total 4CL activity, and loss of 4CL1 leads to reduction in lignin content but no growth defect. The 4cl1 4cl2 and 4cl1 4cl2 4cl3 mutants are both dwarf but do not have further reduced lignin than the 4cl1 mutant, indicating that either 4CL1 or 4CL2 is required for normal plant growth. Although 4CL4 has a limited expression profile, it does make a modest contribution to lignin biosynthesis. Together, these data show that the four isoforms of 4CL in Arabidopsis have overlapping yet distinct roles in phenylpropanoid metabolism. PMID:26491147

  3. Distinct CD55 Isoform Synthesis and Inhibition of Complement-Dependent Cytolysis by Hepatitis C Virus.

    PubMed

    Kwon, Young-Chan; Kim, Hangeun; Meyer, Keith; Di Bisceglie, Adrian M; Ray, Ranjit

    2016-08-15

    CD55/DAF, one of the regulators of complement activation, is known to limit excess complement activation on the host cell surface by accelerating the decay of C3 convertase. We reported previously that hepatitis C virus (HCV) infection or virus core protein expression upregulates CD55 expression. CD55 associates with HCV particles, potentially protecting HCV from lysis in circulation. An increase in CD55 on the surface of HCV-infected cells may inhibit complement-mediated cell killing. In this study, we show that Abs against cancer cell surface proteins induce complement-dependent cytolysis or Ab-dependent cell-mediated cytotoxicity of immortalized human hepatocytes in the presence of CD55-blocking Ab. CD55 has a secreted isoform (sCD55) that is generated by alternative splicing. We observed that sCD55 is induced in HCV-infected or HCV replicon-harboring cells, as well as in liver biopsy samples from chronically HCV-infected patients. Conditioned medium from HCV-infected hepatoma cells (Huh7.5 cells) or immortalized human hepatocytes inhibited C3 convertase activity and complement-dependent cytolysis of sheep blood erythrocytes. Chronically HCV-infected patient sera inhibited C3 convertase activity, further implicating HCV-specific impairment of complement function in infected humans. CD55-blocking Ab inhibited erythrocyte lysis by conditioned medium, suggesting that CD55/sCD55 impairs convertase activity. Together, our data show that HCV infection induces sCD55 expression in HCV-infected cell culture-conditioned medium and inhibits C3 convertase activity. This may have implications for modulating complement-mediated immune function in the microenvironment and on HCV-harboring cells. PMID:27357152

  4. Increasing the complexity of chromatin: functionally distinct roles for replication-dependent histone H2A isoforms in cell proliferation and carcinogenesis

    PubMed Central

    Singh, Rajbir; Mortazavi, Amir; Telu, Kelly H.; Nagarajan, Prabakaran; Lucas, David M.; Thomas-Ahner, Jennifer M.; Clinton, Steven K.; Byrd, John C.; Freitas, Michael A.; Parthun, Mark R.

    2013-01-01

    Replication-dependent histones are encoded by multigene families found in several large clusters in the human genome and are thought to be functionally redundant. However, the abundance of specific replication-dependent isoforms of histone H2A is altered in patients with chronic lymphocytic leukemia. Similar changes in the abundance of H2A isoforms are also associated with the proliferation and tumorigenicity of bladder cancer cells. To determine whether these H2A isoforms can perform distinct functions, expression of several H2A isoforms was reduced by siRNA knockdown. Reduced expression of the HIST1H2AC locus leads to increased rates of cell proliferation and tumorigenicity. We also observe that regulation of replication-dependent histone H2A expression can occur on a gene-specific level. Specific replication-dependent histone H2A genes are either up- or downregulated in chronic lymphocytic leukemia tumor tissue samples. In addition, discreet elements are identified in the 5′ untranslated region of the HIST1H2AC locus that confer translational repression. Taken together, these results indicate that replication-dependent histone isoforms can possess distinct cellular functions and that regulation of these isoforms may play a role in carcinogenesis. PMID:23956221

  5. Glucocorticoid receptor isoforms direct distinct mitochondrial programs to regulate ATP production.

    PubMed

    Morgan, David J; Poolman, Toryn M; Williamson, Andrew J K; Wang, Zichen; Clark, Neil R; Ma'ayan, Avi; Whetton, Anthony D; Brass, Andrew; Matthews, Laura C; Ray, David W

    2016-01-01

    The glucocorticoid receptor (GR), a nuclear receptor and major drug target, has a highly conserved minor splice variant, GRγ, which differs by a single arginine within the DNA binding domain. GRγ, which comprises 10% of all GR transcripts, is constitutively expressed and tightly conserved through mammalian evolution, suggesting an important non-redundant role. However, to date no specific role for GRγ has been reported. We discovered significant differences in subcellular localisation, and nuclear-cytoplasmic shuttling in response to ligand. In addition the GRγ transcriptome and protein interactome was distinct, and with a gene ontology signal for mitochondrial regulation which was confirmed using Seahorse technology. We propose that evolutionary conservation of the single additional arginine in GRγ is driven by a distinct, non-redundant functional profile, including regulation of mitochondrial function. PMID:27226058

  6. Glucocorticoid receptor isoforms direct distinct mitochondrial programs to regulate ATP production

    PubMed Central

    Morgan, David J.; Poolman, Toryn M.; Williamson, Andrew J. K.; Wang, Zichen; Clark, Neil R.; Ma’ayan, Avi; Whetton, Anthony D.; Brass, Andrew; Matthews, Laura C.; Ray, David W.

    2016-01-01

    The glucocorticoid receptor (GR), a nuclear receptor and major drug target, has a highly conserved minor splice variant, GRγ, which differs by a single arginine within the DNA binding domain. GRγ, which comprises 10% of all GR transcripts, is constitutively expressed and tightly conserved through mammalian evolution, suggesting an important non-redundant role. However, to date no specific role for GRγ has been reported. We discovered significant differences in subcellular localisation, and nuclear-cytoplasmic shuttling in response to ligand. In addition the GRγ transcriptome and protein interactome was distinct, and with a gene ontology signal for mitochondrial regulation which was confirmed using Seahorse technology. We propose that evolutionary conservation of the single additional arginine in GRγ is driven by a distinct, non-redundant functional profile, including regulation of mitochondrial function. PMID:27226058

  7. R-subunit Isoform Specificity in Protein Kinase A: Distinct Features of Protein Interfaces in PKA Types I and II by Amide H/2H exchange Mass Spectrometry

    PubMed Central

    Anand, Ganesh S.; Hotchko, Matthew; Brown, Simon H.J.; Ten Eyck, Lynn F.; Komives, Elizabeth A.; Taylor, Susan S.

    2009-01-01

    The two isoforms (RI and RII) of the regulatory (R) subunit of cAMP-dependent protein kinase or protein kinase A (PKA) are similar in sequence yet have different biochemical properties and physiological functions. To further understand the molecular basis for R-isoform-specificity, the interactions of the RIIβ isoform with the PKA catalytic (C) subunit were analyzed by amide H/2H exchange mass spectrometry to compare solvent accessibility of RIIβ and the C subunit in their free and complexed states. Direct mapping of the RIIβ-C interface revealed important differences between the intersubunit interfaces in the type I and type II holoenzyme complexes. These differences are seen in both the R-subunits as well as the C-subunit. Unlike the type I isoform, the type II isoform complexes require both cAMP-binding domains, and ATP is not obligatory for high affinity interactions with the C-subunit. Surprisingly, the C-subunit mediates distinct, overlapping surfaces of interaction with the two R-isoforms despite a strong homology in sequence and similarity in domain organization. Identification of a remote allosteric site on the C-subunit that is essential for interactions with RII, but not RI subunits, further highlights the considerable diversity in interfaces found in higher order protein complexes mediated by the C-subunit of PKA. PMID:17942118

  8. Ubiquitylation of cyclin E requires the sequential function of SCF complexes containing distinct hCdc4 isoforms.

    PubMed

    van Drogen, Frank; Sangfelt, Olle; Malyukova, Aljona; Matskova, Ludmila; Yeh, Elizabeth; Means, Anthony R; Reed, Steven I

    2006-07-01

    Cyclin E, an activator of cyclin-dependent kinase 2 (Cdk2), is targeted for proteasomal degradation by phosphorylation-dependent multiubiquitylation via the ubiquitin ligase SCF(hCdc4). SCF ubiquitin ligases are composed of a core of conserved subunits and one variable subunit (an F box protein) involved in substrate recognition. We show here that multiubiquitylation of cyclin E requires the sequential function of two distinct splice variant isoforms of the F box protein hCdc4 known as alpha and gamma. SCF(hCdc4alpha) binds a complex containing cyclin E, Cdk2, and the prolyl cis/trans isomerase Pin1 and promotes the activity of Pin1 without directly ubiquitylating cyclin E. However, due to the action of this SCF(hCdc4alpha)-Pin1 complex, cyclin E becomes an efficient ubiquitylation substrate of SCF(hCdc4gamma). Furthermore, in the context of Cdc4alpha and cyclin E, mutational data suggest that Pin1 isomerizes a noncanonical proline-proline bond, with the possibility that Cdc4alpha may serve as a cofactor for altering the specificity of Pin1. PMID:16818231

  9. Analysis of the N-terminal region of human MLKL, as well as two distinct MLKL isoforms, reveals new insights into necroptotic cell death

    PubMed Central

    Arnež, Katja Hrovat; Kindlova, Michaela; Bokil, Nilesh J.; Murphy, James M.; Sweet, Matthew J.; Gunčar, Gregor

    2015-01-01

    The pseudokinase mixed lineage kinase domain-like (MLKL) is an essential effector of necroptotic cell death. Two distinct human MLKL isoforms have previously been reported, but their capacities to trigger cell death have not been compared directly. Herein, we examine these two MLKL isoforms, and further probe the features of the human MLKL N-terminal domain that are required for cell death. Expression in HEK293T cells of the N-terminal 201 amino acids (aa) of human MLKL is sufficient to cause cell death, whereas expression of the first 154 aa is not. Given that aa 1–125 are able to initiate necroptosis, our findings indicate that the helix that follows this region restrains necroptotic activity, which is again restored in longer constructs. Furthermore, MLKL isoform 2 (MLKL2), which lacks much of the regulatory pseudokinase domain, is a much more potent inducer of cell death than MLKL isoform 1 (MLKL1) in ectopic expression studies in HEK293T cells. Modelling predicts that a C-terminal helix constrains the activity of MLKL1, but not MLKL2. Although both isoforms are expressed by human monocyte-derived macrophages at the mRNA level, MLKL2 is expressed at much lower levels. We propose that it may have a regulatory role in controlling macrophage survival, either in the steady state or in response to specific stimuli. PMID:26704887

  10. Distinct isoform of FABP7 revealed by screening for retroelement-activated genes in diffuse large B-cell lymphoma.

    PubMed

    Lock, Frances E; Rebollo, Rita; Miceli-Royer, Katharine; Gagnier, Liane; Kuah, Sabrina; Babaian, Artem; Sistiaga-Poveda, Maialen; Lai, C Benjamin; Nemirovsky, Oksana; Serrano, Isabel; Steidl, Christian; Karimi, Mohammad M; Mager, Dixie L

    2014-08-26

    Remnants of ancient transposable elements (TEs) are abundant in mammalian genomes. These sequences harbor multiple regulatory motifs and hence are capable of influencing expression of host genes. In response to environmental changes, TEs are known to be released from epigenetic repression and to become transcriptionally active. Such activation could also lead to lineage-inappropriate activation of oncogenes, as one study described in Hodgkin lymphoma. However, little further evidence for this mechanism in other cancers has been reported. Here, we reanalyzed whole transcriptome data from a large cohort of patients with diffuse large B-cell lymphoma (DLBCL) compared with normal B-cell centroblasts to detect genes ectopically expressed through activation of TE promoters. We have identified 98 such TE-gene chimeric transcripts that were exclusively expressed in primary DLBCL cases and confirmed several in DLBCL-derived cell lines. We further characterized a TE-gene chimeric transcript involving a fatty acid-binding protein gene (LTR2-FABP7), normally expressed in brain, that was ectopically expressed in a subset of DLBCL patients through the use of an endogenous retroviral LTR promoter of the LTR2 family. The LTR2-FABP7 chimeric transcript encodes a novel chimeric isoform of the protein with characteristics distinct from native FABP7. In vitro studies reveal a dependency for DLBCL cell line proliferation and growth on LTR2-FABP7 chimeric protein expression. Taken together, these data demonstrate the significance of TEs as regulators of aberrant gene expression in cancer and suggest that LTR2-FABP7 may contribute to the pathogenesis of DLBCL in a subgroup of patients. PMID:25114248

  11. Distinct isoform of FABP7 revealed by screening for retroelement-activated genes in diffuse large B-cell lymphoma

    PubMed Central

    Lock, Frances E.; Rebollo, Rita; Miceli-Royer, Katharine; Gagnier, Liane; Kuah, Sabrina; Babaian, Artem; Sistiaga-Poveda, Maialen; Lai, C. Benjamin; Nemirovsky, Oksana; Serrano, Isabel; Steidl, Christian; Karimi, Mohammad M.; Mager, Dixie L.

    2014-01-01

    Remnants of ancient transposable elements (TEs) are abundant in mammalian genomes. These sequences harbor multiple regulatory motifs and hence are capable of influencing expression of host genes. In response to environmental changes, TEs are known to be released from epigenetic repression and to become transcriptionally active. Such activation could also lead to lineage-inappropriate activation of oncogenes, as one study described in Hodgkin lymphoma. However, little further evidence for this mechanism in other cancers has been reported. Here, we reanalyzed whole transcriptome data from a large cohort of patients with diffuse large B-cell lymphoma (DLBCL) compared with normal B-cell centroblasts to detect genes ectopically expressed through activation of TE promoters. We have identified 98 such TE-gene chimeric transcripts that were exclusively expressed in primary DLBCL cases and confirmed several in DLBCL-derived cell lines. We further characterized a TE-gene chimeric transcript involving a fatty acid-binding protein gene (LTR2-FABP7), normally expressed in brain, that was ectopically expressed in a subset of DLBCL patients through the use of an endogenous retroviral LTR promoter of the LTR2 family. The LTR2-FABP7 chimeric transcript encodes a novel chimeric isoform of the protein with characteristics distinct from native FABP7. In vitro studies reveal a dependency for DLBCL cell line proliferation and growth on LTR2-FABP7 chimeric protein expression. Taken together, these data demonstrate the significance of TEs as regulators of aberrant gene expression in cancer and suggest that LTR2-FABP7 may contribute to the pathogenesis of DLBCL in a subgroup of patients. PMID:25114248

  12. Co-immunoprecipitation with Tau Isoform-specific Antibodies Reveals Distinct Protein Interactions and Highlights a Putative Role for 2N Tau in Disease.

    PubMed

    Liu, Chang; Song, Xiaomin; Nisbet, Rebecca; Götz, Jürgen

    2016-04-01

    Alternative splicing generates multiple isoforms of the microtubule-associated protein Tau, but little is known about their specific function. In the adult mouse brain, three Tau isoforms are expressed that contain either 0, 1, or 2 N-terminal inserts (0N, 1N, and 2N). We generated Tau isoform-specific antibodies and performed co-immunoprecipitations followed by tandem mass tag multiplexed quantitative mass spectrometry. We identified novel Tau-interacting proteins of which one-half comprised membrane-bound proteins, localized to the plasma membrane, mitochondria, and other organelles. Tau was also found to interact with proteins involved in presynaptic signal transduction. MetaCore analysis revealed one major Tau interaction cluster that contained 33 Tau pulldown proteins. To explore the pathways in which these proteins are involved, we conducted an ingenuity pathway analysis that revealed two significant overlapping pathways, "cell-to-cell signaling and interaction" and "neurological disease." The functional enrichment tool DAVID showed that in particular the 2N Tau-interacting proteins were specifically associated with neurological disease. Finally, for a subset of Tau interactions (apolipoprotein A1 (apoA1), apoE, mitochondrial creatine kinase U-type, β-synuclein, synaptogyrin-3, synaptophysin, syntaxin 1B, synaptotagmin, and synapsin 1), we performed reverse co-immunoprecipitations, confirming the preferential interaction of specific isoforms. For example, apoA1 displayed a 5-fold preference for the interaction with 2N, whereas β-synuclein showed preference for 0N. Remarkably, a reverse immunoprecipitation with apoA1 detected only the 2N isoform. This highlights distinct protein interactions of the different Tau isoforms, suggesting that they execute different functions in brain tissue. PMID:26861879

  13. Co-immunoprecipitation with Tau Isoform-specific Antibodies Reveals Distinct Protein Interactions and Highlights a Putative Role for 2N Tau in Disease*

    PubMed Central

    Liu, Chang; Song, Xiaomin; Nisbet, Rebecca

    2016-01-01

    Alternative splicing generates multiple isoforms of the microtubule-associated protein Tau, but little is known about their specific function. In the adult mouse brain, three Tau isoforms are expressed that contain either 0, 1, or 2 N-terminal inserts (0N, 1N, and 2N). We generated Tau isoform-specific antibodies and performed co-immunoprecipitations followed by tandem mass tag multiplexed quantitative mass spectrometry. We identified novel Tau-interacting proteins of which one-half comprised membrane-bound proteins, localized to the plasma membrane, mitochondria, and other organelles. Tau was also found to interact with proteins involved in presynaptic signal transduction. MetaCore analysis revealed one major Tau interaction cluster that contained 33 Tau pulldown proteins. To explore the pathways in which these proteins are involved, we conducted an ingenuity pathway analysis that revealed two significant overlapping pathways, “cell-to-cell signaling and interaction” and “neurological disease.” The functional enrichment tool DAVID showed that in particular the 2N Tau-interacting proteins were specifically associated with neurological disease. Finally, for a subset of Tau interactions (apolipoprotein A1 (apoA1), apoE, mitochondrial creatine kinase U-type, β-synuclein, synaptogyrin-3, synaptophysin, syntaxin 1B, synaptotagmin, and synapsin 1), we performed reverse co-immunoprecipitations, confirming the preferential interaction of specific isoforms. For example, apoA1 displayed a 5-fold preference for the interaction with 2N, whereas β-synuclein showed preference for 0N. Remarkably, a reverse immunoprecipitation with apoA1 detected only the 2N isoform. This highlights distinct protein interactions of the different Tau isoforms, suggesting that they execute different functions in brain tissue. PMID:26861879

  14. Biophysical characterization of refolded Drosophila Spätzle, a cystine knot protein, reveals distinct properties of three isoforms.

    PubMed

    Hoffmann, Anita; Funkner, Andreas; Neumann, Piotr; Juhnke, Susanne; Walther, Matthias; Schierhorn, Angelika; Weininger, Ulrich; Balbach, Jochen; Reuter, Gunter; Stubbs, Milton T

    2008-11-21

    The Drosophila Spätzle protein, involved in the embryonic development of the dorsal-ventral axis and in the adult immune response, is expressed as a proprotein and is activated by the serine proteinases Easter or Spätzle-processing enzyme. Proteolytic cleavage generates a 106-amino acid COOH-terminal fragment, C106, homologous to the mature form of nerve growth factor NGF, a cystine knot protein. Through alternative splicing, the Spätzle gene encodes for several isoforms that (with one exception, the "propeptide isoform") share C106 but differ in the prosequence. Three isoforms have been expressed recombinantly in Escherichia coli strains. The propeptide isoform could be expressed in soluble form and is unstructured according to CD and NMR measurements. Dimeric full-length Spätzle isoforms have been refolded from insoluble inclusion bodies and are able to rescue Spätzle-deficient embryos. Although the two full-length isoforms exhibit similar far-UV CD spectra, large differences in tryptophan fluorescence quenching by the respective pro-parts are observed. Both full-length isoforms exhibited highly cooperative folding transitions. Proteolytic digestion using trypsin resulted in C106, whose unfolding exhibits lower thermodynamic stability and cooperativity compared with the full-length proteins. The structure of C106 reveals a T-shaped dimer with significant differences to NGF and a deep internal cavity. Substantial beta-sheet formation is observed between the two monomers, whereas a long loop containing the single tryptophan residue is disordered in the crystals. Our results suggest that the propeptides stabilize the tertiary structure of the "mature" Spätzle cystine knot. PMID:18790733

  15. The OsCYP19-4 Gene Is Expressed as Multiple Alternatively Spliced Transcripts Encoding Isoforms with Distinct Cellular Localizations and PPIase Activities under Cold Stress

    PubMed Central

    Lee, Areum; Lee, Sang Sook; Jung, Won Yong; Park, Hyun Ji; Lim, Bo Ra; Kim, Hyun-Soon; Ahn, Jun Cheul; Cho, Hye Sun

    2016-01-01

    Alternative splicing (AS) is an important molecular mechanism by which single genes can generate multiple mRNA isoforms. We reported previously that, in Oryza sativa, the cyclophilin 19-4 (OsCYP19-4.1) transcript was significantly upregulated in response to cold stress, and that transgenic plants were cold tolerant. Here we show that, under cold stress, OsCYP19-4 produces eight transcript variants by intron retention and exon skipping, resulting in production of four distinct protein isoforms. The OsCYP19-4 AS isoforms exhibited different cellular localizations in the epidermal cells: in contrast to OsCYP19-4.1, the OsCYP19-4.2 and OsCYP19-4.3 proteins were primarily targeted to guard and subsidiary cells, whereas OsCYP19-4.5, which consists largely of an endoplasmic reticulum (ER) targeting signal, was co-localized with the RFP-BiP marker in the ER. In OsCYP19-4.2, the key residues of the PPIase domain are altered; consistent with this, recombinant OsCYP19-4.2 had significantly lower PPIase activity than OsCYP19-4.1 in vitro. Specific protein-protein interactions between OsCYP19-4.2/3 and AtRCN1 were verified in yeast two-hybrid (Y2H) and bimolecular fluoresence complementation (BiFC assays), although the OsCYP19-4 isoforms could not bind each other. Based on these results, we propose that two OsCYP19-4 AS isoforms, OsCYP19-4.2 and OsCYP19-4.3, play roles linking auxin transport and cold stress via interactions with RCN1. PMID:27447607

  16. The OsCYP19-4 Gene Is Expressed as Multiple Alternatively Spliced Transcripts Encoding Isoforms with Distinct Cellular Localizations and PPIase Activities under Cold Stress.

    PubMed

    Lee, Areum; Lee, Sang Sook; Jung, Won Yong; Park, Hyun Ji; Lim, Bo Ra; Kim, Hyun-Soon; Ahn, Jun Cheul; Cho, Hye Sun

    2016-01-01

    Alternative splicing (AS) is an important molecular mechanism by which single genes can generate multiple mRNA isoforms. We reported previously that, in Oryza sativa, the cyclophilin 19-4 (OsCYP19-4.1) transcript was significantly upregulated in response to cold stress, and that transgenic plants were cold tolerant. Here we show that, under cold stress, OsCYP19-4 produces eight transcript variants by intron retention and exon skipping, resulting in production of four distinct protein isoforms. The OsCYP19-4 AS isoforms exhibited different cellular localizations in the epidermal cells: in contrast to OsCYP19-4.1, the OsCYP19-4.2 and OsCYP19-4.3 proteins were primarily targeted to guard and subsidiary cells, whereas OsCYP19-4.5, which consists largely of an endoplasmic reticulum (ER) targeting signal, was co-localized with the RFP-BiP marker in the ER. In OsCYP19-4.2, the key residues of the PPIase domain are altered; consistent with this, recombinant OsCYP19-4.2 had significantly lower PPIase activity than OsCYP19-4.1 in vitro. Specific protein-protein interactions between OsCYP19-4.2/3 and AtRCN1 were verified in yeast two-hybrid (Y2H) and bimolecular fluoresence complementation (BiFC assays), although the OsCYP19-4 isoforms could not bind each other. Based on these results, we propose that two OsCYP19-4 AS isoforms, OsCYP19-4.2 and OsCYP19-4.3, play roles linking auxin transport and cold stress via interactions with RCN1. PMID:27447607

  17. Distinct expression and function of whirlin isoforms in the inner ear and retina: an insight into pathogenesis of USH2D and DFNB31.

    PubMed

    Mathur, Pranav Dinesh; Zou, Junhuang; Zheng, Tihua; Almishaal, Ali; Wang, Yong; Chen, Qian; Wang, Le; Vashist, Deepti; Brown, Steve; Park, Albert; Yang, Jun

    2015-11-01

    Usher syndrome (USH) is the most common inherited deaf-blindness with the majority of USH causative genes also involved in nonsyndromic recessive deafness (DFNB). The mechanism underlying this disease variation of USH genes is unclear. Here, we addressed this issue by investigating the DFNB31 gene, whose mutations cause USH2D or DFNB31 depending on their position. We found that the mouse DFNB31 ortholog (Dfnb31) expressed different mRNA variants and whirlin protein isoforms in the cochlea and retina, where these isoforms played different roles spatially and temporally. Full-length (FL-) whirlin in photoreceptors and hair cell stereociliary bases is important for the USH type 2 protein complex, while FL- and C-terminal (C-) whirlins in hair cell stereociliary tips participate in stereociliary elongation. Mutations in the whirlin N-terminal region disrupted FL-whirlin isoform in the inner ear and retina but not C-whirlin in the inner ear, and led to retinal degeneration as well as moderate to severe hearing loss. By contrast, a mutation in the whirlin C-terminal region eliminated all normal whirlin isoforms but generated a truncated N-terminal whirlin protein fragment, which was partially functional in the retina and thus prevented retinal degeneration. Mice with this mutation had profound hearing loss. In summary, disruption of distinct whirlin isoforms by Dfnb31 mutations leads to a variety of phenotype configurations and may explain the mechanism underlying the different disease manifestations of human DFNB31 mutations. Our findings have a potential to improve diagnosis and treatment of USH disease and quality of life in USH patients. PMID:26307081

  18. Novel exons in the tbx5 gene locus generate protein isoforms with distinct expression domains and function.

    PubMed

    Yamak, Abir; Georges, Romain O; Sheikh-Hassani, Massomeh; Morin, Martin; Komati, Hiba; Nemer, Mona

    2015-03-13

    TBX5 is the gene mutated in Holt-Oram syndrome, an autosomal dominant disorder with complex heart and limb deformities. Its protein product is a member of the T-box family of transcription factors and an evolutionarily conserved dosage-sensitive regulator of heart and limb development. Understanding TBX5 regulation is therefore of paramount importance. Here we uncover the existence of novel exons and provide evidence that TBX5 activity may be extensively regulated through alternative splicing to produce protein isoforms with differing N- and C-terminal domains. These isoforms are also present in human heart, indicative of an evolutionarily conserved regulatory mechanism. The newly identified isoforms have different transcriptional properties and can antagonize TBX5a target gene activation. Droplet Digital PCR as well as immunohistochemistry with isoform-specific antibodies reveal differential as well as overlapping expression domains. In particular, we find that the predominant isoform in skeletal myoblasts is Tbx5c, and we show that it is dramatically up-regulated in differentiating myotubes and is essential for myotube formation. Mechanistically, TBX5c antagonizes TBX5a activation of pro-proliferative signals such as IGF-1, FGF-10, and BMP4. The results provide new insight into Tbx5 regulation and function that will further our understanding of its role in health and disease. The finding of new exons in the Tbx5 locus may also be relevant to mutational screening especially in the 30% of Holt-Oram syndrome patients with no mutations in the known TBX5a exons. PMID:25623069

  19. Distinct pharmacological properties and distribution in neurons and endocrine cells of two isoforms of the human vesicular monoamine transporter.

    PubMed Central

    Erickson, J D; Schafer, M K; Bonner, T I; Eiden, L E; Weihe, E

    1996-01-01

    A second isoform of the human vesicular monoamine transporter (hVMAT) has been cloned from a pheochromocytoma cDNA library. The contribution of the two transporter isoforms to monoamine storage in human neuroendocrine tissues was examined with isoform-specific polyclonal antibodies against hVMAT1 and hVMAT2. Central, peripheral, and enteric neurons express only VMAT2. VMAT1 is expressed exclusively in neuroendocrine, including chromaffin and enterochromaffin, cells. VMAT1 and VMAT2 are coexpressed in all chromaffin cells of the adrenal medulla. VMAT2 alone is expressed in histamine-storing enterochromaffin-like cells of the oxyntic mucosa of the stomach. The transport characteristics and pharmacology of each VMAT isoform have been directly compared after expression in digitonin-permeabilized fibroblastic (CV-1) cells, providing information about substrate feature recognition by each transporter and the role of vesicular monoamine storage in the mechanism of action of psychopharmacologic and neurotoxic agents in human. Serotonin has a similar affinity for both transporters. Catecholamines exhibit a 3-fold higher affinity, and histamine exhibits a 30-fold higher affinity, for VMAT2. Reserpine and ketanserin are slightly more potent inhibitors of VMAT2-mediated transport than of VMAT1-mediated transport, whereas tetrabenazine binds to and inhibits only VMAT2. N-methyl-4-phenylpyridinium, phenylethylamine, amphetamine, and methylenedioxymethamphetamine are all more potent inhibitors of VMAT2 than of VMAT1, whereas fenfluramine is a more potent inhibitor of VMAT1-mediated monamine transport than of VMAT2-mediated monoamine transport. The unique distributions of hVMAT1 and hVMAT2 provide new markers for multiple neuroendocrine lineages, and examination of their transport properties provides mechanistic insights into the pharmacology and physiology of amine storage in cardiovascular, endocrine, and central nervous system function. Images Fig. 3 Fig. 4 PMID:8643547

  20. Distinct Roles for the α, β and γ1 isoforms of Protein Phosphatase 1 in the Outside-in αIIbβ3 Integrin Signaling Dependent Functions

    PubMed Central

    Alrehani, Nawaf; Pradhan, Subhashree; Khatlani, Tanvir; Kailasam, Lavanya; Vijayan, K. Vinod

    2013-01-01

    Although protein kinases and phosphatases participate in integrin αIIbβ3 signaling, whether integrin functions are regulated by the catalytic subunit of protein phosphatase 1 (PP1c) isoforms are unclear. We show that siRNA mediated knockdown of all PP1c isoforms (α, β and γ1) in 293 αIIbβ3 cells decreased adhesion to immobilized fibrinogen and fibrin clot retraction. Selective knockdown of only PP1cγ1 did not alter adhesion or clot retraction, while depletion of PP1cβ decreased both functions. Unexpectedly, knockdown of PP1cα enhanced αIIbβ3 adhesion to fibrinogen and clot retraction. Protein interaction studies revealed that all PP1c isoforms can interact with the integrin αIIb subunit. Phosphoprofiling studies revealed an enhanced activation of mitogen-activated protein kinase (MAPK) p38 in the PP1cα depleted cells. Enhanced adhesive phenotype displayed by the PP1cα depleted 293 αIIbβ3 cells was blocked by pharmacological inhibition of p38. Conversely, the decreased adhesion of PP1cα overexpressing cells was rescued by the expression of constitutively active p38α or p38γ. Thus, PP1c isoforms have distinct contribution to the outside-in αIIbβ3 signaling-dependent functions in 293 αIIbβ3 cells. Moreover, PP1cα negatively regulates integrin function by suppressing the p38 pathway. PMID:23197154

  1. Laminin and Type IV Collagen Isoform Substitutions Occur in Temporally and Spatially Distinct Patterns in Developing Kidney Glomerular Basement Membranes

    PubMed Central

    St. John, Patricia L.; Stroganova, Larysa; Zelenchuk, Adrian; Steenhard, Brooke M.

    2013-01-01

    Kidney glomerular basement membranes (GBMs) undergo laminin and type IV collagen isoform substitutions during glomerular development, which are believed to be required for maturation of the filtration barrier. Specifically, GBMs of earliest glomeruli contain laminin α1β1γ1 and collagen α1α2α1(IV), whereas mature glomeruli contain laminin α5β2γ1 and collagen α3α4α5(IV). Here, we used confocal microscopy to simultaneously evaluate expression of different laminin and collagen IV isoforms in newborn mouse GBMs. Our results show loss of laminin α1 from GBMs in early capillary loop stages and continuous linear deposition of laminin bearing the α5 chain thereafter. In contrast, collagen α1α2α1(IV) persisted in linear patterns into late capillary loop stages, when collagen α3α4α5(IV) first appeared in discontinuous, non-linear patterns. This patchy pattern for collagen α3α4α5(IV) continued into maturing glomeruli where there were lengths of linear, laminin α5-positive GBM entirely lacking either isoform of collagen IV. Relative abundance of laminin and collagen IV mRNAs in newborn and 5-week-old mouse kidneys also differed, with those encoding laminin α1, α5, β1, β2, and γ1, and collagen α1(IV) and α2(IV) chains all significantly declining at 5 weeks, but α3(IV) and α4(IV) were significantly upregulated. We conclude that different biosynthetic mechanisms control laminin and type IV collagen expression in developing glomeruli. PMID:23896970

  2. The different large subunit isoforms of Arabidopsis thaliana ADP-glucose pyrophosphorylase confer distinct kinetic and regulatory properties to the heterotetrameric enzyme.

    PubMed

    Crevillén, Pedro; Ballicora, Miguel A; Mérida, Angel; Preiss, Jack; Romero, José M

    2003-08-01

    ADP-glucose pyrophosphorylase catalyzes the first and limiting step in starch biosynthesis and is allosterically regulated by the levels of 3-phosphoglycerate and phosphate in plants. ADP-glucose pyrophosphorylases from plants are heterotetramers composed of two types of subunits (small and large). In this study, the six Arabidopsis thaliana genes coding for ADP-glucose pyrophosphorylase isoforms (two small and four large subunits) have been cloned and expressed in an Escherichia coli mutant deficient in ADP-glucose pyrophosphorylase activity. The co-expression of the small subunit APS1 with the different Arabidopsis large subunits (APL1, APL2, APL3, and APL4) resulted in heterotetramers with different regulatory and kinetic properties. Heterotetramers composed of APS1 and APL1 showed the highest sensitivity to the allosteric effectors as well as the highest apparent affinity for the substrates (glucose-1-phosphate and ATP), whereas heterotetramers formed by APS1 and APL2 showed the lower response to allosteric effectors and the lower affinity for the substrates. No activity was detected for the second gene coding for a small subunit isoform (APS2) annotated in the Arabidopsis genome. This lack of activity is possibly due to the absence of essential amino acids involved in catalysis and/or in the binding of glucose-1-phosphate and 3-phosphoglycerate. Kinetic and regulatory properties of the different heterotetramers, together with sequence analysis has allowed us to make a distinction between sink and source enzymes, because the combination of different large subunits would provide a high plasticity to ADP-glucose pyrophosphorylase activity and regulation. This is the first experimental data concerning the role that all the ADP-glucose pyrophosphorylase isoforms play in a single plant species. This phenomenon could have an important role in vivo, because different large subunits would confer distinct regulatory properties to ADP-glucose pyrophosphorylase according

  3. Neural activity selects myosin IIB and VI with a specific time window in distinct dynamin isoform-mediated synaptic vesicle reuse pathways.

    PubMed

    Hayashida, Michikata; Tanifuji, Shota; Ma, Huan; Murakami, Noriko; Mochida, Sumiko

    2015-06-10

    Presynaptic nerve terminals must maintain stable neurotransmissions via synaptic vesicle (SV) resupply despite encountering wide fluctuations in the number and frequency of incoming action potentials (APs). However, the molecular mechanism linking variation in neural activity to SV resupply is unknown. Myosins II and VI are actin-based cytoskeletal motors that drive dendritic actin dynamics and membrane transport, respectively, at brain synapses. Here we combined genetic knockdown or molecular dysfunction and direct physiological measurement of fast synaptic transmission from paired rat superior cervical ganglion neurons in culture to show that myosins IIB and VI work individually in SV reuse pathways, having distinct dependency and time constants with physiological AP frequency. Myosin VI resupplied the readily releasable pool (RRP) with slow kinetics independently of firing rates but acted quickly within 50 ms after AP. Under high-frequency AP firing, myosin IIB resupplied the RRP with fast kinetics in a slower time window of 200 ms. Knockdown of both myosin and dynamin isoforms by mixed siRNA microinjection revealed that myosin IIB-mediated SV resupply follows amphiphysin/dynamin-1-mediated endocytosis, while myosin VI-mediated SV resupply follows dynamin-3-mediated endocytosis. Collectively, our findings show how distinct myosin isoforms work as vesicle motors in appropriate SV reuse pathways associated with specific firing patterns. PMID:26063922

  4. Client Proteins and Small Molecule Inhibitors Display Distinct Binding Preferences for Constitutive and Stress-Induced HSP90 Isoforms and Their Conformationally Restricted Mutants

    PubMed Central

    Lee, Sunmin; Tsutsumi, Shinji; Yim, Kendrick; Rivas, Candy; Alarcon, Sylvia; Schwartz, Harvey; Khamit-Kush, Kofi; Scroggins, Bradley T.; Beebe, Kristin; Trepel, Jane B.; Neckers, Len

    2015-01-01

    The two cytosolic/nuclear isoforms of the molecular chaperone HSP90, stress-inducible HSP90α and constitutively expressed HSP90β, fold, assemble and maintain the three-dimensional structure of numerous client proteins. Because many HSP90 clients are important in cancer, several HSP90 inhibitors have been evaluated in the clinic. However, little is known concerning possible unique isoform or conformational preferences of either individual HSP90 clients or inhibitors. In this report, we compare the relative interaction strength of both HSP90α and HSP90β with the transcription factors HSF1 and HIF1α, the kinases ERBB2 and MET, the E3-ubiquitin ligases KEAP1 and RHOBTB2, and the HSP90 inhibitors geldanamycin and ganetespib. We observed unexpected differences in relative client and drug preferences for the two HSP90 isoforms, with HSP90α binding each client protein with greater apparent affinity compared to HSP90β, while HSP90β bound each inhibitor with greater relative interaction strength compared to HSP90α. Stable HSP90 interaction was associated with reduced client activity. Using a defined set of HSP90 conformational mutants, we found that some clients interact strongly with a single, ATP-stabilized HSP90 conformation, only transiently populated during the dynamic HSP90 chaperone cycle, while other clients interact equally with multiple HSP90 conformations. These data suggest different functional requirements among HSP90 clientele that, for some clients, are likely to be ATP-independent. Lastly, the two inhibitors examined, although sharing the same binding site, were differentially able to access distinct HSP90 conformational states. PMID:26517842

  5. Distinct roles of isoforms of the heme-liganded nuclear receptor E75, an insect orthologue of the vertebrate Rev-Erb, in mosquito reproduction

    PubMed Central

    Cruz, Josefa; Mane-Padros, Daniel; Zou, Zhen; Raikhel, Alexander S.

    2012-01-01

    Mosquitoes are adapted to using vertebrate blood as a nutrient source to promote egg development and as a consequence serve as disease vectors. Blood-meal activated reproductive events in female mosquitoes are hormonally and nutritionally controlled with an insect steroid hormone 20-hydroxyecdysone (20E) playing a central role. The nuclear receptor E75 is an essential factor in the 20E genetic hierarchy, however functions of its three isoforms - E75A, E75B, and E75C – in mosquito reproduction are unclear. By means of specific RNA interference depletion of E75 isoforms, we identified their distinct roles in regulating the level and timing of expression of key genes involved in vitellogenesis in the fat body (an insect analogue of vertebrate liver and adipose tissue) of the mosquito Aedes aegypti. Heme is required in a high level of expression of 20E-controlled genes in the fat body, and this heme action depends on E75. Thus, in mosquitoes, heme is an important signaling molecule, serving as a sensor of the availability of a protein meal for egg development. Disruption of this signaling pathway could be explored in the design of mosquito control approaches. PMID:22115961

  6. Distinct functionality of dishevelled isoforms on Ca2+/calmodulin-dependent protein kinase 2 (CamKII) in Xenopus gastrulation

    PubMed Central

    Gentzel, Marc; Schille, Carolin; Rauschenberger, Verena; Schambony, Alexandra

    2015-01-01

    Wnt ligands trigger the activation of a variety of β-catenin–dependent and β-catenin–independent intracellular signaling cascades. Despite the variations in intracellular signaling, Wnt pathways share the effector proteins frizzled, dishevelled, and β-arrestin. It is unclear how the specific activation of individual branches and the integration of multiple signals are achieved. We hypothesized that the composition of dishevelled–β-arrestin protein complexes contributes to signal specificity and identified CamKII as an interaction partner of the dishevelled–β-arrestin protein complex by quantitative functional proteomics. Specifically, we found that CamKII isoforms interact differentially with the three vertebrate dishevelled proteins. Dvl1 is required for the activation of CamKII and PKC in the Wnt/Ca2+ pathway. However, CamKII interacts with Dvl2 but not with Dvl1, and Dvl2 is necessary to mediate CamKII function downstream of Dvl1 in convergent extension movements in Xenopus gastrulation. Our findings indicate that the different Dvl proteins and the composition of dishevelled–β-arrestin protein complexes contribute to the specific activation of individual branches of Wnt signaling. PMID:25568338

  7. Profiling Murine Tau with 0N, 1N and 2N Isoform-Specific Antibodies in Brain and Peripheral Organs Reveals Distinct Subcellular Localization, with the 1N Isoform Being Enriched in the Nucleus

    PubMed Central

    Liu, Chang; Götz, Jürgen

    2013-01-01

    In the adult murine brain, the microtubule-associated protein tau exists as three major isoforms, which have four microtubule-binding repeats (4R), with either no (0N), one (1N) or two (2N) amino-terminal inserts. The human brain expresses three additional isoforms with three microtubule-binding repeats (3R) each. However, little is known about the role of the amino-terminal inserts and how the 0N, 1N and 2N tau species differ. In order to investigate this, we generated a series of isoform-specific antibodies and performed a profiling by Western blotting and immunohistochemical analyses using wild-type mice in three age groups: two months, two weeks and postnatal day 0 (P0). This revealed that the brain is the only organ to express tau at significant levels, with 0N4R being the predominant isoform in the two month-old adult. Subcellular fractionation of the brain showed that the 1N isoform is over-represented in the soluble nuclear fraction. This is in agreement with the immunohistochemical analysis as the 1N isoform strongly localizes to the neuronal nucleus, although it is also found in cell bodies and dendrites, but not axons. The 0N isoform is mainly found in cell bodies and axons, whereas nuclei and dendrites are only slightly stained with the 0N antibody. The 2N isoform is highly expressed in axons and in cell bodies, with a detectable expression in dendrites and a very slight expression in nuclei. The 2N isoform that was undetectable at P0, in adult brain was mainly found localized to cell bodies and dendrites. Together these findings reveal significant differences between the three murine tau isoforms that are likely to reflect different neuronal functions. PMID:24386422

  8. The selective post-translational processing of transcription factor Nrf1 yields distinct isoforms that dictate its ability to differentially regulate gene expression.

    PubMed

    Zhang, Yiguo; Li, Shaojun; Xiang, Yuancai; Qiu, Lu; Zhao, Huakan; Hayes, John D

    2015-01-01

    Upon translation, the N-terminal homology box 1 (NHB1) signal anchor sequence of Nrf1 integrates it within the endoplasmic reticulum (ER) whilst its transactivation domains [TADs, including acidic domain 1 (AD1), the flanking Asn/Ser/Thr-rich (NST) domain and AD2] are transiently translocated into the ER lumen, whereupon the NST domain is glycosylated to yield an inactive 120-kDa glycoprotein. Subsequently, these TADs are retrotranslocated into extra-luminal subcellular compartments, where Nrf1 is deglycosylated to yield an active 95-kDa isoform. Herein, we report that AD1 and AD2 are required for the stability of the 120-kDa Nrf1 glycoprotein, but not that of the non-glycosylated/de-glycosylated 95-kDa isoform. Degrons within AD1 do not promote proteolytic degradation of the 120-kDa Nrf1 glycoprotein. However, repositioning of AD2-adjoining degrons (i.e. DSGLS-containing SDS1 and PEST2 sequences) into the cyto/nucleoplasm enables selective topovectorial processing of Nrf1 by the proteasome and/or calpains to generate a cleaved active 85-kDa Nrf1 or a dominant-negative 36-kDa Nrf1γ. Production of Nrf1γ is abolished by removal of SDS1 or PEST2 degrons, whereas production of the cleaved 85-kDa Nrf1 is blocked by deletion of the ER luminal-anchoring NHB2 sequence (aa 81-106). Importantly, Nrf1 activity is positively and/or negatively regulated by distinct doses of proteasome and calpain inhibitors. PMID:26268886

  9. The selective post-translational processing of transcription factor Nrf1 yields distinct isoforms that dictate its ability to differentially regulate gene expression

    PubMed Central

    Zhang, Yiguo; Li, Shaojun; Xiang, Yuancai; Qiu, Lu; Zhao, Huakan; Hayes, John D.

    2015-01-01

    Upon translation, the N-terminal homology box 1 (NHB1) signal anchor sequence of Nrf1 integrates it within the endoplasmic reticulum (ER) whilst its transactivation domains [TADs, including acidic domain 1 (AD1), the flanking Asn/Ser/Thr-rich (NST) domain and AD2] are transiently translocated into the ER lumen, whereupon the NST domain is glycosylated to yield an inactive 120-kDa glycoprotein. Subsequently, these TADs are retrotranslocated into extra-luminal subcellular compartments, where Nrf1 is deglycosylated to yield an active 95-kDa isoform. Herein, we report that AD1 and AD2 are required for the stability of the 120-kDa Nrf1 glycoprotein, but not that of the non-glycosylated/de-glycosylated 95-kDa isoform. Degrons within AD1 do not promote proteolytic degradation of the 120-kDa Nrf1 glycoprotein. However, repositioning of AD2-adjoining degrons (i.e. DSGLS-containing SDS1 and PEST2 sequences) into the cyto/nucleoplasm enables selective topovectorial processing of Nrf1 by the proteasome and/or calpains to generate a cleaved active 85-kDa Nrf1 or a dominant-negative 36-kDa Nrf1γ. Production of Nrf1γ is abolished by removal of SDS1 or PEST2 degrons, whereas production of the cleaved 85-kDa Nrf1 is blocked by deletion of the ER luminal-anchoring NHB2 sequence (aa 81–106). Importantly, Nrf1 activity is positively and/or negatively regulated by distinct doses of proteasome and calpain inhibitors. PMID:26268886

  10. The Flaveria bidentis β-Carbonic Anhydrase Gene Family Encodes Cytosolic and Chloroplastic Isoforms Demonstrating Distinct Organ-Specific Expression Patterns1[OA

    PubMed Central

    Tetu, Sasha G.; Tanz, Sandra K.; Vella, Nicole; Burnell, James N.; Ludwig, Martha

    2007-01-01

    Carbonic anhydrase (CA) catalyzes the interconversion of CO2 and bicarbonate, the forms of inorganic carbon used by the primary carboxylating enzymes of C3 and C4 plants, respectively. Multiple forms of CA are found in both photosynthetic subtypes; however, the number of isoforms and the location and function of each have not been elucidated for any single plant species. Genomic Southern analyses showed that the C4 dicotyledon Flaveria bidentis ‘Kuntze’ contains a small gene family encoding β-CA and cDNAs encoding three distinct β-CAs, named CA1, CA2, and CA3, were isolated. Quantitative reverse transcription-polymerase chain reactions showed that each member of this β-CA family has a specific expression pattern in F. bidentis leaves, roots, and flowers. CA3 transcripts were at least 50 times more abundant than CA2 or CA1 transcripts in leaves. CA2 transcripts were detected in all organs examined and were the most abundant CA transcripts in roots. CA1 mRNA levels were similar to those of CA2 in leaves, but were considerably lower in roots and flowers. In vitro import assays showed CA1 was imported into isolated pea (Pisum sativum) chloroplasts, whereas CA2 and CA3 were not. These results support the following roles for F. bidentis CAs: CA3 is responsible for catalyzing the first step in the C4 pathway in the mesophyll cell cytosol; CA2 provides bicarbonate for anapleurotic reactions involving nonphotosynthetic forms of phosphoenolpyruvate carboxylase in the cytosol of cells in both photosynthetic and nongreen tissues; and CA1 carries out nonphotosynthetic functions demonstrated by C3 chloroplastic β-CAs, including lipid biosynthesis and antioxidant activity. PMID:17496111

  11. DNA signals at isoform promoters

    PubMed Central

    Dai, Zhiming; Xiong, Yuanyan; Dai, Xianhua

    2016-01-01

    Transcriptional heterogeneity is extensive in the genome, and most genes express variable transcript isoforms. However, whether variable transcript isoforms of one gene are regulated by common promoter elements remain to be elucidated. Here, we investigated whether isoform promoters of one gene have separated DNA signals for transcription and translation initiation. We found that TATA box and nucleosome-disfavored DNA sequences are prevalent in distinct transcript isoform promoters of one gene. These DNA signals are conserved among species. Transcript isoform has a RNA-determined unstructured region around its start site. We found that these DNA/RNA features facilitate isoform transcription and translation. These results suggest a DNA-encoded mechanism by which transcript isoform is generated. PMID:27353836

  12. DNA signals at isoform promoters.

    PubMed

    Dai, Zhiming; Xiong, Yuanyan; Dai, Xianhua

    2016-01-01

    Transcriptional heterogeneity is extensive in the genome, and most genes express variable transcript isoforms. However, whether variable transcript isoforms of one gene are regulated by common promoter elements remain to be elucidated. Here, we investigated whether isoform promoters of one gene have separated DNA signals for transcription and translation initiation. We found that TATA box and nucleosome-disfavored DNA sequences are prevalent in distinct transcript isoform promoters of one gene. These DNA signals are conserved among species. Transcript isoform has a RNA-determined unstructured region around its start site. We found that these DNA/RNA features facilitate isoform transcription and translation. These results suggest a DNA-encoded mechanism by which transcript isoform is generated. PMID:27353836

  13. Two Isoforms of Geobacter sulfurreducens PilA Have Distinct Roles in Pilus Biogenesis, Cytochrome Localization, Extracellular Electron Transfer, and Biofilm Formation

    PubMed Central

    Richter, Lubna V.; Sandler, Steven J.

    2012-01-01

    Type IV pili of Geobacter sulfurreducens are composed of PilA monomers and are essential for long-range extracellular electron transfer to insoluble Fe(III) oxides and graphite anodes. A previous analysis of pilA expression indicated that transcription was initiated at two positions, with two predicted ribosome-binding sites and translation start codons, potentially producing two PilA preprotein isoforms. The present study supports the existence of two functional translation start codons for pilA and identifies two isoforms (short and long) of the PilA preprotein. The short PilA isoform is found predominantly in an intracellular fraction. It seems to stabilize the long isoform and to influence the secretion of several outer-surface c-type cytochromes. The long PilA isoform is required for secretion of PilA to the outer cell surface, a process that requires coexpression of pilA with nine downstream genes. The long isoform was determined to be essential for biofilm formation on certain surfaces, for optimum current production in microbial fuel cells, and for growth on insoluble Fe(III) oxides. PMID:22408162

  14. Eukaryotic Initiation Factor eIFiso4G1 and eIFiso4G2 Are Isoforms Exhibiting Distinct Functional Differences in Supporting Translation in Arabidopsis.

    PubMed

    Gallie, Daniel R

    2016-01-15

    The eukaryotic translation initiation factor (eIF) 4G is required during protein synthesis to promote the assembly of several factors involved in the recruitment of a 40S ribosomal subunit to an mRNA. Although many eukaryotes express two eIF4G isoforms that are highly similar, the eIF4G isoforms in plants, referred to as eIF4G and eIFiso4G, are highly divergent in size, sequence, and domain organization but both can interact with eIF4A, eIF4B, eIF4E isoforms, and the poly(A)-binding protein. Nevertheless, eIF4G and eIFiso4G from wheat exhibit preferences in the mRNAs they translate optimally. For example, mRNA containing the 5'-leader (called Ω) of tobacco mosaic virus preferentially uses eIF4G in wheat germ lysate. In this study, the eIF4G isoform specificity of Ω was used to examine functional differences of the eIF4G isoforms in Arabidopsis. As in wheat, Ω-mediated translation was reduced in an eif4g null mutant. Loss of the eIFiso4G1 isoform, which is similar in sequence to wheat eIFiso4G, did not substantially affect Ω-mediated translation. However, loss of the eIFiso4G2 isoform substantially reduced Ω-mediated translation. eIFiso4G2 is substantially divergent from eIFiso4G1 and is present only in the Brassicaceae, suggesting a recent evolution. eIFiso4G2 isoforms exhibit sequence-specific differences in regions representing partner protein and RNA binding sites. Loss of any eIF4G isoform also resulted in a substantial reduction in reporter transcript level. These results suggest that eIFiso4G2 appeared late in plant evolution and exhibits more functional similarity with eIF4G than with eIFiso4G1 during Ω-mediated translation. PMID:26578519

  15. Non-Secreted Clusterin Isoforms Are Translated in Rare Amounts from Distinct Human mRNA Variants and Do Not Affect Bax-Mediated Apoptosis or the NF-κB Signaling Pathway

    PubMed Central

    Prochnow, Hans; Gollan, Rene; Rohne, Philipp; Hassemer, Matthias; Koch-Brandt, Claudia; Baiersdörfer, Markus

    2013-01-01

    Clusterin, also known as apolipoprotein J, is expressed from a variety of tissues and implicated in pathological disorders such as neurodegenerative diseases, ischemia and cancer. In contrast to secretory clusterin (sCLU), which acts as an extracellular chaperone, the synthesis, subcellular localization and function(s) of intracellular CLU isoforms is currently a matter of intense discussion. By investigating human CLU mRNAs we here unravel mechanisms leading to the synthesis of distinct CLU protein isoforms and analyze their subcellular localization and their impact on apoptosis and on NF-κB-activity. Quantitative PCR-analyses revealed the expression of four different stress-inducible CLU mRNA variants in non-cancer and cancer cell lines. In all cell lines variant 1 represents the most abundant mRNA, whereas all other variants collectively account for no more than 0.34% of total CLU mRNA, even under stressed conditions. Overexpression of CLU cDNAs combined with in vitro mutagenesis revealed distinct translational start sites including a so far uncharacterized non-canonical CUG start codon. We show that all exon 2-containing mRNAs encode sCLU and at least three non-glycosylated intracellular isoforms, CLU1‑449, CLU21‑449 and CLU34‑449, which all reside in the cytosol of unstressed and stressed HEK‑293 cells. The latter is the only form expressed from an alternatively spliced mRNA variant lacking exon 2. Functional analysis revealed that none of these cytosolic CLU forms modulate caspase-mediated intrinsic apoptosis or significantly affects TNF-α-induced NF-κB-activity. Therefore our data challenge some of the current ideas regarding the physiological functions of CLU isoforms in pathologies. PMID:24073260

  16. Distinct transcriptional regulation of long-chain acyl-CoA synthetase isoforms and cytosolic thioesterase 1 in the rodent heart by fatty acids and insulin.

    PubMed

    Durgan, David J; Smith, Justin K; Hotze, Margaret A; Egbejimi, Oluwaseun; Cuthbert, Karalyn D; Zaha, Vlad G; Dyck, Jason R B; Abel, E Dale; Young, Martin E

    2006-06-01

    The molecular mechanism(s) responsible for channeling long-chain fatty acids (LCFAs) into oxidative versus nonoxidative pathways is (are) poorly understood in the heart. Intracellular LCFAs are converted to long-chain fatty acyl-CoAs (LCFA-CoAs) by a family of long-chain acyl-CoA synthetases (ACSLs). Cytosolic thioesterase 1 (CTE1) hydrolyzes cytosolic LCFA-CoAs to LCFAs, generating a potential futile cycle at the expense of ATP utilization. We hypothesized that ACSL isoforms and CTE1 are differentially regulated in the heart during physiological and pathophysiological conditions. Using quantitative RT-PCR, we report that the five known acsl isoforms (acsl1, acsl3, acsl4, acsl5, and acsl6) and cte1 are expressed in whole rat and mouse hearts, as well as adult rat cardiomyocytes (ARCs). Streptozotocin-induced insulin-dependent diabetes (4 wk) and fasting (isoforms. In contrast, high-fat feeding (4 wk) induced cte1 without affecting expression of the acsl isoforms in the heart. Investigation into the mechanism(s) responsible for these transcriptional changes uncovered roles for peroxisome proliferator-activated receptor-alpha (PPARalpha) and insulin as regulators of specific acsl isoforms and cte1 in the heart. Culturing ARCs with oleate (0.1-0.4 mM) or the PPARalpha agonists WY-14643 (1 muM) and fenofibrate (10 muM) consistently induced acsl1 and cte1. Conversely, PPARalpha null mouse hearts exhibited decreased acsl1 and cte1 expression. Culturing ARCs with insulin (10 nM) induced acsl6, whereas specific loss of insulin signaling within the heart (cardiac-specific insulin receptor knockout mice) caused decreased acsl6 expression. Our data expose differential regulation of acsl isoforms and cte1 in the heart, where acsl1 and cte1 are PPARalpha-regulated genes, whereas acsl6 is an insulin-regulated gene. PMID:16428347

  17. Alternative mRNA Splicing from the Glial Fibrillary Acidic Protein (GFAP) Gene Generates Isoforms with Distinct Subcellular mRNA Localization Patterns in Astrocytes

    PubMed Central

    Thomsen, Rune; Daugaard, Tina F.; Holm, Ida E.; Nielsen, Anders Lade

    2013-01-01

    The intermediate filament network of astrocytes includes Glial fibrillary acidic protein (Gfap) as a major component. Gfap mRNA is alternatively spliced resulting in generation of different protein isoforms where Gfapα is the most predominant isoform. The Gfapδ isoform is expressed in proliferating neurogenic astrocytes of the developing human brain and in the adult human and mouse brain. Here we provide a characterization of mouse Gfapδ mRNA and Gfapδ protein. RT-qPCR analysis showed that Gfapδ mRNA and Gfapα mRNA expression is coordinately increased in the post-natal period. Immunohistochemical staining of developing mouse brain samples showed that Gfapδ is expressed in the sub-ventricular zones in accordance with the described localization in the developing and adult human brain. Immunofluorescence analysis verified incorporation of Gfapδ into the Gfap intermediate filament network and overlap in Gfapδ and Gfapα subcellular localization. Subcellular mRNA localization studies identified different localization patterns of Gfapδ and Gfapα mRNA in mouse primary astrocytes. A larger fraction of Gfapα mRNA showed mRNA localization to astrocyte protrusions compared to Gfapδ mRNA. The differential mRNA localization patterns were dependent on the different 3′-exon sequences included in Gfapδ and Gfapα mRNA. The presented results show that alternative Gfap mRNA splicing results in isoform-specific mRNA localization patterns with resulting different local mRNA concentration ratios which have potential to participate in subcellular region-specific intermediate filament dynamics during brain development, maintenance and in disease. PMID:23991052

  18. Endopeptidase Cleavage Generates a Functionally Distinct Isoform of C1q/Tumor Necrosis Factor-related Protein-12 (CTRP12) with an Altered Oligomeric State and Signaling Specificity*

    PubMed Central

    Wei, Zhikui; Lei, Xia; Seldin, Marcus M.; Wong, G. William

    2012-01-01

    Adipose tissue-derived adipokines are an important class of secreted metabolic regulators that mediate tissue cross-talk to control systemic energy balance. We recently described C1q/TNF-related protein-12 (CTRP12), a novel insulin-sensitizing adipokine that regulates glucose metabolism in liver and adipose tissue. However, the biochemical properties of CTRP12 and its naturally occurring cleaved isoform have not been characterized. Here, we show that CTRP12 is a secreted hormone subjected to multiple functionally relevant posttranslational modifications at highly conserved residues. For example, Asn39 is glycosylated, whereas Cys85 mediates the assembly of higher order oligomeric structure. Endopeptidase cleavage at Lys91 generates a cleaved globular gCTRP12 isoform, the expression of which is increased by insulin. PCSK3/furin was identified as the major proprotein convertase expressed by adipocytes that mediates the endogenous cleavage of CTRP12. Cleavage at Lys91 is context-dependent: mutation of the charged Arg93 to Ala on the P2′ position enhanced cleavage, and triple mutations (K90A/K91A/R93A) abolished cleavage. Importantly, the two isoforms of CTRP12 differ in oligomeric structures and are functionally distinct. The full-length protein forms trimers and larger complexes, and the cleaved isoform consisted of predominantly dimers. Whereas full-length fCTRP12 strongly activated Akt signaling in H4IIE hepatocytes and 3T3-L1 adipocytes, gCTRP12 preferentially activated MAP kinase (ERK1/2 and p38 MAPK) signaling. Further, only fCTRP12 improved insulin-stimulated glucose uptake in adipocytes. These results reveal a novel mechanism controlling signaling specificity and function of a hormone via cleavage-dependent alteration in oligomeric state. PMID:22942287

  19. Distinct PKC isoforms mediate the activation of cPLA2 and adenylyl cyclase by phorbol ester in RAW264.7 macrophages

    PubMed Central

    Lin, Wan-W; Chen, Bin C

    1998-01-01

    The modulatory effects of protein kinase C (PKC) on the activation of cytosolic phospholipase A2 (cPLA2) and adenylyl cyclase (AC) have recently been described. Since the signalling cascades associated with these events play critical roles in various functions of macrophages, we set out to investigate the crosstalk between PKC and the cPLA2 and AC pathways in mouse RAW 264.7 macrophages and to determine the involvement of individual PKC isoforms. The cPLA2 and AC pathways were studied by measuring the potentiation by the phorbol ester PMA of ionomycin-induced arachidonic acid (AA) release and prostagladin E1 (PGE1)-stimulated cyclic AMP production, respectively.PMA at 1 μM caused a significant increase in AA release both in the presence (371%) and absence (67%) of ionomycin induction, while exposure of RAW 264.7 cells to PMA increased PGE1 stimulation of cyclic AMP levels by 208%.Treatment of cells with staurosporine and Ro 31-8220 inhibited the PMA-induced potentiation of both AA release and cyclic AMP accumulation, while Go 6976 (an inhibitor of classical PKC isoforms) and LY 379196 (a specific inhibitor of PKCβ) inhibited the AA response but failed to affect the enhancement of the cyclic AMP response by PMA.Long term pretreatment of cells with PMA abolished the subsequent effect of PMA in potentiating AA release, but only inhibited the cyclic AMP response by 42%.Neither PD 98059, an inhibitor of MEK, nor genistein, an inhibitor of tyrosine kinases, had any effect on the ability of PMA to potentiate AA or cyclic AMP production.The potentiation of AA release, but not of cyclic AMP formation, by PMA was sensitive to inhibition by wortmannin. This effect was unrelated to the inhibition of PKC activation as deduced from the translocation of PKC activity to the cell membrane.Western blot analysis revealed the presence of eight PKC isoforms (α, βI, βII, δ, ε, μ λ and ξ) in RAW 264.7 cells and PMA was shown to induce the translocation of the α, βI, βII,

  20. A splicing alteration of 4.1R pre-mRNA generates 2 protein isoforms with distinct assembly to spindle poles in mitotic cells.

    PubMed

    Delhommeau, François; Vasseur-Godbillon, Corinne; Leclerc, Philippe; Schischmanoff, Pierre-Olivier; Croisille, Laure; Rince, Patricia; Morinière, Madeleine; Benz, Edward J; Tchernia, Gil; Tamagnini, Gabriel; Ribeiro, Leticia; Delaunay, Jean; Baklouti, Faouzi

    2002-10-01

    The C-terminal region of erythroid cytoskeletal protein 4.1R, encoded by exons 20 and 21, contains a binding site for nuclear mitotic apparatus protein (NuMA), a protein needed for the formation and stabilization of the mitotic spindle. We have previously described a splicing mutation of 4.1R that yields 2 isoforms: One, CO.1, lacks most of exon 20-encoded peptide and carries a missense C-terminal sequence. The other, CO.2, lacks exon 20-encoded C-terminal sequence, but retains the normal exon 21-encoded C-terminal sequence. Knowing that both shortened proteins are expressed in red cells and assemble to the membrane skeleton, we asked whether they would ensure 4.1R mitotic function in dividing cells. We show here that CO.2, but not CO.1, assembles to spindle poles, and colocalizes with NuMA in erythroid and lymphoid mutated cells, but none of these isoforms interact with NuMA in vitro. In microtubule-destabilizing conditions, again only CO.2 localizes to the centrosomes. These data suggest that the stability of 4.1R association with centrosomes requires an intact C-terminal end, either for a proper conformation of the protein, for a direct binding to an unknown centrosome-cytoskeletal network, or for both. We also found that 4.1G, a ubiquitous homolog of 4.1R, is present in mutated as well as control cells and that its C-terminal region binds efficiently to NuMA, suggesting that in fact mitotic spindles host a mixture of the two 4.1 family members. These findings led to the postulate that the coexpression at the spindle poles of 2 related proteins, 4.1R and 4.1G, might reflect a functional redundancy in mitotic cells. PMID:12239178

  1. Differential effects of retinoic acid on the growth of isogenic metastatic and non-metastatic breast cancer cell lines and their association with distinct expression of retinoic acid receptor beta isoforms 2 and 4.

    PubMed

    Hayashi, Ken; Goodison, Steven; Urquidi, Virginia; Tarin, David; Lotan, Reuben; Tahara, Eiichi

    2003-03-01

    The human retinoic acid receptor beta (RARbeta) has three isoforms (beta1, beta2, and beta4), which play important, distinct roles in mediating the effects of retinoic acid on cell growth and apoptosis. Whereas RARbeta2 is a potent inhibitor of breast cancer cell proliferation, RARbeta4 can act as a dominant-negative repressor of RARbeta2-mediated growth suppression. In this study we investigated the effects of all-trans-retinoic acid (ATRA) on two clones derived from the breast cancer cell line MDA-MB-435: a non-metastatic clone (NM-2C5) and a metastatic clone (M-4A4). ATRA treatment of the NM-2C5 cells resulted in growth inhibition and apoptosis, whereas the M-4A4 cells were resistant to ATRA. Analyses of the expression of RARbeta isoforms revealed that the sensitive NM-2C5 clone expressed only RARbeta2, whereas the resistant M-4A4 cells expressed both RARbeta2 and RARbeta4 mRNA and protein. ATRA treatment increased RARbeta2 mRNA level in NM-2C5 cells, whereas the same treatment of the M-4A4 cells resulted in an increase in RARbeta4 and a decrease in RARbeta2 mRNA. ATRA treatment of NM-2C5 cells increased the protein levels of the histone acetyl transferases p300 and CBP, suppressed the level of histone deacetylase and increased the level of acetylated histone H4. ATRA also decreased Bcl-2 and increased Bax and decreased VEGF. In contrast, the same treatment of the M-4A4 cells resulted in opposite effects. These results suggest that the effects of ATRA on the growth of the metastatic and non-metastatic breast cancer cell lines depend on the expression of RARbeta isoforms and that the expression of RARbeta4 may contribute to metastatic properties. PMID:12579317

  2. Purification and characterization of high- and low-molecular-mass isoforms of phosphoenolpyruvate carboxylase from Chlamydomonas reinhardtii. Kinetic, structural and immunological evidence that the green algal enzyme is distinct from the prokaryotic and higher plant enzymes.

    PubMed Central

    Rivoal, J; Plaxton, W C; Turpin, D H

    1998-01-01

    Phosphoenolpyruvate carboxylase (PEPC) is a key enzyme in the supply of carbon skeletons for the assimilation of nitrogen by green algae. Two PEPC isoforms with respective native molecular masses of 400 (PEPC1) and 650 (PEPC2) kDa have been purified from Chlamydomonas reinhardtii CW-15 cc1883 (Chlorophyceae). SDS/PAGE, immunoblot and CNBr peptide-mapping analyses indicate the presence of the same 100 kDa PEPC catalytic subunit in both isoforms. PEPC1 is a homotetramer, whereas PEPC2 seems to be a complex between the PEPC catalytic subunit and other immunologically unrelated polypeptides of 50-70 kDa. Kinetic analyses indicate that these PEPC isoforms are (1) differentially regulated by pH, (2) activated by glutamine and dihydroxyacetone phosphate and (3) inhibited by glutamate, aspartate, 2-oxoglutarate and malate. These results are consistent with the current model for the regulation of anaplerotic carbon fixation in green algae, and demonstrate that green algal PEPCs are uniquely regulated by glutamine. Several techniques were used to assess the structural relationships between C. reinhardtii PEPC and the higher plant or prokaryotic enzyme. Immunoblot studies using anti-(green algal or higher plant PEPC) IgGs suggested that green algal (C. reinhardtii, Selenastrum minutum), higher plant (maize, banana fruit, tobacco) and prokaryotic (Synechococcus leopoliensis, Escherichia coli) PEPCs have little or no immunological relatedness. Moreover, the N-terminal amino acid sequence of the C. reinhardtii PEPC subunit did not have significant similarity to the highly conserved corresponding region in enzymes from higher plants, and CNBr cleavage patterns of green algal PEPCs were distinct from those of higher plant and cyanobacterial PEPCs. These results point to significant evolutionary divergence between green algal, higher plant and prokaryotic PEPCs. PMID:9512480

  3. Adenine nucleotide translocator isoforms 1 and 2 are differently distributed in the mitochondrial inner membrane and have distinct affinities to cyclophilin D.

    PubMed Central

    Vyssokikh, M Y; Katz, A; Rueck, A; Wuensch, C; Dörner, A; Zorov, D B; Brdiczka, D

    2001-01-01

    Different isoforms of the adenine nucleotide translocase (ANT) are expressed in a tissue-specific manner. It was assumed that ANT-1 and ANT-2 co-exist in every single mitochondrion and might be differently distributed within the membrane structures that constitute the peripheral inner membrane or the crista membrane. To discriminate between ANT originating from peripheral or from cristal inner membranes we made use of the fact that complexes between porin, the outer-membrane pore protein, and the ANT can be generated. Such complexes between porin and the ANT in the peripheral inner membrane were induced in rat heart mitochondria and isolated from rat brain and kidney. Using ANT-isotype-specific antibodies and sequence analysis of the N-terminal end, it was discovered that the peripheral inner membrane contained ANT-1 and ANT-2, whereas the cristal membrane contained exclusively ANT-2. Cyclophilin was co-purified with the porin-ANT complexes, whereas it was absent in the crista-derived ANT. This suggested that ANT-1 might have a higher affinity for cyclophilin. This specific intra-mitochondrial distribution of the two ANT isotypes and cyclophilin D suggests specific functions of the peripheral and crista-forming parts of the inner membrane and the two ANT isotypes therein. PMID:11513733

  4. Full-Length Human Placental sFlt-1-e15a Isoform Induces Distinct Maternal Phenotypes of Preeclampsia in Mice

    PubMed Central

    Szalai, Gabor; Romero, Roberto; Chaiworapongsa, Tinnakorn; Xu, Yi; Wang, Bing; Ahn, Hyunyoung; Xu, Zhonghui; Chiang, Po Jen; Sundell, Birgitta; Wang, Rona; Jiang, Yang; Plazyo, Olesya; Olive, Mary; Tarca, Adi L.; Dong, Zhong; Qureshi, Faisal; Papp, Zoltan; Hassan, Sonia S.; Hernandez-Andrade, Edgar; Than, Nandor Gabor

    2015-01-01

    Objective Most anti-angiogenic preeclampsia models in rodents utilized the overexpression of a truncated soluble fms-like tyrosine kinase-1 (sFlt-1) not expressed in any species. Other limitations of mouse preeclampsia models included stressful blood pressure measurements and the lack of postpartum monitoring. We aimed to 1) develop a mouse model of preeclampsia by administering the most abundant human placental sFlt-1 isoform (hsFlt-1-e15a) in preeclampsia; 2) determine blood pressures in non-stressed conditions; and 3) develop a survival surgery that enables the collection of fetuses and placentas and postpartum (PP) monitoring. Methods Pregnancy status of CD-1 mice was evaluated with high-frequency ultrasound on gestational days (GD) 6 and 7. Telemetry catheters were implanted in the carotid artery on GD7, and their positions were verified by ultrasound on GD13. Mice were injected through tail-vein with adenoviruses expressing hsFlt-1-e15a (n = 11) or green fluorescent protein (GFP; n = 9) on GD8/GD11. Placentas and pups were delivered by cesarean section on GD18 allowing PP monitoring. Urine samples were collected with cystocentesis on GD6/GD7, GD13, GD18, and PPD8, and albumin/creatinine ratios were determined. GFP and hsFlt-1-e15a expression profiles were determined by qRT-PCR. Aortic ring assays were performed to assess the effect of hsFlt-1-e15a on endothelia. Results Ultrasound predicted pregnancy on GD7 in 97% of cases. Cesarean section survival rate was 100%. Mean arterial blood pressure was higher in hsFlt-1-e15a-treated than in GFP-treated mice (∆MAP = 13.2 mmHg, p = 0.00107; GD18). Focal glomerular changes were found in hsFlt-1-e15a -treated mice, which had higher urine albumin/creatinine ratios than controls (109.3±51.7μg/mg vs. 19.3±5.6μg/mg, p = 4.4x10-2; GD18). Aortic ring assays showed a 46% lesser microvessel outgrowth in hsFlt-1-e15a-treated than in GFP-treated mice (p = 1.2x10-2). Placental and fetal weights did not differ between the

  5. Distinct or shared actions of peptide family isoforms: I. Peptide-specific actions of pyrokinins in the lobster cardiac neuromuscular system.

    PubMed

    Dickinson, Patsy S; Sreekrishnan, Anirudh; Kwiatkowski, Molly A; Christie, Andrew E

    2015-09-01

    Although the crustacean heart is modulated by a large number of peptides and amines, few of these molecules have been localized to the cardiac ganglion itself; most appear to reach the cardiac ganglion only by hormonal routes. Immunohistochemistry in the American lobster Homarus americanus indicates that pyrokinins are present not only in neuroendocrine organs (pericardial organ and sinus gland), but also in the cardiac ganglion itself, where pyrokinin-positive terminals were found in the pacemaker cell region, as well as surrounding the motor neurons. Surprisingly, the single pyrokinin peptide identified from H. americanus, FSPRLamide, which consists solely of the conserved FXPRLamide residues that characterize pyrokinins, did not alter the activity of the cardiac neuromuscular system. However, a pyrokinin from the shrimp Litopenaeus vannamei [ADFAFNPRLamide, also known as Penaeus vannamei pyrokinin 2 (PevPK2)] increased both the frequency and amplitude of heart contractions when perfused through the isolated whole heart. None of the other crustacean pyrokinins tested (another from L. vannamei and two from the crab Cancer borealis) had any effect on the lobster heart. Similarly, altering the PevPK2 sequence either by truncation or by the substitution of single amino acids resulted in much lower or no activity in all cases; only the conservative substitution of serine for alanine at position 1 resulted in any activity on the heart. Thus, in contrast to other systems (cockroach and crab) in which all tested pyrokinins elicit similar bioactivities, activation of the pyrokinin receptor in the lobster heart appears to be highly isoform specific. PMID:26206360

  6. Distinct or shared actions of peptide family isoforms: I. Peptide-specific actions of pyrokinins in the lobster cardiac neuromuscular system

    PubMed Central

    Dickinson, Patsy S.; Sreekrishnan, Anirudh; Kwiatkowski, Molly A.; Christie, Andrew E.

    2015-01-01

    ABSTRACT Although the crustacean heart is modulated by a large number of peptides and amines, few of these molecules have been localized to the cardiac ganglion itself; most appear to reach the cardiac ganglion only by hormonal routes. Immunohistochemistry in the American lobster Homarus americanus indicates that pyrokinins are present not only in neuroendocrine organs (pericardial organ and sinus gland), but also in the cardiac ganglion itself, where pyrokinin-positive terminals were found in the pacemaker cell region, as well as surrounding the motor neurons. Surprisingly, the single pyrokinin peptide identified from H. americanus, FSPRLamide, which consists solely of the conserved FXPRLamide residues that characterize pyrokinins, did not alter the activity of the cardiac neuromuscular system. However, a pyrokinin from the shrimp Litopenaeus vannamei [ADFAFNPRLamide, also known as Penaeus vannamei pyrokinin 2 (PevPK2)] increased both the frequency and amplitude of heart contractions when perfused through the isolated whole heart. None of the other crustacean pyrokinins tested (another from L. vannamei and two from the crab Cancer borealis) had any effect on the lobster heart. Similarly, altering the PevPK2 sequence either by truncation or by the substitution of single amino acids resulted in much lower or no activity in all cases; only the conservative substitution of serine for alanine at position 1 resulted in any activity on the heart. Thus, in contrast to other systems (cockroach and crab) in which all tested pyrokinins elicit similar bioactivities, activation of the pyrokinin receptor in the lobster heart appears to be highly isoform specific. PMID:26206360

  7. Development and characterization of a polyclonal antibody against rat liver mitochondrial overt carnitine palmitoyltransferase (CPT I). Distinction of CPT I from CPT II and of isoforms of CPT I in different tissues.

    PubMed Central

    Kolodziej, M P; Crilly, P J; Corstorphine, C G; Zammit, V A

    1992-01-01

    The [3H]tetradecylglycidyl-CoA (TDG-CoA)-binding protein (Mr approx. 88,000) of purified outer membranes from rat liver mitochondria was identified by SDS/PAGE. The region in which it migrated was shown to contain another protein which stained strongly with periodic acid-Schiff reagent and could be removed from membrane extracts by incubation with Sepharose-concanavalin A. Amounts of TDG-CoA-binding protein were prepared from lectin-treated extracts using preparative SDS/PAGE and used to raise a polyclonal antibody in a sheep. The IgG fraction purified from this anti-serum reacted strongly with a protein of Mr approximately 88,000 on Western blots, and much more weakly with two other proteins of Mr approximately 76,000 and Mr approximately 53,000 in extracts of rat liver mitochondrial outer membranes. The crude IgG fraction and immunopurified IgG both removed carnitine palmitoyltransferase (CPT) I activity from very pure outer membrane extracts, suggesting that the TDG-CoA-binding protein against which the antiserum was raised also expresses CPT I activity. This was confirmed by the demonstration of a strong positive correlation between CPT I activity and the amount of immunoreactive protein of Mr approximately 88,000 in mitochondria prepared from rats in different physiological states. By contrast, the antibody did not react with CPT II either in mitochondria or in purified form. Similarly, an anti-(CPT II) antibody did not cross-react with CPT I on Western blots, proving conclusively that CPT I and CPT II are immunologically distinct proteins, as well as being of different functional molecular sizes [Zammit, Corstophine & Kelliher (1988) Biochem. J. 250, 415-420]. Immunoblots of mitochondrial proteins obtained from different tissues indicated that, of the rat tissues tested, only kidney cortex mitochondria contain the same isoform of CPT I as that in liver. Heart, skeletal muscle and brown adipose tissue mitochondria contain a slightly smaller isoform which was

  8. Isoform distinct time-, dose-, and castration-dependent alterations in flavin-containing monooxygenase expression in mouse liver after 2,3,7,8-tetrachlorodibenzo-p-dioxin treatment

    PubMed Central

    Novick, Rachel M.; Vezina, Chad M.; Elfarra, Adnan A.

    2010-01-01

    Flavin-containing monooxygenase (FMO) expression in male mouse liver is altered after 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure or castration. Because TCDD is slowly eliminated from the body, we examined hepatic Fmo mRNA alterations for up to 32 days following 10 or 64 µg/kg TCDD exposure by oral gavage in male C57BL/6J mice. Fmo2 mRNA was significantly induced at 1, 4, and 8 days whereas Fmo3 mRNA was also induced at 32 days relative to controls. Fmo3 mRNA levels exhibited a dose-dependent increase at 4, 8, and 32 days after exposure; Fmo1, Fmo4, and Fmo5 mRNA did not exhibit clear trends. Because castration alone also increased Fmo2, Fmo3, and Fmo4 mRNA we examined the combined effects of castration and TCDD treatment on FMO expression. A greater than additive effect was observed with Fmo2 and Fmo3 mRNA expression. Fmo2 mRNA exhibited a 3–5 fold increase after castration or 10 µg/kg TCDD exposure by oral gavage, whereas an approximately 20-fold increase was observed between the sham-castrated control and castrated TCDD-treated mice. Similarly, treatment with 10 µg/kg TCDD alone increased Fmo3 mRNA 130- and 180-fold in the sham-castrated and castrated mice compared to their controls respectively, whereas, Fmo3 mRNA increased approximately 1900-fold between the sham control and castrated TCDD-treated mice. An increase in hepatic Fmo3 protein in TCDD treated mice was observed by immunoblotting and assaying methionine S-oxidase activity. Collectively, these results provide evidence for isoform distinct time-, dose-, and castration-dependent effects of TCDD on FMO expression and suggest cross-talk between TCDD and testosterone signal transduction pathways. PMID:20036217

  9. Isoforms of Melanopsin Mediate Different Behavioral Responses to Light.

    PubMed

    Jagannath, Aarti; Hughes, Steven; Abdelgany, Amr; Pothecary, Carina A; Di Pretoro, Simona; Pires, Susana S; Vachtsevanos, Athanasios; Pilorz, Violetta; Brown, Laurence A; Hossbach, Markus; MacLaren, Robert E; Halford, Stephanie; Gatti, Silvia; Hankins, Mark W; Wood, Matthew J A; Foster, Russell G; Peirson, Stuart N

    2015-09-21

    Melanopsin (OPN4) is a retinal photopigment that mediates a wide range of non-image-forming (NIF) responses to light including circadian entrainment, sleep induction, the pupillary light response (PLR), and negative masking of locomotor behavior (the acute suppression of activity in response to light). How these diverse NIF responses can all be mediated by a single photopigment has remained a mystery. We reasoned that the alternative splicing of melanopsin could provide the basis for functionally distinct photopigments arising from a single gene. The murine melanopsin gene is indeed alternatively spliced, producing two distinct isoforms, a short (OPN4S) and a long (OPN4L) isoform, which differ only in their C terminus tails. Significantly, both isoforms form fully functional photopigments. Here, we show that different isoforms of OPN4 mediate different behavioral responses to light. By using RNAi-mediated silencing of each isoform in vivo, we demonstrated that the short isoform (OPN4S) mediates light-induced pupillary constriction, the long isoform (OPN4L) regulates negative masking, and both isoforms contribute to phase-shifting circadian rhythms of locomotor behavior and light-mediated sleep induction. These findings demonstrate that splice variants of a single receptor gene can regulate strikingly different behaviors. PMID:26320947

  10. Isoforms of Melanopsin Mediate Different Behavioral Responses to Light

    PubMed Central

    Jagannath, Aarti; Hughes, Steven; Abdelgany, Amr; Pothecary, Carina A.; Di Pretoro, Simona; Pires, Susana S.; Vachtsevanos, Athanasios; Pilorz, Violetta; Brown, Laurence A.; Hossbach, Markus; MacLaren, Robert E.; Halford, Stephanie; Gatti, Silvia; Hankins, Mark W.; Wood, Matthew J.A.; Foster, Russell G.; Peirson, Stuart N.

    2015-01-01

    Summary Melanopsin (OPN4) is a retinal photopigment that mediates a wide range of non-image-forming (NIF) responses to light [1, 2] including circadian entrainment [3], sleep induction [4], the pupillary light response (PLR) [5], and negative masking of locomotor behavior (the acute suppression of activity in response to light) [6]. How these diverse NIF responses can all be mediated by a single photopigment has remained a mystery. We reasoned that the alternative splicing of melanopsin could provide the basis for functionally distinct photopigments arising from a single gene. The murine melanopsin gene is indeed alternatively spliced, producing two distinct isoforms, a short (OPN4S) and a long (OPN4L) isoform, which differ only in their C terminus tails [7]. Significantly, both isoforms form fully functional photopigments [7]. Here, we show that different isoforms of OPN4 mediate different behavioral responses to light. By using RNAi-mediated silencing of each isoform in vivo, we demonstrated that the short isoform (OPN4S) mediates light-induced pupillary constriction, the long isoform (OPN4L) regulates negative masking, and both isoforms contribute to phase-shifting circadian rhythms of locomotor behavior and light-mediated sleep induction. These findings demonstrate that splice variants of a single receptor gene can regulate strikingly different behaviors. PMID:26320947

  11. Tropomyosin isoforms and reagents

    PubMed Central

    Schevzov, Galina; Whittaker, Shane P; Fath, Thomas; Lin, Jim JC

    2011-01-01

    Tropomyosins are rod-like dimers which form head-to-tail polymers along the length of actin filaments and regulate the access of actin binding proteins to the filaments.1 The diversity of tropomyosin isoforms, over 40 in mammals, and their role in an increasing number of biological processes presents a challenge both to experienced researchers and those new to this field. The increased appreciation that the role of these isoforms expands beyond that of simply stabilizing actin filaments has lead to a surge of reagents and techniques to study their function and mechanisms of action. This report is designed to provide a basic guide to the genes and proteins and the availability of reagents which allow effective study of this family of proteins. We highlight the value of combining multiple techniques to better evaluate the function of different tm isoforms and discuss the limitations of selected reagents. Brief background material is included to demystify some of the unfortunate complexity regarding this multi-gene family of proteins including the unconventional nomenclature of the isoforms and the evolutionary relationships of isoforms between species. Additionally, we present step-by-step detailed experimental protocols used in our laboratory to assist new comers to the field and experts alike. PMID:22069507

  12. Distinct Activation Mechanisms of NF-κB Regulator Inhibitor of NF-κB Kinase (IKK) by Isoforms of the Cell Death Regulator Cellular FLICE-like Inhibitory Protein (cFLIP)*

    PubMed Central

    Baratchian, Mehdi; Davis, Christopher A.; Shimizu, Akira; Escors, David; Bagnéris, Claire; Barrett, Tracey; Collins, Mary K.

    2016-01-01

    The viral FLICE-like inhibitory protein (FLIP) protein from Kaposi sarcoma-associated herpesvirus activates the NF-κB pathway by forming a stable complex with a central region (amino acids 150–272) of the inhibitor of NF-κB kinase (IKK) γ subunits, thereby activating IKK. Cellular FLIP (cFLIP) forms are also known to activate the NF-κB pathway via IKK activation. Here we demonstrate that cFLIPL, cFLIPS, and their proteolytic product p22-FLIP all require the C-terminal region of NEMO/IKKγ (amino acids 272–419) and its ubiquitin binding function for activation of the IKK kinase (or kinase complex), but none form a stable complex with IKKγ. Our results further reveal that cFLIPL requires the linear ubiquitin chain assembly complex and the kinase TAK1 for activation of the IKK kinase. Similarly, cFLIPS and p22-FLIP also require TAK1 but do not require LUBAC. In contrast, these isoforms are both components of complexes that incorporate Fas-associated death domain and RIP1, which appear essential for kinase activation. This conservation of IKK activation among the cFLIP family using different mechanisms suggests that the mechanism plays a critical role in their function. PMID:26865630

  13. Selective glucocorticoid receptor translational isoforms reveal glucocorticoid-induced apoptotic transcriptomes

    PubMed Central

    Wu, I; Shin, S C; Cao, Y; Bender, I K; Jafari, N; Feng, G; Lin, S; Cidlowski, J A; Schleimer, R P; Lu, N Z

    2013-01-01

    Induction of T-cell apoptosis contributes to the anti-inflammatory and antineoplastic benefits of glucocorticoids. The glucocorticoid receptor (GR) translational isoforms have distinct proapoptotic activities in osteosarcoma cells. Here we determined whether GR isoforms selectively induce apoptosis in Jurkat T lymphoblastic leukemia cells. Jurkat cells stably expressing individual GR isoforms were generated and treated with vehicle or dexamethasone (DEX). DEX induced apoptosis in cells expressing the GR-A, -B, or -C, but not the GR-D, isoform. cDNA microarray analyses of cells sensitive (GR-C3) and insensitive (GR-D3) to DEX revealed glucocorticoid-induced proapoptotic transcriptomes. Genes that were regulated by the proapoptotic GR-C3, but not by the GR-D3, isoform likely contributed to glucocorticoid-induced apoptosis. The identified genes include those that are directly involved in apoptosis and those that facilitate cell killing. Chromatin immunoprecipitation assays demonstrated that distinct chromatin modification abilities may underlie the distinct functions of GR isoforms. Interestingly, all GR isoforms, including the GR-D3 isoform, suppressed mitogen-stimulated cytokines. Furthermore, the GR-C isoforms were selectively upregulated in mitogen-activated primary T cells and DEX treatment induced GR-C target genes in activated T cells. Cell-specific expressions and functions of GR isoforms may help to explain the tissue- and individual-selective actions of glucocorticoids and may provide a basis for developing improved glucocorticoids. PMID:23303127

  14. Selective glucocorticoid receptor translational isoforms reveal glucocorticoid-induced apoptotic transcriptomes.

    PubMed

    Wu, I; Shin, S C; Cao, Y; Bender, I K; Jafari, N; Feng, G; Lin, S; Cidlowski, J A; Schleimer, R P; Lu, N Z

    2013-01-01

    Induction of T-cell apoptosis contributes to the anti-inflammatory and antineoplastic benefits of glucocorticoids. The glucocorticoid receptor (GR) translational isoforms have distinct proapoptotic activities in osteosarcoma cells. Here we determined whether GR isoforms selectively induce apoptosis in Jurkat T lymphoblastic leukemia cells. Jurkat cells stably expressing individual GR isoforms were generated and treated with vehicle or dexamethasone (DEX). DEX induced apoptosis in cells expressing the GR-A, -B, or -C, but not the GR-D, isoform. cDNA microarray analyses of cells sensitive (GR-C3) and insensitive (GR-D3) to DEX revealed glucocorticoid-induced proapoptotic transcriptomes. Genes that were regulated by the proapoptotic GR-C3, but not by the GR-D3, isoform likely contributed to glucocorticoid-induced apoptosis. The identified genes include those that are directly involved in apoptosis and those that facilitate cell killing. Chromatin immunoprecipitation assays demonstrated that distinct chromatin modification abilities may underlie the distinct functions of GR isoforms. Interestingly, all GR isoforms, including the GR-D3 isoform, suppressed mitogen-stimulated cytokines. Furthermore, the GR-C isoforms were selectively upregulated in mitogen-activated primary T cells and DEX treatment induced GR-C target genes in activated T cells. Cell-specific expressions and functions of GR isoforms may help to explain the tissue- and individual-selective actions of glucocorticoids and may provide a basis for developing improved glucocorticoids. PMID:23303127

  15. Apolipoprotein E isoform-dependent microglia migration

    PubMed Central

    Cudaback, Eiron; Li, Xianwu; Montine, Kathleen S.; Montine, Thomas J.; Keene, C. Dirk

    2011-01-01

    Complement component C5a and ATP are potent effectors of microglial movement and are increased in diverse neurodegenerative diseases and at sites of injury. Apolipoprotein E (apoE) influences microglial function, and different human apoE isoforms confer variable risk for development of neurodegenerative disorders, especially Alzheimer's disease. The purpose of this investigation was to test the hypothesis that mouse apoE and human apoE isoforms influence microglial migration. Using primary wild-type and apoE-deficient microglia, we show that C5a- and ATP-stimulated chemotaxis are largely apoE-dependent processes with different molecular bases. Although the C5a-dependent chemotaxis of wild-type microglia was completely blocked by receptor-associated protein (RAP), suggesting apoE receptor involvement, ATP-stimulated migration was unaffected by RAP but was associated with differential ERK phosphorylation. Studies using primary microglia derived from targeted replacement mice “humanized” for the coding exons (protein isoform) of human ε2 (apoE2), ε3 (apoE3), or ε4 (apoE4) allele of APOE revealed that primary mouse microglia expressing apoE4 or apoE2 exhibited significantly reduced C5a- and ATP-stimulated migration compared with microglia expressing human apoE3. This study, for the first time, demonstrates apoE dependence and apoE isoform-specific modulation of microglial migration in response to distinct chemotactic stimuli commonly associated with neurodegenerative disease.—Cudaback, E., Li, X., Montine, K. S., Montine, T. J., Keene, C. D. Apolipoprotein E isoform-dependent microglia migration. PMID:21385991

  16. Targeted Proteomics Enables Simultaneous Quantification of Folate Receptor Isoforms and Potential Isoform-based Diagnosis in Breast Cancer

    PubMed Central

    Yang, Ting; Xu, Feifei; Fang, Danjun; Chen, Yun

    2015-01-01

    The distinct roles of protein isoforms in cancer are becoming increasingly evident. FRα and FRβ, two major isoforms of the folate receptor family, generally have different cellular distribution and tissue specificity. However, the presence of FRβ in breast tumors, where FRα is normally expressed, complicates this situation. Prior to applying any FR isoform-based diagnosis and therapeutics, it is essential to monitor the expression profile of FR isoforms in a more accurate manner. An LC-MS/MS-based targeted proteomics assay was developed and validated in this study because of the lack of suitable methodology for the simultaneous and specific measurement of highly homologous isoforms occurring at low concentrations. FRα and FRβ monitoring was achieved by measuring their surrogate isoform-specific peptides. Five human breast cell lines, isolated macrophages and 60 matched pairs of breast tissue samples were subjected to the analysis. The results indicated that FRβ was overexpressed in tumor-associated macrophages (TAMs) but not epithelial cells, in addition to an enhanced level of FRα in breast cancer cells and tissue samples. Moreover, the levels of the FR isoforms were evaluated according to the histology, histopathological features and molecular subtypes of breast cancer. Several positive associations with PR/ER and HER2 status and metastasis were revealed. PMID:26573433

  17. Structural Basis of Dscam Isoform Specificity

    SciTech Connect

    Meijers,R.; Puettmann-Holgado, R.; Skiniotis, G.; Liu, J.; Walz, T.; Wang, J.; Schmucker, D.

    2007-01-01

    The Dscam gene gives rise to thousands of diverse cell surface receptors1 thought to provide homophilic and heterophilic recognition specificity for neuronal wiring and immune responses. Mutually exclusive splicing allows for the generation of sequence variability in three immunoglobulin ecto-domains, D2, D3 and D7. We report X-ray structures of the amino-terminal four immunoglobulin domains (D1-D4) of two distinct Dscam isoforms. The structures reveal a horseshoe configuration, with variable residues of D2 and D3 constituting two independent surface epitopes on either side of the receptor. Both isoforms engage in homo-dimerization coupling variable domain D2 with D2, and D3 with D3. These interactions involve symmetric, antiparallel pairing of identical peptide segments from epitope I that are unique to each isoform. Structure-guided mutagenesis and swapping of peptide segments confirm that epitope I, but not epitope II, confers homophilic binding specificity of full-length Dscam receptors. Phylogenetic analysis shows strong selection of matching peptide sequences only for epitope I. We propose that peptide complementarity of variable residues in epitope I of Dscam is essential for homophilic binding specificity.

  18. N Termini of apPDE4 Isoforms Are Responsible for Targeting the Isoforms to Different Cellular Membranes

    ERIC Educational Resources Information Center

    Jang, Deok-Jin; Park, Soo-Won; Lee, Jin-A; Lee, Changhoon; Chae, Yeon-Su; Park, Hyungju; Kim, Min-Jeong; Choi, Sun-Lim; Lee, Nuribalhae; Kim, Hyoung; Kaang, Bong-Kiun

    2010-01-01

    Phosphodiesterases (PDEs) are known to play a key role in the compartmentalization of cAMP signaling; however, the molecular mechanisms underlying intracellular localization of different PDE isoforms are not understood. In this study, we have found that each of the supershort, short, and long forms of apPDE4 showed distinct localization in the…

  19. Identification and characterization of novel NuMA isoforms

    SciTech Connect

    Wu, Jin; Xu, Zhe; He, Dacheng; Lu, Guanting

    2014-11-21

    Highlights: • Seven NuMA isoforms generated by alternative splicing were categorized into 3 groups: long, middle and short. • Both exons 15 and 16 in long NuMA were “hotspot” for alternative splicing. • Lower expression of short NuMA was observed in cancer cells compared with nonneoplastic controls. • Distinct localization pattern of short isoforms indicated different function from that of long and middle NuMA. - Abstract: The large nuclear mitotic apparatus (NuMA) has been investigated for over 30 years with functions related to the formation and maintenance of mitotic spindle poles during mitosis. However, the existence and functions of NuMA isoforms generated by alternative splicing remains unclear. In the present work, we show that at least seven NuMA isoforms (categorized into long, middle and short groups) generated by alternative splicing from a common NuMA mRNA precursor were discovered in HeLa cells and these isoforms differ mainly at the carboxyl terminus and the coiled-coil domains. Two “hotspot” exons with molecular mass of 3366-nt and 42-nt tend to be spliced during alternative splicing in long and middle groups. Furthermore, full-length coding sequences of long and middle NuMA obtained by using fusion PCR were constructed into GFP-tagged vector to illustrate their cellular localization. Long NuMA mainly localized in the nucleus with absence from nucleoli during interphase and translocated to the spindle poles in mitosis. Middle NuMA displayed the similar cell cycle-dependent distribution pattern as long NuMA. However, expression of NuMA short isoforms revealed a distinct subcellular localization. Short NuMA were present in the cytosol during the whole cycle, without colocalization with mitotic apparatus. These results have allowed us tentatively to explore a new research direction for NuMA’s various functions.

  20. Differential Roles of PML Isoforms.

    PubMed

    Nisole, Sébastien; Maroui, Mohamed Ali; Mascle, Xavier H; Aubry, Muriel; Chelbi-Alix, Mounira K

    2013-01-01

    The tumor suppressor promyelocytic leukemia (PML) protein is fused to the retinoic acid receptor alpha in patients suffering from acute promyelocytic leukemia (APL). Treatment of APL patients with arsenic trioxide (As2O3) reverses the disease phenotype by a process involving the degradation of the fusion protein via its PML moiety. Several PML isoforms are generated from a single PML gene by alternative splicing. They share the same N-terminal region containing the RBCC/tripartite motif but differ in their C-terminal sequences. Recent studies of all the PML isoforms reveal the specific functions of each. Here, we review the nomenclature and structural organization of the PML isoforms in order to clarify the various designations and classifications found in different databases. The functions of the PML isoforms and their differential roles in antiviral defense also are reviewed. Finally, the key players involved in the degradation of the PML isoforms in response to As2O3 or other inducers are discussed. PMID:23734343

  1. Identification of signals that facilitate isoform specific nucleolar localization of myosin IC

    SciTech Connect

    Schwab, Ryan S.; Ihnatovych, Ivanna; Yunus, Sharifah Z.S.A.; Domaradzki, Tera; Hofmann, Wilma A.

    2013-05-01

    Myosin IC is a single headed member of the myosin superfamily that localizes to the cytoplasm and the nucleus, where it is involved in transcription by RNA polymerases I and II, intranuclear transport, and nuclear export. In mammalian cells, three isoforms of myosin IC are expressed that differ only in the addition of short isoform-specific N-terminal peptides. Despite the high sequence homology, the isoforms show differences in cellular distribution, in localization to nuclear substructures, and in their interaction with nuclear proteins through yet unknown mechanisms. In this study, we used EGFP-fusion constructs that express truncated or mutated versions of myosin IC isoforms to detect regions that are involved in isoform-specific localization. We identified two nucleolar localization signals (NoLS). One NoLS is located in the myosin IC isoform B specific N-terminal peptide, the second NoLS is located upstream of the neck region within the head domain. We demonstrate that both NoLS are functional and necessary for nucleolar localization of specifically myosin IC isoform B. Our data provide a first mechanistic explanation for the observed functional differences between the myosin IC isoforms and are an important step toward our understanding of the underlying mechanisms that regulate the various and distinct functions of myosin IC isoforms. - Highlights: ► Two NoLS have been identified in the myosin IC isoform B sequence. ► Both NoLS are necessary for myosin IC isoform B specific nucleolar localization. ► First mechanistic explanation of functional differences between the isoforms.

  2. Identification and characterization of a novel retinal isoform of dystrophin

    SciTech Connect

    D`Souza, V.N.; Sigesmund, D.A.; Man, N.

    1994-09-01

    We have shown that dystrophin is required for normal function of the retina as measured by electroretinography (ERG). In these studies a genotype/phenotype correlation was found in which DMD/BMD patients with deletions in the central to distal region of the gene had abnormal ERGs, while patients with deletions in the 5{prime} end of the gene had a mild or normal retinal phenotype. A similar correlation was also observed in the mouse in which the mdx mouse having a mutation in exon 23 had a normal retinal phenotype, whereas the mdx{sup Cv3} mouse (mutation in intron 65) had an abnormal phenotype. Molecular analysis of both human and mouse retina indicated that at least two isoforms of dystrophin are expressed in the retina and localize to the outer plexiform layer, the synaptic junction between the photoreceptors, the bipolar cells, and the horizontal cells. Using a panel of monoclonal dystrophin antisera to analyze mdx mouse retina which does not contain full length dystrophin antisera, we showed that a shorter dystrophin isoform (approximately 260 kDa) was present and contained part of the rod, the cysteine-rich and C-terminal domains. The 5{prime} end of the transcript giving rise to this isoform was characterized and cloned using 5{prime}RACE. Sequence analysis indicated that this transcript contained a novel exon 1 consisting of 240 nucleotides and coded for a unique N-terminus of 13 amino acids. This isoform is distinct from the DP116 dystrophin isoform identified in peripheral nerve. From the functional analysis of DMD patients and dystrophic mice we conclude that this 260 kDa dystrophin isoform is required for normal retinal electrophysiology.

  3. Expression of Phosphoinositide-Specific Phospholipase C Isoforms in Native Endothelial Cells

    PubMed Central

    Béziau, Delphine M.; Toussaint, Fanny; Blanchette, Alexandre; Dayeh, Nour R.; Charbel, Chimène; Tardif, Jean-Claude; Dupuis, Jocelyn; Ledoux, Jonathan

    2015-01-01

    Phospholipase C (PLC) comprises a superfamily of enzymes that play a key role in a wide array of intracellular signalling pathways, including protein kinase C and intracellular calcium. Thirteen different mammalian PLC isoforms have been identified and classified into 6 families (PLC-β, γ, δ, ε, ζ and η) based on their biochemical properties. Although the expression of PLC isoforms is tissue-specific, concomitant expression of different PLC has been reported, suggesting that PLC family is involved in multiple cellular functions. Despite their critical role, the PLC isoforms expressed in native endothelial cells (ECs) remains undetermined. A conventional PCR approach was initially used to elucidate the mRNA expression pattern of PLC isoforms in 3 distinct murine vascular beds: mesenteric (MA), pulmonary (PA) and middle cerebral arteries (MCA). mRNA encoding for most PLC isoforms was detected in MA, MCA and PA with the exception of η2 and β2 (only expressed in PA), δ4 (only expressed in MCA), η1 (expressed in all but MA) and ζ (not detected in any vascular beds tested). The endothelial-specific PLC expression was then sought in freshly isolated ECs. Interestingly, the PLC expression profile appears to differ across the investigated arterial beds. While mRNA for 8 of the 13 PLC isoforms was detected in ECs from MA, two additional PLC isoforms were detected in ECs from PA and MCA. Co-expression of multiple PLC isoforms in ECs suggests an elaborate network of signalling pathways: PLC isoforms may contribute to the complexity or diversity of signalling by their selective localization in cellular microdomains. However in situ immunofluorescence revealed a homogeneous distribution for all PLC isoforms probed (β3, γ2 and δ1) in intact endothelium. Although PLC isoforms play a crucial role in endothelial signal transduction, subcellular localization alone does not appear to be sufficient to determine the role of PLC in the signalling microdomains found in the

  4. P120-catenin isoforms 1A and 3A differently affect invasion and proliferation of lung cancer cells

    SciTech Connect

    Liu Yang; Dong Qianze; Zhao Yue; Dong Xinjun; Miao Yuan; Dai Shundong; Yang Zhiqiang; Zhang Di; Wang Yan; Li Qingchang; Zhao Chen; Wang Enhua

    2009-03-10

    Different isoforms of p120-catenin (p120ctn), a member of the Armadillo gene family, are variably expressed in different tissues as a result of alternative splicing and the use of multiple translation initiation codons. When expressed in cancer cells, these isoforms may confer different properties with respect to cell adhesion and invasion. We have previously reported that the p120ctn isoforms 1 and 3 were the most highly expressed isoforms in normal lung tissues, and their expression level was reduced in lung tumor cells. To precisely define their biological roles, we transfected p120ctn isoforms 1A and 3A into the lung cancer cell lines A549 and NCI-H460. Enhanced expression of p120ctn isoform 1A not only upregulated E-cadherin and {beta}-catenin, but also downregulated the Rac1 activity, and as a result, inhibited the ability of cells to invade. In contrast, overexpression of p120ctn isoform 3A led to the inactivation of Cdc42 and the activation of RhoA, and had a smaller influence on invasion. However, we found that isoform 3A had a greater ability than isoform 1A in both inhibiting the cell cycle and reducing tumor cell proliferation. The present study revealed that p120ctn isoforms 1A and 3A differently regulated the adhesive, proliferative, and invasive properties of lung cancer cells through distinct mechanisms.

  5. Antagonistic functions of LMNA isoforms in energy expenditure and lifespan.

    PubMed

    Lopez-Mejia, Isabel C; de Toledo, Marion; Chavey, Carine; Lapasset, Laure; Cavelier, Patricia; Lopez-Herrera, Celia; Chebli, Karim; Fort, Philippe; Beranger, Guillaume; Fajas, Lluis; Amri, Ez Z; Casas, Francois; Tazi, Jamal

    2014-05-01

    Alternative RNA processing of LMNA pre-mRNA produces three main protein isoforms, that is, lamin A, progerin, and lamin C. De novo mutations that favor the expression of progerin over lamin A lead to Hutchinson-Gilford progeria syndrome (HGPS), providing support for the involvement of LMNA processing in pathological aging. Lamin C expression is mutually exclusive with the splicing of lamin A and progerin isoforms and occurs by alternative polyadenylation. Here, we investigate the function of lamin C in aging and metabolism using mice that express only this isoform. Intriguingly, these mice live longer, have decreased energy metabolism, increased weight gain, and reduced respiration. In contrast, progerin-expressing mice show increased energy metabolism and are lipodystrophic. Increased mitochondrial biogenesis is found in adipose tissue from HGPS-like mice, whereas lamin C-only mice have fewer mitochondria. Consistently, transcriptome analyses of adipose tissues from HGPS and lamin C-only mice reveal inversely correlated expression of key regulators of energy expenditure, including Pgc1a and Sfrp5. Our results demonstrate that LMNA encodes functionally distinct isoforms that have opposing effects on energy metabolism and lifespan in mammals. PMID:24639560

  6. Characterization of multiple nestin isoforms in the goldfish brain.

    PubMed

    Venables, Maddie J; Navarro-Martín, Laia; Basak, Ajoy; Baum, Bernard R; Zhang, Dapeng; Trudeau, Vance L

    2016-09-01

    Nestin is an intermediate filament protein involved in neurogenesis in fish, mice, and humans. In this study we used rapid amplification of cDNA ends PCR to isolate goldfish nestin (nes). PCR analysis and sequencing revealed three different nes transcripts of 4003, 2446, and 2126 nucleotides, which are predicted to generate proteins of 860, 274, and 344 amino acids in length. Sequence analysis suggests that these nes transcripts are likely a result of alternative splicing. We next applied a multiple-antigenic peptide strategy to generate a goldfish-specific nestin antibody. Western blotting with this antibody together with mass spectrometry verified the presence of major nestin protein isoforms with differing molecular weights (~70, 40 and 30kDa). We further examined expression patterns of these nestin protein isoforms in different parts of the goldfish brain and pituitary and found the telencephalon to express all three isoforms at a distinct level and abundance. We report that multiple nestin isoforms are present indicating another level of complexity for the regulation of intermediate filaments in comparison to mammals. Studying the differential roles and regulation of these nestins could lead to a better understanding of cellular remodeling during neurogenesis and the unparalleled regenerative abilities after damage in the teleost CNS. PMID:27254106

  7. Tropomyosin-binding properties modulate competition between tropomodulin isoforms.

    PubMed

    Colpan, Mert; Moroz, Natalia A; Gray, Kevin T; Cooper, Dillon A; Diaz, Christian A; Kostyukova, Alla S

    2016-06-15

    The formation and fine-tuning of cytoskeleton in cells are governed by proteins that influence actin filament dynamics. Tropomodulin (Tmod) regulates the length of actin filaments by capping the pointed ends in a tropomyosin (TM)-dependent manner. Tmod1, Tmod2 and Tmod3 are associated with the cytoskeleton of non-muscle cells and their expression has distinct consequences on cell morphology. To understand the molecular basis of differences in the function and localization of Tmod isoforms in a cell, we compared the actin filament-binding abilities of Tmod1, Tmod2 and Tmod3 in the presence of Tpm3.1, a non-muscle TM isoform. Tmod3 displayed preferential binding to actin filaments when competing with other isoforms. Mutating the second or both TM-binding sites of Tmod3 destroyed its preferential binding. Our findings clarify how Tmod1, Tmod2 and Tmod3 compete for binding actin filaments. Different binding mechanisms and strengths of Tmod isoforms for Tpm3.1 contribute to their divergent functional capabilities. PMID:27091317

  8. Inference of Isoforms from Short Sequence Reads

    NASA Astrophysics Data System (ADS)

    Feng, Jianxing; Li, Wei; Jiang, Tao

    Due to alternative splicing events in eukaryotic species, the identification of mRNA isoforms (or splicing variants) is a difficult problem. Traditional experimental methods for this purpose are time consuming and cost ineffective. The emerging RNA-Seq technology provides a possible effective method to address this problem. Although the advantages of RNA-Seq over traditional methods in transcriptome analysis have been confirmed by many studies, the inference of isoforms from millions of short sequence reads (e.g., Illumina/Solexa reads) has remained computationally challenging. In this work, we propose a method to calculate the expression levels of isoforms and infer isoforms from short RNA-Seq reads using exon-intron boundary, transcription start site (TSS) and poly-A site (PAS) information. We first formulate the relationship among exons, isoforms, and single-end reads as a convex quadratic program, and then use an efficient algorithm (called IsoInfer) to search for isoforms. IsoInfer can calculate the expression levels of isoforms accurately if all the isoforms are known and infer novel isoforms from scratch. Our experimental tests on known mouse isoforms with both simulated expression levels and reads demonstrate that IsoInfer is able to calculate the expression levels of isoforms with an accuracy comparable to the state-of-the-art statistical method and a 60 times faster speed. Moreover, our tests on both simulated and real reads show that it achieves a good precision and sensitivity in inferring isoforms when given accurate exon-intron boundary, TSS and PAS information, especially for isoforms whose expression levels are significantly high.

  9. Direct Activation of Epac by Sulfonylurea is Isoform Selective

    PubMed Central

    Herbst, Katie J.; Coltharp, Carla; Amzel, L. Mario; Zhang, Jin

    2011-01-01

    Summary Commonly used as a treatment for Type II diabetes, sulfonylureas (SUs) stimulate insulin secretion from pancreatic β cells by binding to sulfonylurea receptors. Recently, SUs have been shown to also activate exchange protein directly activated by cAMP 2 (Epac2), however little is known about this molecular action. Using biosensor imaging and biochemical analysis, we show that SUs activate Epac2 and the downstream signaling via direct binding to Epac2. We further identify R447 of Epac2 to be critically involved in SU binding. This distinct binding site from cAMP points to a new mode of allosteric activation of Epac2. We also show that SUs selectively activate Epac2 isoform, but not the closely related Epac1, further establishing SUs as a new class of isoform-selective enzyme activators. PMID:21338921

  10. VEGF-A isoforms program differential VEGFR2 signal transduction, trafficking and proteolysis

    PubMed Central

    Fearnley, Gareth W.; Smith, Gina A.; Abdul-Zani, Izma; Yuldasheva, Nadira; Mughal, Nadeem A.; Homer-Vanniasinkam, Shervanthi; Kearney, Mark T.; Zachary, Ian C.; Tomlinson, Darren C.; Harrison, Michael A.; Wheatcroft, Stephen B.; Ponnambalam, Sreenivasan

    2016-01-01

    ABSTRACT Vascular endothelial growth factor A (VEGF-A) binding to the receptor tyrosine kinase VEGFR2 triggers multiple signal transduction pathways, which regulate endothelial cell responses that control vascular development. Multiple isoforms of VEGF-A can elicit differential signal transduction and endothelial responses. However, it is unclear how such cellular responses are controlled by isoform-specific VEGF-A–VEGFR2 complexes. Increasingly, there is the realization that the membrane trafficking of receptor–ligand complexes influences signal transduction and protein turnover. By building on these concepts, our study shows for the first time that three different VEGF-A isoforms (VEGF-A165, VEGF-A121 and VEGF-A145) promote distinct patterns of VEGFR2 endocytosis for delivery into early endosomes. This differential VEGFR2 endocytosis and trafficking is linked to VEGF-A isoform-specific signal transduction events. Disruption of clathrin-dependent endocytosis blocked VEGF-A isoform-specific VEGFR2 activation, signal transduction and caused substantial depletion in membrane-bound VEGFR1 and VEGFR2 levels. Furthermore, such VEGF-A isoforms promoted differential patterns of VEGFR2 ubiquitylation, proteolysis and terminal degradation. Our study now provides novel insights into how different VEGF-A isoforms can bind the same receptor tyrosine kinase and elicit diverse cellular outcomes. PMID:27044325

  11. VEGF-A isoforms program differential VEGFR2 signal transduction, trafficking and proteolysis.

    PubMed

    Fearnley, Gareth W; Smith, Gina A; Abdul-Zani, Izma; Yuldasheva, Nadira; Mughal, Nadeem A; Homer-Vanniasinkam, Shervanthi; Kearney, Mark T; Zachary, Ian C; Tomlinson, Darren C; Harrison, Michael A; Wheatcroft, Stephen B; Ponnambalam, Sreenivasan

    2016-01-01

    Vascular endothelial growth factor A (VEGF-A) binding to the receptor tyrosine kinase VEGFR2 triggers multiple signal transduction pathways, which regulate endothelial cell responses that control vascular development. Multiple isoforms of VEGF-A can elicit differential signal transduction and endothelial responses. However, it is unclear how such cellular responses are controlled by isoform-specific VEGF-A-VEGFR2 complexes. Increasingly, there is the realization that the membrane trafficking of receptor-ligand complexes influences signal transduction and protein turnover. By building on these concepts, our study shows for the first time that three different VEGF-A isoforms (VEGF-A165, VEGF-A121 and VEGF-A145) promote distinct patterns of VEGFR2 endocytosis for delivery into early endosomes. This differential VEGFR2 endocytosis and trafficking is linked to VEGF-A isoform-specific signal transduction events. Disruption of clathrin-dependent endocytosis blocked VEGF-A isoform-specific VEGFR2 activation, signal transduction and caused substantial depletion in membrane-bound VEGFR1 and VEGFR2 levels. Furthermore, such VEGF-A isoforms promoted differential patterns of VEGFR2 ubiquitylation, proteolysis and terminal degradation. Our study now provides novel insights into how different VEGF-A isoforms can bind the same receptor tyrosine kinase and elicit diverse cellular outcomes. PMID:27044325

  12. Laminin isoforms: biological roles and effects on the intracellular distribution of nuclear proteins in intestinal epithelial cells

    SciTech Connect

    Turck, Natacha; Gross, Isabelle; Gendry, Patrick; Stutzmann, Jeanne; Freund, Jean-Noel; Kedinger, Michele; Simon-Assmann, Patricia; Launay, Jean-Francois . E-mail: Jean-Francois.Launay@inserm.u-strasbg.fr

    2005-02-15

    Laminins are structurally and functionally major components of the extracellular matrix. Four isoforms of laminins (laminin-1, -2, -5 and -10) are expressed in a specific pattern along the crypt-villus axis of the intestine. Previous works indicated that expression of these isoforms is developmentally regulated and that laminins could modulate the behaviour of intestinal cells, but the exact role of each isoform remained unclear. Here, we report the first systematic analysis of the cellular functions of the four isoforms using the human colon adenocarcinoma Caco2/TC7 cell line as a model. We compared the respective abilities of each isoform to modulate adhesion, proliferation and differentiation of intestinal epithelial cells. We found that the isoforms were functionally distinct, with laminin-10 being the most adhesive substratum, laminin-2, laminin-5 and laminin-10 enhancing cellular proliferation and at the opposite, laminin-1 stimulating intestinal cell differentiation. To begin to characterise the molecular events induced by the different isoforms, we examined by immunofluorescence the intracellular distribution of several nuclear proteins, recently highlighted by a nuclear proteomic approach. We observed clear nucleocytoplasmic redistribution of these proteins, which depended on the laminin isoform. These results provide evidence for a distinct functional role of laminins in intestinal cell functions characterised by specific localisation of nuclear proteins.

  13. Glucocorticoid receptor translational isoforms underlie maturational stage-specific glucocorticoid sensitivities of dendritic cells in mice and humans.

    PubMed

    Cao, Yun; Bender, Ingrid K; Konstantinidis, Athanasios K; Shin, Soon Cheon; Jewell, Christine M; Cidlowski, John A; Schleimer, Robert P; Lu, Nick Z

    2013-02-28

    Although glucocorticoids are a profoundly important class of anti-inflammatory and immunosuppressive agents, their actions in dendritic cells (DCs) are not well understood. We found that dexamethasone, a potent glucocorticoid, selectively induced apoptosis in mature, but not in immature, DCs in healthy mice, in mice with experimental airway inflammation, and in vitro in bone marrow–derived DCs. Distinct glucocorticoid receptor (GR) translational isoforms expressed in immature and mature DCs probably contribute to the DC maturational stage-specific glucocorticoid sensitivity. The GR-D isoforms were the predominant isoforms in immature DCs, whereas the proapoptotic GR-A isoform was the main isoform in mature DCs. Ectopic expression of the GR-A isoform in immature DCs increased glucocorticoid sensitivity and RU486, a selective GR antagonist, inhibited the glucocorticoid sensitivity of mature DCs. Furthermore, the distinct expression pattern of GR isoforms in immature and mature murine DCs was also observed in human monocyte–derived DCs. These studies suggest that glucocorticoids may spare immature DCs and suppress mature DCs and inflammation via differential expression of GR translational isoforms. PMID:23297131

  14. Glucocorticoid receptor translational isoforms underlie maturational stage-specific glucocorticoid sensitivities of dendritic cells in mice and humans

    PubMed Central

    Cao, Yun; Bender, Ingrid K.; Konstantinidis, Athanasios K.; Shin, Soon Cheon; Jewell, Christine M.; Cidlowski, John A.; Schleimer, Robert P.

    2013-01-01

    Although glucocorticoids are a profoundly important class of anti-inflammatory and immunosuppressive agents, their actions in dendritic cells (DCs) are not well understood. We found that dexamethasone, a potent glucocorticoid, selectively induced apoptosis in mature, but not in immature, DCs in healthy mice, in mice with experimental airway inflammation, and in vitro in bone marrow–derived DCs. Distinct glucocorticoid receptor (GR) translational isoforms expressed in immature and mature DCs probably contribute to the DC maturational stage-specific glucocorticoid sensitivity. The GR-D isoforms were the predominant isoforms in immature DCs, whereas the proapoptotic GR-A isoform was the main isoform in mature DCs. Ectopic expression of the GR-A isoform in immature DCs increased glucocorticoid sensitivity and RU486, a selective GR antagonist, inhibited the glucocorticoid sensitivity of mature DCs. Furthermore, the distinct expression pattern of GR isoforms in immature and mature murine DCs was also observed in human monocyte–derived DCs. These studies suggest that glucocorticoids may spare immature DCs and suppress mature DCs and inflammation via differential expression of GR translational isoforms. PMID:23297131

  15. Enhanced protein electrophoresis technique for separating human skeletal muscle myosin heavy chain isoforms

    NASA Technical Reports Server (NTRS)

    Bamman, M. M.; Clarke, M. S.; Talmadge, R. J.; Feeback, D. L.

    1999-01-01

    Talmadge and Roy (J. Appl. Physiol. 1993, 75, 2337-2340) previously established a sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) protocol for separating all four rat skeletal muscle myosin heavy chain (MHC) isoforms (MHC I, IIa, IIx, IIb); however, when applied to human muscle, the type II MHC isoforms (Ila, IIx) are not clearly distinguished. In this brief paper we describe a modification of the SDS-PAGE protocol which yields distinct and consistent separation of all three adult human MHC isoforms (MHC I, IIa, IIx) in a minigel system. MHC specificity of each band was confirmed by Western blot using three monoclonal IgG antibodies (mAbs) immunoreactive against MHCI (mAb MHCs, Novacastra Laboratories), MHCI+IIa (mAb BF-35), and MHCIIa+IIx (mAb SC-71). Results provide a valuable SDS-PAGE minigel technique for separating MHC isoforms in human muscle without the difficult task of casting gradient gels.

  16. Substrate specificity, kinetic properties and inhibition by fumonisin B1 of ceramide synthase isoforms from Arabidopsis.

    PubMed

    Luttgeharm, Kyle D; Cahoon, Edgar B; Markham, Jonathan E

    2016-03-01

    Ceramide makes up the acyl-backbone of sphingolipids and plays a central role in determining the function of these essential membrane lipids. In Arabidopsis, the varied chemical composition of ceramide is determined by the specificity of three different isoforms of ceramide synthase, denoted LAG one homologue 1, -2 and -3 (LOH1, LOH2 and LOH3), for a range of long-chain base (LCB) and acyl-CoA substrates. The contribution of each of these isoforms to the synthesis of ceramide was investigated by in vitro ceramide synthase assays. The plant LCB phytosphingosine was efficiently used by the LOH1 and LOH3 isoforms, with LOH1 having the lowest Km for the LCB substrate of the three isoforms. In contrast, sphinganine was used efficiently only by the LOH2 isoform. Acyl-CoA specificity was also distinguished between the three isoforms with LOH2 almost completely specific for palmitoyl-CoA whereas the LOH1 isoform showed greatest activity with lignoceroyl- and hexacosanoyl-CoAs. Interestingly, unsaturated acyl-CoAs were not used efficiently by any isoform whereas unsaturated LCB substrates were preferred by LOH2 and 3. Fumonisin B1 (FB1) is a general inhibitor of ceramide synthases but LOH1 was found to have a much lower Ki than the other isoforms pointing towards the origin of FB1 sensitivity in plants. Overall, the data suggest distinct roles and modes of regulation for each of the ceramide synthases in Arabidopsis sphingolipid metabolism. PMID:26635357

  17. SMRT has tissue-specific isoform profiles that include a form containing one CoRNR box

    SciTech Connect

    Short, Stephen; Malartre, Marianne; Sharpe, Colin . E-mail: colin.sharpe@port.ac.uk

    2005-09-02

    SMRT acts as a corepressor for a range of transcription factors. The amino-terminal part of the protein includes domains that mainly mediate transcriptional repression whilst the carboxy-terminal part includes domains that interact with nuclear receptors using up to three motifs called CoRNR boxes. The region of the SMRT primary transcript encoding the interaction domains is subject to alternative splicing that varies the inclusion of the third CoRNR box. The profile in mice includes an abundant, novel SMRT isoform that possesses just one CoRNR box. Mouse tissues therefore express SMRT isoforms containing one, two or three CoRNR boxes. In frogs, the SMRT isoform profile is tissue-specific. The mouse also shows distinct profiles generated by differential expression levels of the SMRT transcript isoforms. The formation of multiple SMRT isoforms and their tissue-specific regulation indicates a mechanism, whereby cells can define the repertoire of transcription factors regulated by SMRT.

  18. Isoform and Splice-Variant Specific Functions of Dynamin-2 Revealed by Analysis of Conditional Knock-Out Cells

    PubMed Central

    Liu, Ya-Wen; Surka, Mark C.; Schroeter, Thomas; Lukiyanchuk, Vasyl

    2008-01-01

    Dynamin (Dyn) is a multifunctional GTPase implicated in several cellular events, including endocytosis, intracellular trafficking, cell signaling, and cytokinesis. The mammalian genome encodes three isoforms, Dyn1, Dyn2, and Dyn3, and several splice variants of each, leading to the suggestion that distinct isoforms and/or distinct splice variants might mediate distinct cellular functions. We generated a conditional Dyn2 KO cell line and performed knockout and reconstitution experiments to explore the isoform- and splice variant specific cellular functions of ubiquitously expressed Dyn2. We find that Dyn2 is required for clathrin-mediated endocytosis (CME), p75 export from the Golgi, and PDGF-stimulated macropinocytosis and cytokinesis, but not for other endocytic pathways. Surprisingly, CME and p75 exocytosis were efficiently rescued by reintroduction of Dyn2, but not Dyn1, suggesting that these two isoforms function differentially in vesicular trafficking in nonneuronal cells. Both isoforms rescued macropinocytosis and cytokinesis, suggesting that dynamin function in these processes might be mechanistically distinct from its role in CME. Although all four Dyn2 splice variants could equally restore CME, Dyn2ba and -bb were more effective at restoring p75 exocytosis. This splice variant specificity correlated with their differential targeting to the Golgi. These studies reveal isoform and splice-variant specific functions for Dyn2. PMID:18923138

  19. Isoform and splice-variant specific functions of dynamin-2 revealed by analysis of conditional knock-out cells.

    PubMed

    Liu, Ya-Wen; Surka, Mark C; Schroeter, Thomas; Lukiyanchuk, Vasyl; Schmid, Sandra L

    2008-12-01

    Dynamin (Dyn) is a multifunctional GTPase implicated in several cellular events, including endocytosis, intracellular trafficking, cell signaling, and cytokinesis. The mammalian genome encodes three isoforms, Dyn1, Dyn2, and Dyn3, and several splice variants of each, leading to the suggestion that distinct isoforms and/or distinct splice variants might mediate distinct cellular functions. We generated a conditional Dyn2 KO cell line and performed knockout and reconstitution experiments to explore the isoform- and splice variant specific cellular functions of ubiquitously expressed Dyn2. We find that Dyn2 is required for clathrin-mediated endocytosis (CME), p75 export from the Golgi, and PDGF-stimulated macropinocytosis and cytokinesis, but not for other endocytic pathways. Surprisingly, CME and p75 exocytosis were efficiently rescued by reintroduction of Dyn2, but not Dyn1, suggesting that these two isoforms function differentially in vesicular trafficking in nonneuronal cells. Both isoforms rescued macropinocytosis and cytokinesis, suggesting that dynamin function in these processes might be mechanistically distinct from its role in CME. Although all four Dyn2 splice variants could equally restore CME, Dyn2ba and -bb were more effective at restoring p75 exocytosis. This splice variant specificity correlated with their differential targeting to the Golgi. These studies reveal isoform and splice-variant specific functions for Dyn2. PMID:18923138

  20. Differential Regulation of Aromatase Isoforms and Tissue Responses to Environmental Chemicals in Fish

    EPA Science Inventory

    As in mammals, aromatase plays a basic role in fish reproduction. Unlike most mammals, with only one form of aromatase, fish have two distinct forms. One isoform, P450aromA, predominates in ovaries. Ovarian aromatase activity controls circulating levels of estrogens and is critic...

  1. HPLC analysis of discrete haptoglobin isoform N-linked oligosaccharides following 2D-PAGE isolation.

    PubMed

    He, Zhicong; Aristoteli, Lina P; Kritharides, Leonard; Garner, Brett

    2006-05-01

    Glycosylation is a common but variable modification that regulates glycoprotein structure and function. We combined small format 2D-PAGE with HPLC to analyse discrete human haptoglobin isoform N-glycans. Seven major and several minor haptoglobin isoforms were detected by 2D-PAGE. N-Glycans released from Coomassie-stained gel spots using PNGase were labeled at their reducing termini with 2-aminobenzamide. HPLC analysis of selected major isoform N-glycans indicated that sialic acid composition determined their separation by isoelectric focussing. N-Glycans from two doublets of quantitatively minor isoforms were also analysed. Although separation of each pair of doublets was influenced by sialylation, individual spots within each doublet contained identical N-glycans. Thus, heterogeneity in minor haptoglobin isoforms was due to modifications distinct from N-glycan structure. These studies describe a simple method for analysing low abundance protein N-glycans and provide details of discrete haptoglobin isoform N-glycan structures which will be useful in proteomic analysis of human plasma samples. PMID:16546121

  2. Osteopontin (OPN/SPP1) isoforms collectively enhance tumor cell invasion and dissemination in esophageal adenocarcinoma

    PubMed Central

    Lin, Jules; Myers, Amy L.; Wang, Zhuwen; Nancarrow, Derek J.; Ferrer-Torres, Daysha; Handlogten, Amy; Leverenz, Kimmy; Bao, Julia; Thomas, Dafydd G.; Wang, Thomas D.; Orringer, Mark B.; Reddy, Rishindra M.; Chang, Andrew C.; Beer, David G.; Lin, Lin

    2015-01-01

    Esophageal adenocarcinoma (EAC) is often diagnosed at an advanced stage, thus understanding the molecular basis for EAC invasion and metastasis is critical. Here we report that SPP1/OPN was highly overexpressed in primary EACs and intracellularly localized to tumor cells. We further demonstrate that all known OPN isoforms (OPNa, b, c, 4 and 5) were frequently co-overexpressed in primary EACs. Distinct pro-invasion and dissemination phenotypes of isoform-specific OPNb and OPNc stable transfectants were observed. Expression of OPNb significantly enhanced cell migration and adhesion to laminin. In contrast, OPNc cells showed significantly decreased cell migration yet increased cell detachment. Enhanced invasion, both in vitro and in vivo, was observed for OPNb- but not OPNc-expressing cells. Inhibition of RGD integrins, one family of OPN receptors, attenuated OPNb cell migration, abrogated OPNb cell adhesion and significantly reduced OPNb cell clonogenic survival but did not affect OPNc phenotypes, indicating that OPNb but not OPNc acts through integrin-dependent signaling. Differential expression of vimentin, E-cadherin and β-catenin in OPN stable cells may account for the variation in cell adhesion and detachment between these isoforms. We conclude that while all OPN isoforms are frequently co-overexpressed in primary EACs, isoforms OPNb and OPNc enhance invasion and dissemination through collective yet distinct mechanisms. PMID:26068949

  3. Secretion of PDGF isoforms during osteoclastogenesis and its modulation by anti-osteoclast drugs.

    PubMed

    Rahman, M Motiur; Matsuoka, Kazuhiko; Takeshita, Sunao; Ikeda, Kyoji

    2015-06-26

    In an attempt to identify secretory products of osteoclasts that mediate the coupling of bone formation to resorption, we found that along with osteoclast differentiation, PDGF-A gene expression increase occurred first, by 12 h after stimulation of bone marrow macrophages with M-CSF and RANKL, and peaked at 36 h. This was next followed by a progressive increase in PDGF-B gene expression until a peak at 60 h, when mature osteoclasts formed. Isoform-specific ELISA of the conditioned medium collected every 24 h revealed that all three of the isoforms of PDGF-AA, AB and BB were secreted, in this temporal order as differentiation proceeded. Their secretion was enhanced when osteoclasts were activated by placing them on dentin slices. The secretion of all three isoforms was decreased in cathepsin K-deficient osteoclasts compared with wild-type osteoclasts. Pharmacological inhibition of cathepsin K with odanacatib also inhibited the secretion of all three isoforms, as was also the case with alendronate treatment. The secretion of sphingosine-1-phosphate, which increased during osteoclastogenesis, was reduced from cathepsin K-deficient osteoclasts, and was inhibited by treatment with odanacatib more profoundly than with alendronate. Thus, all three isoforms of PDGF, which are secreted at distinct differentiation stages of osteoclasts, appear to have distinct roles in the cell-cell communication that takes place in the microenvironment of bone remodeling, especially from the osteoclast lineage to mesenchymal cells and vascular cells, thereby stimulating osteogenesis and angiogenesis. PMID:25951977

  4. Crystal structures of a halophilic archaeal malate synthase from Haloferax volcanii and comparisons with isoforms A and G

    PubMed Central

    2011-01-01

    Background Malate synthase, one of the two enzymes unique to the glyoxylate cycle, is found in all three domains of life, and is crucial to the utilization of two-carbon compounds for net biosynthetic pathways such as gluconeogenesis. In addition to the main isoforms A and G, so named because of their differential expression in E. coli grown on either acetate or glycolate respectively, a third distinct isoform has been identified. These three isoforms differ considerably in size and sequence conservation. The A isoform (MSA) comprises ~530 residues, the G isoform (MSG) is ~730 residues, and this third isoform (MSH-halophilic) is ~430 residues in length. Both isoforms A and G have been structurally characterized in detail, but no structures have been reported for the H isoform which has been found thus far only in members of the halophilic Archaea. Results We have solved the structure of a malate synthase H (MSH) isoform member from Haloferax volcanii in complex with glyoxylate at 2.51 Å resolution, and also as a ternary complex with acetyl-coenzyme A and pyruvate at 1.95 Å. Like the A and G isoforms, MSH is based on a β8/α8 (TIM) barrel. Unlike previously solved malate synthase structures which are all monomeric, this enzyme is found in the native state as a trimer/hexamer equilibrium. Compared to isoforms A and G, MSH displays deletion of an N-terminal domain and a smaller deletion at the C-terminus. The MSH active site is closely superimposable with those of MSA and MSG, with the ternary complex indicating a nucleophilic attack on pyruvate by the enolate intermediate of acetyl-coenzyme A. Conclusions The reported structures of MSH from Haloferax volcanii allow a detailed analysis and comparison with previously solved structures of isoforms A and G. These structural comparisons provide insight into evolutionary relationships among these isoforms, and also indicate that despite the size and sequence variation, and the truncated C-terminal domain of the H

  5. A New View of Ras Isoforms in Cancers.

    PubMed

    Nussinov, Ruth; Tsai, Chung-Jung; Chakrabarti, Mayukh; Jang, Hyunbum

    2016-01-01

    Does small GTPase K-Ras4A have a single state or two states, one resembling K-Ras4B and the other N-Ras? A recent study of K-Ras4A made the remarkable observation that even in the absence of the palmitoyl, K-Ras4A can be active at the plasma membrane. Importantly, this suggests that K-Ras4A may exist in two distinct signaling states. In state 1, K-Ras4A is only farnesylated, like K-Ras4B; in state 2, farnesylated and palmitoylated, like N-Ras. The K-Ras4A hypervariable region sequence is positively charged, in between K-Ras4B and N-Ras. Taken together, this raises the possibility that the farnesylated but nonpalmitoylated state 1, like K-Ras4B, binds calmodulin and is associated with colorectal and other adenocarcinomas like lung cancer and pancreatic ductal adenocarcinoma. On the other hand, state 2 may be associated with melanoma and other cancers where N-Ras is a major contributor, such as acute myeloid leukemia. Importantly, H-Ras has two, singly and doubly, palmitoylated states that may also serve distinct functional roles. The multiple signaling states of palmitoylated Ras isoforms question the completeness of small GTPase Ras isoform statistics in different cancer types and call for reevaluation of concepts and protocols. They may also call for reconsideration of oncogenic Ras therapeutics. PMID:26659836

  6. Isoform specificity of cardiac glycosides binding to human Na+,K+-ATPase α1β1, α2β1 and α3β1

    PubMed Central

    Hauck, Christian; Potter, Tatjana; Bartz, Michaela; Wittwer, Thorsten; Wahlers, Thorsten; Mehlhorn, Uwe; Scheiner-Bobis, Georgios; McDonough, Alicia A.; Bloch, Wilhelm; Schwinger, Robert H.G.; Müller-Ehmsen, Jochen

    2016-01-01

    Cardiac glycosides inhibit the Na+,K+-ATPase and are used for the treatment of symptomatic heart failure and atrial fibrillation. In human heart three isoforms of Na+,K+-ATPase are expressed: α1β1, α2β1 and α3β1. It is unknown, if clinically used cardiac glycosides differ in isoform specific affinities, and if the isoforms have specific subcellular localization in human cardiac myocytes. Human Na+,K+-ATPase isoforms α1β1, α2β1 and α3β1 were expressed in yeast which has no endogenous Na+,K+-ATPase. Isoform specific affinities of digoxin, digitoxin, β-acetyldigoxin, methyldigoxin and ouabain were assessed in [3H]-ouabain binding assays in the absence or presence of K+ (each n=5). The subcellular localizations of the Na+,K+-ATPase isoforms were investigated in isolated human atrial cardiomyocytes by immunohistochemistry. In the absence of K+, methyldigoxin (α1>α3>α2) and ouabain (α1=α3>α2) showed distinct isoform specific affinities, while for digoxin, digitoxin and β-acetyldigoxin no differences were found. In the presence of K+, also digoxin (α2=α3>α1) and β-acetyldigoxin (α1>α3) had isoform specificities. A comparison between the cardiac glycosides demonstrated highly different affinity profiles for the isoforms. Immunohistochemistry showed that all three isoforms are located in the plasma membrane and in intracellular membranes, but only α1β1 and α2β1 are located in the T-tubuli. Cardiac glycosides show distinct isoform specific affinities and different affinity profiles to Na+,K+-ATPase isoforms which have different subcellular localizations in human cardiomyocytes. Thus, in contrast to current notion, different cardiac glycoside agents may significantly differ in their pharmacological profile which could be of hitherto unknown clinical relevance. PMID:19751721

  7. Multiple isoforms of myofibrillar proteins in crustacean muscle: evidence for two slow fiber types

    SciTech Connect

    Mykles, D.L.

    1986-01-01

    Four distinct patterns of myofibrillar proteins, extracted from fast and slow muscles of the lobster, Homarus americanus, are distinguished by different assemblages of regulatory and contractile protein variants. Multiple isoforms of troponin-T, -I, and -C, paramyosin, and myosin light chains occur in six muscles of the claws and abdomen. Analysis of glycerinated fibers from the claws of lobster and land crab, Gecarcinus lateralis, show that more than one isoform is expressed in a single fiber, forming unique assemblages by which subgroups can be discriminated within the broader categories of fast and slow fibers. 9 refs., 3 figs.

  8. The Drosophila pumilio gene encodes two functional protein isoforms that play multiple roles in germline development, gonadogenesis, oogenesis and embryogenesis.

    PubMed Central

    Parisi, M; Lin, H

    1999-01-01

    The pumilio (pum) gene plays an essential role in embryonic patterning and germline stem cell (GSC) maintenance during oogenesis in Drosophila. Here we report on a phenotypic analysis using pum(ovarette) mutations, which reveals multiple functions of pum in primordial germ cell proliferation, larval ovary formation, GSC division, and subsequent oogenic processes, as well as in oviposition. Specifically, by inducing pum(-) GSC clones at the onset of oogenesis, we show that pum is directly involved in GSC division, a function that is distinct from its requirement in primordial germ cells. Furthermore, we show that pum encodes 156- and 130-kD proteins, both of which are functional isoforms. Among pum(ovarette) mutations, pum(1688) specifically eliminates the 156-kD isoform but not the 130-kD isoform, while pum(2003) and pum(4277) specifically affect the 130-kD isoform but not the 156-kD isoform. Normal doses of both isoforms are required for the zygotic function of pum, yet either isoform alone at a normal dose is sufficient for the maternal effect function of pum. A pum cDNA transgene that contains the known open reading frame encodes only the 156-kD isoform and rescues the phenotype of both pum(1688) and pum(2003) mutants. These observations suggest that the 156- and 130-kD isoforms can compensate for each other's function in a dosage-dependent manner. Finally, we present molecular evidence suggesting that the two PUM isoforms share some of their primary structures. PMID:10471709

  9. Isoform Specificity of Protein Kinase Cs in Synaptic Plasticity

    ERIC Educational Resources Information Center

    Sossin, Wayne S.

    2007-01-01

    Protein kinase Cs (PKCs) are implicated in many forms of synaptic plasticity. However, the specific isoform(s) of PKC that underlie(s) these events are often not known. We have used "Aplysia" as a model system in order to investigate the isoform specificity of PKC actions due to the presence of fewer isoforms and a large number of documented…

  10. PKC Isoform Expression in Modeled Microgravity

    NASA Technical Reports Server (NTRS)

    Risin, Diana; Sundaresan, Alamelu; Pellis, Neal R.; Dawson, David L. (Technical Monitor)

    1999-01-01

    Our previous studies showed that modeled (MMG) and true (USA Space Shuttle Missions STS-54 and STS-56) microgravity (MG) inhibit human lymphocyte locomotion, Modeled MG also suppressed polyclonal and antigen-specific lymphocyte activation. Activation of PKC by phorbol myristate acetate (PMA) restored the microgravity-inhibited lymphocyte locomotion as well as activation by phytohaemagglutinin (PHA), whereas calcium ionophore (ionomycin) was unable to restore these functions. Based on these results we hypothesized that MG-induced changes in lymphocyte functions are caused by a fundamental defect in signal transduction mechanism. This defect may be localized either at the PKC level or upstream of PKC, most likely, at the cell membrane level. In this study we examined the expression of PKC isoforms alpha, epsilon and delta in PBMC cultured in rotating wall vessel bioreactor, developed at NASA JSC, which models microgravity by sustaining cells in continuous free fall. The assessment of the isoforms was performed by FACS analysis following cell permeabilization. A decrease in the expression of isoforms epsilon and delta, but not isoform a, was observed in PBMC cultured in microgravity conditions. These data suggest that MMG might selectively affect the expression of Ca2+ independent isoforms of PKC Molecular analysis confirm selective suppression of Ca2+ independent isoforms of PKC.

  11. A truncated, activin-induced Smad3 isoform acts as a transcriptional repressor of FSHβ expression in mouse pituitary.

    PubMed

    Kim, So-Youn; Zhu, Jie; Woodruff, Teresa K

    2011-08-01

    The receptor-regulated protein Smad3 is key player in the signaling cascade stimulated by the binding of activin to its cell surface receptor. Upon phosphorylation, Smad3 forms a heterocomplex with Smad2 and Smad4, translocates to the nucleus and acts as a transcriptional co-activator. We have identified a unique isoform of Smad3 that is expressed in mature pituitary gonadotropes. 5' RACE revealed that this truncated Smad3 isoform is transcribed from an ATG site within exon 4 and consists of 7 exons encoding half of the linker region and the MH2 region. In pituitary cells, the truncated Smad3 isoform was phosphorylated upon activin treatment, in a manner that was temporally distinct from the phosphorylation of full-length Smad3. Activin-induced phosphorylation of Smad3 and the truncated Smad3 isoform was blocked by both follistatin and siRNA-mediated knockdown of Smad3. The truncated Smad3 isoform antagonized Smad3-mediated, activin-responsive promoter activity. We propose that the pituitary gonadotrope contains an ultra-short, activin-responsive feedback loop utilizing two different isoforms of Smad3, one which acts as an agonist (Smad3) and another that acts as an intracrine antagonist (truncated Smad3 isoform) to regulate FSHβ production. PMID:21664424

  12. Gene Duplication and the Evolution of Hemoglobin Isoform Differentiation in Birds*

    PubMed Central

    Grispo, Michael T.; Natarajan, Chandrasekhar; Projecto-Garcia, Joana; Moriyama, Hideaki; Weber, Roy E.; Storz, Jay F.

    2012-01-01

    The majority of bird species co-express two functionally distinct hemoglobin (Hb) isoforms in definitive erythrocytes as follows: HbA (the major adult Hb isoform, with α-chain subunits encoded by the αA-globin gene) and HbD (the minor adult Hb isoform, with α-chain subunits encoded by the αD-globin gene). The αD-globin gene originated via tandem duplication of an embryonic α-like globin gene in the stem lineage of tetrapod vertebrates, which suggests the possibility that functional differentiation between the HbA and HbD isoforms may be attributable to a retained ancestral character state in HbD that harkens back to a primordial, embryonic function. To investigate this possibility, we conducted a combined analysis of protein biochemistry and sequence evolution to characterize the structural and functional basis of Hb isoform differentiation in birds. Functional experiments involving purified HbA and HbD isoforms from 11 different bird species revealed that HbD is characterized by a consistently higher O2 affinity in the presence of allosteric effectors such as organic phosphates and Cl− ions. In the case of both HbA and HbD, analyses of oxygenation properties under the two-state Monod-Wyman-Changeux allosteric model revealed that the pH dependence of Hb-O2 affinity stems primarily from changes in the O2 association constant of deoxy (T-state)-Hb. Ancestral sequence reconstructions revealed that the amino acid substitutions that distinguish the adult-expressed Hb isoforms are not attributable to the retention of an ancestral (pre-duplication) character state in the αD-globin gene that is shared with the embryonic α-like globin gene. PMID:22962007

  13. Characterization of Sin1 Isoforms Reveals an mTOR-Dependent and Independent Function of Sin1γ

    PubMed Central

    Yuan, Yuanyang; Pan, Bangfen; Sun, Haipeng; Chen, Guoqiang; Su, Bing; Huang, Ying

    2015-01-01

    Sin1 or MAPKAP1 is a key component of mTORC2 signaling complex which is necessary for AKT phosphorylation at the S473 and T450 sites, and also for AKT downstream signaling as well. A number of Sin1 splicing variants have been reported that can produce different Sin1 isoforms due to exon skipping or alternative transcription initiation. In this report, we characterized four Sin1 isoforms, including a novel Sin1 isoform due to alternative 3’ termination of the exon 9a, termed Sin1γ. Sin1γ expression can be detected in multiple adult mouse tissues, and it encodes a C-terminal truncated protein comparing to the full length Sin1β isoform. In contrast to Sin1β, Sin1γ overexpression in Sin1 deficient mouse embryonic fibroblasts has no significant impact on mTORC2 activity or mTORC2 subunits protein level, although it still can interact with mTORC2 components. More interestingly, Sin1γ was detected in a specific cytosolic location with a distinct feature in structure, and its localization was transiently disrupted during cell cycle. Therefore, Sin1γ is a novel Sin1 isoform and may have distinct properties in cell signaling and intracellular localization from other Sin1 isoforms. PMID:26263164

  14. Alternative Splicing Regulates the Subcellular Localization of Divalent Metal Transporter 1 Isoforms

    PubMed Central

    Tabuchi, Mitsuaki; Tanaka, Naotaka; Nishida-Kitayama, Junko; Ohno, Hiroshi; Kishi, Fumio

    2002-01-01

    Divalent metal transporter 1 (DMT1) is responsible for dietary-iron absorption from apical plasma membrane in the duodenum and iron acquisition from the transferrin cycle endosomes in peripheral tissues. Two isoforms of the DMT1 transcript generated by alternative splicing of the 3′ exons have been identified in mouse, rat, and human. These isoforms can be distinguished by the different C-terminal amino acid sequences and by the presence (DMT1A) or absence (DMT1B) of an iron response element located in the 3′ untranslated region of the mRNA. However, it has been still unknown whether the structural differences between the two DMT1 isoforms is functionally important. Here, we report that each DMT1 isoform exhibits a differential cell type–specific expression patterns and distinct subcellular localizations. DMT1A is predominantly expressed by epithelial cell lines, whereas DMT1B is expressed by the blood cell lines. In HEp-2 cells, GFP-tagged DMT1A is localized in late endosomes and lysosomes, whereas GFP-tagged DMT1B is localized in early endosomes. Using site-directed mutagenesis, a Y555XLXX sequence in the cytoplasmic tail of DMT1B has been identified as an important signal sequence for the early endosomal-targeting of DMT1B. In polarized MDCK cells, GFP-tagged DMT1A and DMT1B are localized in the apical plasma membrane and their respective specific endosomes. Disruption of the N-glycosylation sites in each of the DMT1 isoforms affects their polarized distribution into the apical plasma membrane but not their correct endosomal localization. Our data indicate that the cell type–specific expression patterns and the distinct subcellular localizations of two DMT1 isoforms may be involved in the different iron acquisition steps from the subcellular membranes in various cell types. PMID:12475959

  15. Impact of individual acute phase serum amyloid A isoforms on HDL metabolism in mice.

    PubMed

    Kim, Myung-Hee; de Beer, Maria C; Wroblewski, Joanne M; Charnigo, Richard J; Ji, Ailing; Webb, Nancy R; de Beer, Frederick C; van der Westhuyzen, Deneys R

    2016-06-01

    The acute phase (AP) reactant serum amyloid A (SAA), an HDL apolipoprotein, exhibits pro-inflammatory activities, but its physiological function(s) are poorly understood. Functional differences between SAA1.1 and SAA2.1, the two major SAA isoforms, are unclear. Mice deficient in either isoform were used to investigate plasma isoform effects on HDL structure, composition, and apolipoprotein catabolism. Lack of either isoform did not affect the size of HDL, normally enlarged in the AP, and did not significantly change HDL composition. Plasma clearance rates of HDL apolipoproteins were determined using native HDL particles. The fractional clearance rates (FCRs) of apoA-I, apoA-II, and SAA were distinct, indicating that HDL is not cleared as intact particles. The FCRs of SAA1.1 and SAA2.1 in AP mice were similar, suggesting that the selective deposition of SAA1.1 in amyloid plaques is not associated with a difference in the rates of plasma clearance of the isoforms. Although the clearance rate of SAA was reduced in the absence of the HDL receptor, scavenger receptor class B type I (SR-BI), it remained significantly faster compared with that of apoA-I and apoA-II, indicating a relatively minor role of SR-BI in SAA's rapid clearance. These studies enhance our understanding of SAA metabolism and SAA's effects on AP-HDL composition and catabolism. PMID:27018443

  16. Most highly expressed protein-coding genes have a single dominant isoform.

    PubMed

    Ezkurdia, Iakes; Rodriguez, Jose Manuel; Carrillo-de Santa Pau, Enrique; Vázquez, Jesús; Valencia, Alfonso; Tress, Michael L

    2015-04-01

    Although eukaryotic cells express a wide range of alternatively spliced transcripts, it is not clear whether genes tend to express a range of transcripts simultaneously across cells, or produce dominant isoforms in a manner that is either tissue-specific or regardless of tissue. To date, large-scale investigations into the pattern of transcript expression across distinct tissues have produced contradictory results. Here, we attempt to determine whether genes express a dominant splice variant at the protein level. We interrogate peptides from eight large-scale human proteomics experiments and databases and find that there is a single dominant protein isoform, irrespective of tissue or cell type, for the vast majority of the protein-coding genes in these experiments, in partial agreement with the conclusions from the most recent large-scale RNAseq study. Remarkably, the dominant isoforms from the experimental proteomics analyses coincided overwhelmingly with the reference isoforms selected by two completely orthogonal sources, the consensus coding sequence variants, which are agreed upon by separate manual genome curation teams, and the principal isoforms from the APPRIS database, predicted automatically from the conservation of protein sequence, structure, and function. PMID:25732134

  17. Insulin receptor isoforms: an integrated view focused on gestational diabetes mellitus.

    PubMed

    Westermeier, F; Sáez, T; Arroyo, P; Toledo, F; Gutiérrez, J; Sanhueza, C; Pardo, F; Leiva, A; Sobrevia, L

    2016-05-01

    The human insulin receptor (IR) exists in two isoforms that differ by the absence (IR-A) or the presence (IR-B) of a 12-amino acid segment encoded by exon 11. Both isoforms are functionally distinct regarding their binding affinities and intracellular signalling. However, the underlying mechanisms related to their cellular functions in several tissues are only partially understood. In this review, we summarize the current knowledge in this field regarding the alternative splicing of IR isoform, tissue-specific distribution and signalling both in physiology and disease, with an emphasis on the human placenta in gestational diabetes mellitus (GDM). Furthermore, we discuss the clinical relevance of IR isoforms highlighted by findings that show altered insulin signalling due to differential IR-A and IR-B expression in human placental endothelium in GDM pregnancies. Future research and clinical studies focused on the role of IR isoform signalling might provide novel therapeutic targets for treating GDM to improve the adverse maternal and neonatal outcomes. Copyright © 2015 John Wiley & Sons, Ltd. PMID:26431063

  18. HIF-1alpha and HIF-2alpha Are Differentially Activated in Distinct Cell Populations in Retinal Ischaemia

    PubMed Central

    Mowat, Freya M.; Luhmann, Ulrich F. O.; Smith, Alexander J.; Lange, Clemens; Duran, Yanai; Harten, Sarah; Shukla, Deepa; Maxwell, Patrick H.; Ali, Robin R.; Bainbridge, James W. B.

    2010-01-01

    Background Hypoxia plays a key role in ischaemic and neovascular disorders of the retina. Cellular responses to oxygen are mediated by hypoxia-inducible transcription factors (HIFs) that are stabilised in hypoxia and induce the expression of a diverse range of genes. The purpose of this study was to define the cellular specificities of HIF-1alpha and HIF-2alpha in retinal ischaemia, and to determine their correlation with the pattern of retinal hypoxia and the expression profiles of induced molecular mediators. Methodology/Principal Findings We investigated the tissue distribution of retinal hypoxia during oxygen-induced retinopathy (OIR) in mice using the bio-reductive drug pimonidazole. We measured the levels of HIF-1alpha and HIF-2alpha proteins by Western blotting and determined their cellular distribution by immunohistochemistry during the development of OIR. We measured the temporal expression profiles of two downstream mediators, vascular endothelial growth factor (VEGF) and erythropoietin (Epo) by ELISA. Pimonidazole labelling was evident specifically in the inner retina. Labelling peaked at 2 hours after the onset of hypoxia and gradually declined thereafter. Marked binding to Müller glia was evident during the early hypoxic stages of OIR. Both HIF-1alpha and HIF-2alpha protein levels were significantly increased during retinal hypoxia but were evident in distinct cellular distributions; HIF-1alpha stabilisation was evident in neuronal cells throughout the inner retinal layers whereas HIF-2alpha was restricted to Müller glia and astrocytes. Hypoxia and HIF-alpha stabilisation in the retina were closely followed by upregulated expression of the downstream mediators VEGF and EPO. Conclusions/Significance Both HIF-1alpha and HIF-2alpha are activated in close correlation with retinal hypoxia but have contrasting cell specificities, consistent with differential roles in retinal ischaemia. Our findings suggest that HIF-2alpha activation plays a key role in

  19. Correlation analysis of p53 protein isoforms with NPM1/FLT3 mutations and therapy response in acute myeloid leukemia.

    PubMed

    Ånensen, N; Hjelle, S M; Van Belle, W; Haaland, I; Silden, E; Bourdon, J-C; Hovland, R; Taskén, K; Knappskog, S; Lønning, P E; Bruserud, Ø; Gjertsen, B T

    2012-03-22

    The wild-type tumor-suppressor gene TP53 encodes several isoforms of the p53 protein. However, while the role of p53 in controlling normal cell cycle progression and tumor suppression is well established, the clinical significance of p53 isoform expression is unknown. A novel bioinformatic analysis of p53 isoform expression in 68 patients with acute myeloid leukemia revealed distinct p53 protein biosignatures correlating with clinical outcome. Furthermore, we show that mutated FLT3, a prognostic marker for short survival in AML, is associated with expression of full-length p53. In contrast, mutated NPM1, a prognostic marker for long-term survival, correlated with p53 isoforms β and γ expression. In conclusion, p53 biosignatures contain useful information for cancer evaluation and prognostication. PMID:21860418

  20. Antiangiogenic VEGF Isoform in Inflammatory Myopathies

    PubMed Central

    Volpi, Nila; Pecorelli, Alessandra; Lorenzoni, Paola; Di Lazzaro, Francesco; Belmonte, Giuseppe; Aglianò, Margherita; Giannini, Fabio; Grasso, Giovanni

    2013-01-01

    Objective. To investigate expression of vascular endothelial growth factor (VEGF) antiangiogenic isoform A-165b on human muscle in idiopathic inflammatory myopathies (IIM) and to compare distribution of angiogenic/antiangiogenic VEGFs, as isoforms shifts are described in other autoimmune disorders. Subjects and Methods. We analyzed VEGF-A165b and VEGF-A by western blot and immunohistochemistry on skeletal muscle biopsies from 21 patients affected with IIM (polymyositis, dermatomyositis, and inclusion body myositis) and 6 control muscle samples. TGF-β, a prominent VEGF inductor, was analogously evaluated. Intergroup differences of western blot bands density were statistically examined. Endomysial vascularization, inflammatory score, and muscle regeneration, as pathological parameters of IIM, were quantitatively determined and their levels were confronted with VEGF expression. Results. VEGF-A165b was significantly upregulated in IIM, as well as TGF-β. VEGF-A was diffusely expressed on unaffected myofibers, whereas regenerating/atrophic myofibres strongly reacted for both VEGF-A isoforms. Most inflammatory cells and endomysial vessels expressed both isoforms. VEGF-A165b levels were in positive correlation to inflammatory score, endomysial vascularization, and TGF-β. Conclusions. Our findings indicate skeletal muscle expression of antiangiogenic VEGF-A165b and preferential upregulation in IIM, suggesting that modulation of VEGF-A isoforms may occur in myositides. PMID:23840094

  1. Axonal and dendritic synaptotagmin isoforms revealed by a pHluorin-syt functional screen

    PubMed Central

    Dean, Camin; Dunning, F. Mark; Liu, Huisheng; Bomba-Warczak, Ewa; Martens, Henrik; Bharat, Vinita; Ahmed, Saheeb; Chapman, Edwin R.

    2012-01-01

    The synaptotagmins (syts) are a family of molecules that regulate membrane fusion. There are 17 mammalian syt isoforms, most of which are expressed in the brain. However, little is known regarding the subcellular location and function of the majority of these syts in neurons, largely due to a lack of isoform-specific antibodies. Here we generated pHluorin-syt constructs harboring a luminal domain pH sensor, which reports localization, pH of organelles to which syts are targeted, and the kinetics and sites of exocytosis and endocytosis. Of interest, only syt-1 and 2 are targeted to synaptic vesicles, whereas other isoforms selectively recycle in dendrites (syt-3 and 11), axons (syt-5, 7, 10, and 17), or both axons and dendrites (syt-4, 6, 9, and 12), where they undergo exocytosis and endocytosis with distinctive kinetics. Hence most syt isoforms localize to distinct secretory organelles in both axons and dendrites and may regulate neuropeptide/neurotrophin release to modulate neuronal function. PMID:22398727

  2. Smad phospho-isoforms direct context-dependent TGF-β signaling.

    PubMed

    Matsuzaki, Koichi

    2013-08-01

    Better understanding of TGF-β signaling has deepened our appreciation of normal epithelial cell homeostasis and its dysfunction in such human disorders as cancer and fibrosis. Smad proteins, which convey signals from TGF-β receptors to the nucleus, possess intermediate linker regions connecting Mad homology domains. Membrane-bound, cytoplasmic, and nuclear protein kinases differentially phosphorylate Smad2 and Smad3 to create C-tail (C), the linker (L), or dually (L/C) phosphorylated (p, phospho-) isoforms. According to domain-specific phosphorylation, distinct transcriptional responses, and selective metabolism, Smad phospho-isoform pathways can be grouped into 4 types: cytostatic pSmad3C signaling, mitogenic pSmad3L (Ser-213) signaling, invasive/fibrogenic pSmad2L (Ser-245/250/255)/C or pSmad3L (Ser-204)/C signaling, and mitogenic/migratory pSmad2/3L (Thr-220/179)/C signaling. We outline how responses to TGF-β change through the multiple Smad phospho-isoforms as normal epithelial cells mature from stem cells through progenitors to differentiated cells, and further reflect upon how constitutive Ras-activating mutants favor the Smad phospho-isoform pathway promoting tumor progression. Finally, clinical analyses of reversible Smad phospho-isoform signaling during human carcinogenesis could assess effectiveness of interventions aimed at reducing human cancer risk. Spatiotemporally separate, functionally different Smad phospho-isoforms have been identified in specific cells and tissues, answering long-standing questions about context-dependent TGF-β signaling. PMID:23871609

  3. The α and Δ Isoforms of CREB1 Are Required to Maintain Normal Pulmonary Vascular Resistance

    PubMed Central

    Sands, Michelle; Banahan, Mark; Frohlich, Stephen; Rowan, Simon C.; Neary, Roisín; Ryan, Donal; McLoughlin, Paul

    2013-01-01

    Chronic hypoxia causes pulmonary hypertension associated with structural alterations in pulmonary vessels and sustained vasoconstriction. The transcriptional mechanisms responsible for these distinctive changes are unclear. We have previously reported that CREB1 is activated in the lung in response to alveolar hypoxia but not in other organs. To directly investigate the role of α and Δ isoforms of CREB1 in the regulation of pulmonary vascular resistance we examined the responses of mice in which these isoforms of CREB1 had been inactivated by gene mutation, leaving only the β isoform intact (CREBαΔ mice). Here we report that expression of CREB regulated genes was altered in the lungs of CREBαΔ mice. CREBαΔ mice had greater pulmonary vascular resistance than wild types, both basally in normoxia and following exposure to hypoxic conditions for three weeks. There was no difference in rho kinase mediated vasoconstriction between CREBαΔ and wild type mice. Stereological analysis of pulmonary vascular structure showed characteristic wall thickening and lumen reduction in hypoxic wild-type mice, with similar changes observed in CREBαΔ. CREBαΔ mice had larger lungs with reduced epithelial surface density suggesting increased pulmonary compliance. These findings show that α and Δ isoforms of CREB1 regulate homeostatic gene expression in the lung and that normal activity of these isoforms is essential to maintain low pulmonary vascular resistance in both normoxic and hypoxic conditions and to maintain the normal alveolar structure. Interventions that enhance the actions of α and Δ isoforms of CREB1 warrant further investigation in hypoxic lung diseases. PMID:24349008

  4. Effects of Isoform-selective Phosphatidylinositol 3-Kinase Inhibitors on Osteoclasts

    PubMed Central

    Shugg, Ryan P. P.; Thomson, Ashley; Tanabe, Natsuko; Kashishian, Adam; Steiner, Bart H.; Puri, Kamal D.; Pereverzev, Alexey; Lannutti, Brian J.; Jirik, Frank R.; Dixon, S. Jeffrey; Sims, Stephen M.

    2013-01-01

    Phosphatidylinositol 3-kinases (PI3K) participate in numerous signaling pathways, and control distinct biological functions. Studies using pan-PI3K inhibitors suggest roles for PI3K in osteoclasts, but little is known about specific PI3K isoforms in these cells. Our objective was to determine effects of isoform-selective PI3K inhibitors on osteoclasts. The following inhibitors were investigated (targets in parentheses): wortmannin and LY294002 (pan-p110), PIK75 (α), GDC0941 (α, δ), TGX221 (β), AS252424 (γ), and IC87114 (δ). In addition, we characterized a new potent and selective PI3Kδ inhibitor, GS-9820, and explored roles of PI3K isoforms in regulating osteoclast function. Osteoclasts were isolated from long bones of neonatal rats and rabbits. Wortmannin, LY294002, GDC0941, IC87114, and GS-9820 induced a dramatic retraction of osteoclasts within 15–20 min to 65–75% of the initial area. In contrast, there was no significant retraction in response to vehicle, PIK75, TGX221, or AS252424. Moreover, wortmannin and GS-9820, but not PIK75 or TGX221, disrupted actin belts. We examined effects of PI3K inhibitors on osteoclast survival. Whereas PIK75, TGX221, and GS-9820 had no significant effect on basal survival, all blocked RANKL-stimulated survival. When studied on resorbable substrates, osteoclastic resorption was suppressed by wortmannin and inhibitors of PI3Kβ and PI3Kδ, but not other isoforms. These data are consistent with a critical role for PI3Kδ in regulating osteoclast cytoskeleton and resorptive activity. In contrast, multiple PI3K isoforms contribute to the control of osteoclast survival. Thus, the PI3Kδ isoform, which is predominantly expressed in cells of hematopoietic origin, is an attractive target for anti-resorptive therapeutics. PMID:24133210

  5. Short and long-term memory are modulated by multiple isoforms of the fragile X mental retardation protein

    PubMed Central

    Banerjee, Paromita; Schoenfeld, Brian P.; Bell, Aaron J.; Choi, Catherine H.; Bradley, Michael P.; Hinchey, Paul; Kollaros, Maria; Park, Jae H.; McBride, Sean M.J.; Dockendorff, Thomas C.

    2010-01-01

    The diversity of protein isoforms arising from alternative splicing is thought to modulate fine-tuning of synaptic plasticity. Fragile X mental retardation protein (FMRP), a neuronal RNA binding protein, exists in isoforms as a result of alternative splicing, but the contribution of these isoforms to neural plasticity are not well understood. We show that two isoforms of D. melanogaster FMRP (dFMR1) have differential roles in mediating neural development and behavior functions conferred by the dfmr1 gene. These isoforms differ in the presence of a protein interaction module that is related to prion domains and is functionally conserved between FMRPs. Expression of both isoforms is necessary for optimal performance in tests of short and long-term memory of courtship training. The presence or absence of the protein interaction domain may govern the types of ribonucleoprotein (RNP) complexes dFMR1 assembles into, with different RNPs regulating gene expression in a manner necessary for establishing distinct phases of memory formation. PMID:20463240

  6. Identification of T-cell factor-4 isoforms that contribute to the malignant phenotype of hepatocellular carcinoma cells

    SciTech Connect

    Tsedensodnom, Orkhontuya; Koga, Hironori; Rosenberg, Stephen A.; Nambotin, Sarah B.; Carroll, John J.; Wands, Jack R.; Kim, Miran

    2011-04-15

    The Wnt/{beta}-catenin signaling pathway is frequently activated in hepatocellular carcinoma (HCC). Downstream signaling events involving the Wnt/{beta}-catenin cascade occur through T-cell factor (TCF) proteins. The human TCF-4 gene is composed of 17 exons with multiple alternative splicing sites. However, the role of different TCF-4 isoforms in the pathogenesis of HCC is unknown. The purpose of this study was to identify and characterize TCF-4 isoforms in HCC. We identified 14 novel TCF-4 isoforms from four HCC cell lines. Functional analysis following transfection and expression in HCC cells revealed distinct effects on the phenotype. The TCF-4J isoform expression produced striking features of malignant transformation characterized by high cell proliferation rate, migration and colony formation even though its transcriptional activity was low. In contrast, the TCF-4K isoform displayed low TCF transcriptional activity; cell proliferation rate and colony formation were reduced as well. Interestingly, TCF-4J and TCF-4K differed by only five amino acids (the SxxSS motif). Thus, these studies suggest that conserved splicing motifs may have a major influence on the transcriptional activity and functional properties of TCF-4 isoforms and alter the characteristics of the malignant phenotype.

  7. Isoform-specific monobody inhibitors of small ubiquitin-related modifiers engineered using structure-guided library design

    SciTech Connect

    Gilbreth, Ryan N.; Truong, Khue; Madu, Ikenna; Koide, Akiko; Wojcik, John B.; Li, Nan-Sheng; Piccirilli, Joseph A.; Chen, Yuan; Koide, Shohei

    2011-07-25

    Discriminating closely related molecules remains a major challenge in the engineering of binding proteins and inhibitors. Here we report the development of highly selective inhibitors of small ubiquitin-related modifier (SUMO) family proteins. SUMOylation is involved in the regulation of diverse cellular processes. Functional differences between two major SUMO isoforms in humans, SUMO1 and SUMO2/3, are thought to arise from distinct interactions mediated by each isoform with other proteins containing SUMO-interacting motifs (SIMs). However, the roles of such isoform-specific interactions are largely uncharacterized due in part to the difficulty in generating high-affinity, isoform-specific inhibitors of SUMO/SIM interactions. We first determined the crystal structure of a 'monobody,' a designed binding protein based on the fibronectin type III scaffold, bound to the yeast homolog of SUMO. This structure illustrated a mechanism by which monobodies bind to the highly conserved SIM-binding site while discriminating individual SUMO isoforms. Based on this structure, we designed a SUMO-targeted library from which we obtained monobodies that bound to the SIM-binding site of human SUMO1 with K{sub d} values of approximately 100 nM but bound to SUMO2 400 times more weakly. The monobodies inhibited SUMO1/SIM interactions and, unexpectedly, also inhibited SUMO1 conjugation. These high-affinity and isoform-specific inhibitors will enhance mechanistic and cellular investigations of SUMO biology.

  8. Plectin isoform 1-dependent nuclear docking of desmin networks affects myonuclear architecture and expression of mechanotransducers

    PubMed Central

    Staszewska, Ilona; Fischer, Irmgard; Wiche, Gerhard

    2015-01-01

    Plectin is a highly versatile cytoskeletal protein that acts as a mechanical linker between intermediate filament (IF) networks and various cellular structures. The protein is crucial for myofiber integrity. Its deficiency leads to severe pathological changes in skeletal muscle fibers of patients suffering from epidermolysis bullosa simplex with muscular dystrophy (EBS-MD). Skeletal muscle fibers express four major isoforms of plectin which are distinguished solely by alternative, relatively short, first exon-encoded N-terminal sequences. Each one of these isoforms is localized to a different subcellular compartment and plays a specific role in maintaining integrity and proper function(s) of myofibers. The unique role of individual isoforms is supported by distinct phenotypes of isoform-specific knockout mice and recently discovered mutations in first coding exons of plectin that lead to distinct, tissue-specific, pathological abnormalities in humans. In this study, we demonstrate that the lack of plectin isoform 1 (P1) in myofibers of mice leads to alterations of nuclear morphology, similar to those observed in various forms of MD. We show that P1-mediated targeting of desmin IFs to myonuclei is essential for maintenance of their typically spheroidal architecture as well as their proper positioning and movement along the myofiber. Furthermore, we show that P1 deficiency affects chromatin modifications and the expression of genes involved in various cellular functions, including signaling pathways mediating mechanotransduction. Mechanistically, P1 is shown to specifically interact with the myonuclear membrane-associated (BAR domain-containing) protein endophilin B. Our results open a new perspective on cytoskeleton-nuclear crosstalk via specific cytolinker proteins. PMID:26487297

  9. Sequence-function correlations and dynamics of ERG isoforms. ERG8 is the black sheep of the family.

    PubMed

    Hoesel, Bastian; Malkani, Naila; Hochreiter, Bernhard; Basílio, José; Sughra, Kalsoom; Ilyas, Muhammad; Schmid, Johannes A

    2016-02-01

    The transcription factor ERG is known to have divergent roles. On one hand, it acts as differentiation factor of endothelial cells. On the other hand, it has pathological roles in various cancers. Genomic analyses of the ERG gene show that it gives rise to several isoforms. However, functional differences between these isoforms, representing potential reasons for distinct effects in diverse cell types have not been addressed in detail so far. We set out to investigate the major protein isoforms and found that ERG8 contains a unique C-terminus. This isoform, when expressed as GFP-fusion protein, localized mainly to the cytosol, whereas the other major isoforms (ERG1-4) were predominantly nuclear. Using site directed mutagenesis and laser scanning microscopy of live cells, we could identify nuclear localization (NLS) and nuclear export sequences (NES). These analyses indicated that ERG8 lacks a classical NLS and the DNA-binding domain, but holds an additional NES within its distinctive C-terminus. All the tested isoforms were shuttling between nucleus and cytosol and showed a high degree of mobility. ERG's 1 to 4 were transcriptionally active on ERG-promoter elements whereas ERG8 was inactive, which is in line with the absence of a DNA-binding domain. Fluorescence resonance energy transfer (FRET) microscopy revealed that ERG8 can bind to the transcriptionally active ERG's. Knockdown of ERG8 in endothelial cells resulted in upregulation of endogenous ERG-transcriptional activity implying ERG8 as an inhibitor of the active ERG isoforms. Quantitative PCR revealed a different ratio of active ERG's to ERG8 in cancer- versus non-transformed cells. PMID:26554849

  10. Sequence-function correlations and dynamics of ERG isoforms. ERG8 is the black sheep of the family

    PubMed Central

    Hochreiter, Bernhard; Basílio, José; Sughra, Kalsoom; Ilyas, Muhammad; Schmid, Johannes A.

    2015-01-01

    The transcription factor ERG is known to have divergent roles. On one hand, it acts as differentiation factor of endothelial cells. On the other hand, it has pathological roles in various cancers. Genomic analyses of the ERG gene show that it gives rise to several isoforms. However, functional differences between these isoforms, representing potential reasons for distinct effects in diverse cell types have not been addressed in detail so far. We set out to investigate the major protein isoforms and found that ERG8 contains a unique C-terminus. This isoform, when expressed as GFP-fusion protein, localized mainly to the cytosol, whereas the other major isoforms (ERG1-4) were predominantly nuclear. Using site directed mutagenesis and laser scanning microscopy of live cells, we could identify nuclear localization (NLS) and nuclear export sequences (NES). These analyses indicated that ERG8 lacks a classical NLS and the DNA-binding domain, but holds an additional NES within its distinctive C-terminus. All the tested isoforms were shuttling between nucleus and cytosol and showed a high degree of mobility. ERG’s 1 to 4 were transcriptionally active on ERG-promoter elements whereas ERG8 was inactive, which is in line with the absence of a DNA-binding domain. Fluorescence resonance energy transfer (FRET) microscopy revealed that ERG8 can bind to the transcriptionally active ERG’s. Knockdown of ERG8 in endothelial cells resulted in upregulation of endogenous ERG-transcriptional activity implying ERG8 as an inhibitor of the active ERG isoforms. Quantitative PCR revealed a different ratio of active ERG’s to ERG8 in cancer- versus non-transformed cells. PMID:26554849

  11. Cryptocephal, the Drosophila melanogaster ATF4, Is a Specific Coactivator for Ecdysone Receptor Isoform B2

    PubMed Central

    Gauthier, Sebastien A.; VanHaaften, Eric; Cherbas, Lucy; Cherbas, Peter; Hewes, Randall S.

    2012-01-01

    The ecdysone receptor is a heterodimer of two nuclear receptors, the Ecdysone receptor (EcR) and Ultraspiracle (USP). In Drosophila melanogaster, three EcR isoforms share common DNA and ligand-binding domains, but these proteins differ in their most N-terminal regions and, consequently, in the activation domains (AF1s) contained therein. The transcriptional coactivators for these domains, which impart unique transcriptional regulatory properties to the EcR isoforms, are unknown. Activating transcription factor 4 (ATF4) is a basic-leucine zipper transcription factor that plays a central role in the stress response of mammals. Here we show that Cryptocephal (CRC), the Drosophila homolog of ATF4, is an ecdysone receptor coactivator that is specific for isoform B2. CRC interacts with EcR-B2 to promote ecdysone-dependent expression of ecdysis-triggering hormone (ETH), an essential regulator of insect molting behavior. We propose that this interaction explains some of the differences in transcriptional properties that are displayed by the EcR isoforms, and similar interactions may underlie the differential activities of other nuclear receptors with distinct AF1-coactivators. PMID:22912598

  12. A mitotic SKAP isoform regulates spindle positioning at astral microtubule plus ends.

    PubMed

    Kern, David M; Nicholls, Peter K; Page, David C; Cheeseman, Iain M

    2016-05-01

    The Astrin/SKAP complex plays important roles in mitotic chromosome alignment and centrosome integrity, but previous work found conflicting results for SKAP function. Here, we demonstrate that SKAP is expressed as two distinct isoforms in mammals: a longer, testis-specific isoform that was used for the previous studies in mitotic cells and a novel, shorter mitotic isoform. Unlike the long isoform, short SKAP rescues SKAP depletion in mitosis and displays robust microtubule plus-end tracking, including localization to astral microtubules. Eliminating SKAP microtubule binding results in severe chromosome segregation defects. In contrast, SKAP mutants specifically defective for plus-end tracking facilitate proper chromosome segregation but display spindle positioning defects. Cells lacking SKAP plus-end tracking have reduced Clasp1 localization at microtubule plus ends and display increased lateral microtubule contacts with the cell cortex, which we propose results in unbalanced dynein-dependent cortical pulling forces. Our work reveals an unappreciated role for the Astrin/SKAP complex as an astral microtubule mediator of mitotic spindle positioning. PMID:27138257

  13. Thick Filament Length and Isoform Composition Determine Self-Organized Contractile Units in Actomyosin Bundles

    PubMed Central

    Thoresen, Todd; Lenz, Martin; Gardel, Margaret L.

    2013-01-01

    Diverse myosin II isoforms regulate contractility of actomyosin bundles in disparate physiological processes by variations in both motor mechanochemistry and the extent to which motors are clustered into thick filaments. Although the role of mechanochemistry is well appreciated, the extent to which thick filament length regulates actomyosin contractility is unknown. Here, we study the contractility of minimal actomyosin bundles formed in vitro by mixtures of F-actin and thick filaments of nonmuscle, smooth, and skeletal muscle myosin isoforms with varied length. Diverse myosin II isoforms guide the self-organization of distinct contractile units within in vitro bundles with shortening rates similar to those of in vivo myofibrils and stress fibers. The tendency to form contractile units increases with the thick filament length, resulting in a bundle shortening rate proportional to the length of constituent myosin thick filament. We develop a model that describes our data, providing a framework in which to understand how diverse myosin II isoforms regulate the contractile behaviors of disordered actomyosin bundles found in muscle and nonmuscle cells. These experiments provide insight into physiological processes that use dynamic regulation of thick filament length, such as smooth muscle contraction. PMID:23442916

  14. Inter-isoform-dependent Regulation of the Drosophila Master Transcriptional Regulator SIN3.

    PubMed

    Chaubal, Ashlesha; Todi, Sokol V; Pile, Lori A

    2016-05-27

    SIN3 is a transcriptional corepressor that acts as a scaffold for a histone deacetylase (HDAC) complex. The SIN3 complex regulates various biological processes, including organ development, cell proliferation, and energy metabolism. Little is known, however, about the regulation of SIN3 itself. There are two major isoforms of Drosophila SIN3, 187 and 220, which are differentially expressed. Intrigued by the developmentally timed exchange of SIN3 isoforms, we examined whether SIN3 187 controls the fate of the 220 counterpart. Here, we show that in developing tissue, there is interplay between SIN3 isoforms: when SIN3 187 protein levels increase, SIN3 220 protein decreases concomitantly. SIN3 187 has a dual effect on SIN3 220. Expression of 187 leads to reduced 220 transcript, while also increasing the turnover of SIN3 220 protein by the proteasome. These data support the presence of a novel, inter-isoform-dependent mechanism that regulates the amount of SIN3 protein, and potentially the level of specific SIN3 complexes, during distinct developmental stages. PMID:27129248

  15. Both Myosin-10 isoforms are required for radial neuronal migration in the developing cerebral cortex.

    PubMed

    Ju, Xing-Da; Guo, Ye; Wang, Nan-Nan; Huang, Ying; Lai, Ming-Ming; Zhai, Yan-Hua; Guo, Yu-Guang; Zhang, Jian-Hua; Cao, Rang-Juan; Yu, Hua-Li; Cui, Lei; Li, Yu-Ting; Wang, Xing-Zhi; Ding, Yu-Qiang; Zhu, Xiao-Juan

    2014-05-01

    During embryonic development of the mammalian cerebral cortex, postmitotic cortical neurons migrate radially from the ventricular zone to the cortical plate. Proper migration involves the correct orientation of migrating neurons and the transition from a multipolar to a mature bipolar morphology. Herein, we report that the 2 isoforms of Myosin-10 (Myo10) play distinct roles in the regulation of radial migration in the mouse cortex. We show that the full-length Myo10 (fMyo10) isoform is located in deeper layers of the cortex and is involved in establishing proper migration orientation. We also demonstrate that fMyo10-dependent orientation of radial migration is mediated at least in part by the netrin-1 receptor deleted in colorectal cancer. Moreover, we show that the headless Myo10 (hMyo10) isoform is required for the transition from multipolar to bipolar morphologies in the intermediate zone. Our study reveals divergent functions for the 2 Myo10 isoforms in controlling both the direction of migration and neuronal morphogenesis during radial cortical neuronal migration. PMID:23300110

  16. Functional Cooperativity between ABCG4 and ABCG1 Isoforms.

    PubMed

    Hegyi, Zoltán; Homolya, László

    2016-01-01

    ABCG4 belongs to the ABCG subfamily, the members of which are half transporters composed of a single transmembrane and a single nucleotide-binding domain. ABCG proteins have a reverse domain topology as compared to other mammalian ABC transporters, and have to form functional dimers, since the catalytic sites for ATP binding and hydrolysis, as well as the transmembrane domains are composed of distinct parts of the monomers. Here we demonstrate that ABCG4 can form homodimers, but also heterodimers with its closest relative, ABCG1. Both the full-length and the short isoforms of ABCG1 can dimerize with ABCG4, whereas the ABCG2 multidrug transporter is unable to form a heterodimer with ABCG4. We also show that contrary to that reported in some previous studies, ABCG4 is predominantly localized to the plasma membrane. While both ABCG1 and ABCG4 have been suggested to be involved in lipid transport or regulation, in accordance with our previous results regarding the long version of ABCG1, here we document that the expression of both the short isoform of ABCG1 as well as ABCG4 induce apoptosis in various cell types. This apoptotic effect, as a functional read-out, allowed us to demonstrate that the dimerization between these half transporters is not only a physical interaction but functional cooperativity. Given that ABCG4 is predominantly expressed in microglial-like cells and endothelial cells in the brain, our finding of ABCG4-induced apoptosis may implicate a new role for this protein in the clearance mechanisms within the central nervous system. PMID:27228027

  17. Functional Cooperativity between ABCG4 and ABCG1 Isoforms

    PubMed Central

    Hegyi, Zoltán

    2016-01-01

    ABCG4 belongs to the ABCG subfamily, the members of which are half transporters composed of a single transmembrane and a single nucleotide-binding domain. ABCG proteins have a reverse domain topology as compared to other mammalian ABC transporters, and have to form functional dimers, since the catalytic sites for ATP binding and hydrolysis, as well as the transmembrane domains are composed of distinct parts of the monomers. Here we demonstrate that ABCG4 can form homodimers, but also heterodimers with its closest relative, ABCG1. Both the full-length and the short isoforms of ABCG1 can dimerize with ABCG4, whereas the ABCG2 multidrug transporter is unable to form a heterodimer with ABCG4. We also show that contrary to that reported in some previous studies, ABCG4 is predominantly localized to the plasma membrane. While both ABCG1 and ABCG4 have been suggested to be involved in lipid transport or regulation, in accordance with our previous results regarding the long version of ABCG1, here we document that the expression of both the short isoform of ABCG1 as well as ABCG4 induce apoptosis in various cell types. This apoptotic effect, as a functional read-out, allowed us to demonstrate that the dimerization between these half transporters is not only a physical interaction but functional cooperativity. Given that ABCG4 is predominantly expressed in microglial-like cells and endothelial cells in the brain, our finding of ABCG4-induced apoptosis may implicate a new role for this protein in the clearance mechanisms within the central nervous system. PMID:27228027

  18. Identification of a Novel C-Terminal Truncated WT1 Isoform with Antagonistic Effects against Major WT1 Isoforms

    PubMed Central

    Tatsumi, Naoya; Hojo, Nozomi; Sakamoto, Hiroyuki; Inaba, Rena; Moriguchi, Nahoko; Matsuno, Keiko; Fukuda, Mari; Matsumura, Akihide; Hayashi, Seiji; Morimoto, Soyoko; Nakata, Jun; Fujiki, Fumihiro; Nishida, Sumiyuki; Nakajima, Hiroko; Tsuboi, Akihiro; Oka, Yoshihiro; Hosen, Naoki; Sugiyama, Haruo; Oji, Yusuke

    2015-01-01

    The Wilms’ tumor gene WT1 consists of 10 exons and encodes a zinc finger transcription factor. There are four major WT1 isoforms resulting from alternative splicing at two sites, exon 5 (17AA) and exon 9 (KTS). All major WT1 isoforms are overexpressed in leukemia and solid tumors and play oncogenic roles such as inhibition of apoptosis, and promotion of cell proliferation, migration and invasion. In the present study, a novel alternatively spliced WT1 isoform that had an extended exon 4 (designated as exon 4a) with an additional 153 bp (designated as 4a sequence) at the 3’ end was identified and designated as an Ex4a(+)WT1 isoform. The insertion of exon 4a resulted in the introduction of premature translational stop codons in the reading frame in exon 4a and production of C-terminal truncated WT1 proteins lacking zinc finger DNA-binding domain. Overexpression of the truncated Ex4a(+)WT1 isoform inhibited the major WT1-mediated transcriptional activation of anti-apoptotic Bcl-xL gene promoter and induced mitochondrial damage and apoptosis. Conversely, suppression of the Ex4a(+)WT1 isoform by Ex4a-specific siRNA attenuated apoptosis. These results indicated that the Ex4a(+)WT1 isoform exerted dominant negative effects on anti-apoptotic function of major WT1 isoforms. Ex4a(+)WT1 isoform was endogenously expressed as a minor isoform in myeloid leukemia and solid tumor cells and increased regardless of decrease in major WT1 isoforms during apoptosis, suggesting the dominant negative effects on anti-apoptotic function of major WT1 isoforms. These results indicated that Ex4a(+)WT1 isoform had an important physiological function that regulated oncogenic function of major WT1 isoforms. PMID:26090994

  19. Correlation between Saliva and Plasma Levels of Endothelin Isoforms ET-1, ET-2, and ET-3

    PubMed Central

    Gurusankar, Roma; Kumarathasan, Prem; Saravanamuthu, Anusha; Thomson, Errol M.

    2015-01-01

    Although saliva endothelins are emerging as valuable noninvasive cardiovascular biomarkers, reports on the relationship between isoforms in saliva and plasma remain scarce. We measured endothelins in concurrent saliva and plasma samples (n = 30 males; age 18–63) by HPLC-fluorescence. Results revealed statistically significant positive correlations among all isoforms between saliva and plasma: big endothelin-1 (BET-1, 0.55 ± 0.27 versus 3.35 ± 1.28 pmol/mL; r = 0.38, p = 0.041), endothelin-1 (ET-1, 0.52 ± 0.21 versus 3.45 ± 1.28 pmol/mL; r = 0.53, p = 0.003), endothelin-2 (ET-2, 0.21 ± 0.07 versus 1.63 ± 0.66 pmol/mL; r = 0.51, p = 0.004), and endothelin-3 (ET-3, 0.39 ± 0.19 versus 2.32 ± 1.44 pmol/mL; r = 0.75, p < 0.001). Correlations of BET-1, ET-1, and ET-3 within each compartment were positive in both plasma (p < 0.05) and saliva (p ≤ 0.1), whereas ET-2 was not significantly correlated with other isoforms in either plasma or saliva. For all isoforms, concentrations varied on average fivefold between individuals (90th/10th percentiles); individuals with high plasma endothelin levels generally had high saliva endothelin levels. Our results reveal that salivary ET isoform profiles portray the plasmatic profiles and support the view of coordinated regulation of ET-1 and ET-3, but distinct regulatory pathways for ET-2. PMID:25972900

  20. ApoE isoform-specific regulation of regeneration in the peripheral nervous system

    PubMed Central

    Comley, Laura H.; Fuller, Heidi R.; Wishart, Thomas M.; Mutsaers, Chantal A.; Thomson, Derek; Wright, Ann K.; Ribchester, Richard R.; Morris, Glenn E.; Parson, Simon H.; Horsburgh, Karen; Gillingwater, Thomas H.

    2011-01-01

    Apolipoprotein E (apoE) is a 34 kDa glycoprotein with three distinct isoforms in the human population (apoE2, apoE3 and apoE4) known to play a major role in differentially influencing risk to, as well as outcome from, disease and injury in the central nervous system. In general, the apoE4 allele is associated with poorer outcomes after disease or injury, whereas apoE3 is associated with better responses. The extent to which different apoE isoforms influence degenerative and regenerative events in the peripheral nervous system (PNS) is still to be established, and the mechanisms through which apoE exerts its isoform-specific effects remain unclear. Here, we have investigated isoform-specific effects of human apoE on the mouse PNS. Experiments in mice ubiquitously expressing human apoE3 or human apoE4 on a null mouse apoE background revealed that apoE4 expression significantly disrupted peripheral nerve regeneration and subsequent neuromuscular junction re-innervation following nerve injury compared with apoE3, with no observable effects on normal development, maturation or Wallerian degeneration. Proteomic isobaric tag for relative and absolute quantitation (iTRAQ) screens comparing healthy and regenerating peripheral nerves from mice expressing apoE3 or apoE4 revealed significant differences in networks of proteins regulating cellular outgrowth and regeneration (myosin/actin proteins), as well as differences in expression levels of proteins involved in regulating the blood–nerve barrier (including orosomucoid 1). Taken together, these findings have identified isoform-specific roles for apoE in determining the protein composition of peripheral nerve as well as regulating nerve regeneration pathways in vivo. PMID:21478199

  1. Sequence Discrimination by Alternatively Spliced Isoforms of a DNA Binding Zinc Finger Domain

    NASA Astrophysics Data System (ADS)

    Gogos, Joseph A.; Hsu, Tien; Bolton, Jesse; Kafatos, Fotis C.

    1992-09-01

    Two major developmentally regulated isoforms of the Drosophila chorion transcription factor CF2 differ by an extra zinc finger within the DNA binding domain. The preferred DNA binding sites were determined and are distinguished by an internal duplication of TAT in the site recognized by the isoform with the extra finger. The results are consistent with modular interactions between zinc fingers and trinucleotides and also suggest rules for recognition of AT-rich DNA sites by zinc finger proteins. The results show how modular finger interactions with trinucleotides can be used, in conjunction with alternative splicing, to alter the binding specificity and increase the spectrum of sites recognized by a DNA binding domain. Thus, CF2 may potentially regulate distinct sets of target genes during development.

  2. Isoform-specific inhibitors of ACATs: recent advances and promising developments.

    PubMed

    Ohshiro, Taichi; Tomoda, Hiroshi

    2011-12-01

    Acyl-CoA:cholesterol acyltransferase (ACAT) is a promising therapeutic target for cardiovascular diseases. Although a number of synthetic ACAT inhibitors have been developed, they have failed to show efficacy in clinical trials. Now, the presence of two ACAT isoforms with distinct functions, ACAT1 and ACAT2, has been discovered. Thus, the selectivity of ACAT inhibitors toward the two isoforms is important for their development as novel anti-atherosclerotic agents. The selectivity study indicated that fungal pyripyropene A (PPPA) is only an ACAT2-specific inhibitor. Furthermore, PPPA proved orally active in atherogenic mouse models, indicating it possessed cholesterol-lowering and atheroprotective activities. Certain PPPA derivatives, semi-synthetically prepared, possessed more potent and selective in vitro activity than PPPA against ACAT2. This review covers these studies and describes the future prospects of ACAT2-specific inhibitors. PMID:22098352

  3. Detection of Diverse and High Molecular Weight Nesprin-1 and Nesprin-2 Isoforms Using Western Blotting.

    PubMed

    Carthew, James; Karakesisoglou, Iakowos

    2016-01-01

    Heavily utilized in cell and molecular biology, western blotting is considered a crucial technique for the detection and quantification of proteins within complex mixtures. In particular, the detection of members of the nesprin (nuclear envelope spectrin repeat protein) family has proven difficult to analyze due to their substantial isoform diversity, molecular weight variation, and the sheer size of both nesprin-1 and nesprin-2 giant protein variants (>800 kDa). Nesprin isoforms contain distinct domain signatures, perform differential cytoskeletal associations, occupy different subcellular compartments, and vary in their tissue expression profiles. This structural and functional variance highlights the need to distinguish between the full range of proteins within the nesprin protein family, allowing for greater understanding of their specific roles in cell biology and disease. Herein, we describe a western blotting protocol modified for the detection of low to high molecular weight (50-1000 kDa) nesprin proteins. PMID:27147045

  4. The UNC-73/Trio RhoGEF-2 domain is required in separate isoforms for the regulation of pharynx pumping and normal neurotransmission in C. elegans.

    PubMed

    Steven, Robert; Zhang, Lijia; Culotti, Joseph; Pawson, Tony

    2005-09-01

    In both Caenorhabditis elegans and Drosophila, UNC-73/Trio functions in axon guidance by signaling through the Rac GTPase to regulate cytoskeletal rearrangements necessary for growth cone migrations. Here, we show that the complex C. elegans unc-73 gene encodes at least eight differentially expressed UNC-73 intracellular protein isoforms. Previously reported mutations affecting UNC-73 isoforms encoding the Rac-specific RhoGEF-1 domain cause uncoordinated movement, correlating with defects in axon guidance. Mutations in isoforms encoding the Rho-specific RhoGEF-2 domain, which we describe here, result in L1 stage larval lethality with no associated axon guidance defects. Isoform-specific rescue experiments reveal separate functions for the various RhoGEF-2-containing UNC-73 isoforms, which would not likely be discovered by conventional genetic screening. UNC-73 D1 and D2 appear to function redundantly in pharynx muscle to regulate the rate and strength of pharynx pumping, and in the HSN neurons and vulval muscles to control egg laying. Isoforms C1, C2, E, and F act redundantly within the nervous system to regulate the speed of locomotion. The multiple UNC-73 isoforms containing Rac- and Rho-specific RhoGEF domains therefore have distinct physiological functions. In addition to its previously identified role involving RhoGEF-1 in migrating cells and growth cones, our data indicate that UNC-73 signals through RhoGEF-2 to regulate pharynx and vulva musculature and to modulate synaptic neurotransmission. PMID:16140983

  5. Retinal and choroidal TGF-beta in the tree shrew model of myopia: isoform expression, activation and effects on function.

    PubMed

    Jobling, Andrew Ian; Wan, Ran; Gentle, Alex; Bui, Bang Viet; McBrien, Neville Anthony

    2009-03-01

    A visually evoked signalling cascade, which begins in the retina, transverses the choroid, and mediates scleral remodelling, is considered to control eye growth. The ubiquitous cytokine TGF-beta has been associated with alterations in ocular growth, where alterations in scleral TGF-beta isoforms mediate the scleral remodelling that results in myopia. However, while the TGF-beta isoforms have been implicated in the scleral change during myopia development, it is unclear whether alterations in retinal and choroidal isoforms constitute part of the retinoscleral cascade. This study characterised the retinal and choroidal TGF-beta isoform profiles and TGF-beta2 activation during different stages of myopia development, as induced by form deprivation, in a mammalian model of eye growth. Using quantitative real-time PCR, the mRNA for all three mammalian isoforms of TGF-beta was detected in tree shrew retina and choroid. Distinct tissue-specific isoform profiles were observed for the retina (TGF-beta1:TGF-beta2:TGF-beta3=20:2085:1) and choroid (TGF-beta1:TGF-beta2:TGF-beta3=16:23:1), which remained constant over the development period under investigation. The active and latent pools of retinal TGF-beta2 were quantified using ELISA with the majority (>94%) of total TGF-beta2 found in the latent form. Unlike previous scleral data showing early and continuous decreases in TGF-beta isoform expression during myopia development, the levels of the three isoforms remained within normal ranges for retinal (TGF-beta1, -14 to +14%; TGF-beta2, -2 to +20%; TGF-beta3, -10 to +26%) and choroidal (TGF-beta1, -19 to +21%; TGF-beta2, -26 to +8%; TGF-beta3, -11 to +28%) tissues during myopia development (induction times of 3h, 7h, 11h, 24h, and 5 days). A 40% decrease in retinal TGF-beta2 activation was observed after 5 days of myopia development, however, there was no functional correlate of altered TGF-beta2 activity, as assessed by the retinal ERG response. Overall, these data highlight

  6. Herpes simplex virus 1 ubiquitin ligase ICP0 interacts with PML isoform I and induces its SUMO-independent degradation.

    PubMed

    Cuchet-Lourenço, Delphine; Vanni, Emilia; Glass, Mandy; Orr, Anne; Everett, Roger D

    2012-10-01

    Herpes simplex virus 1 (HSV-1) immediate-early protein ICP0 localizes to cellular structures known as promyelocytic leukemia protein (PML) nuclear bodies or ND10 and disrupts their integrity by inducing the degradation of PML. There are six PML isoforms with different C-terminal regions in ND10, of which PML isoform I (PML.I) is the most abundant. Depletion of all PML isoforms increases the plaque formation efficiency of ICP0-null mutant HSV-1, and reconstitution of expression of PML.I and PML.II partially reverses this improved replication. ICP0 also induces widespread degradation of SUMO-conjugated proteins during HSV-1 infection, and this activity is linked to its ability to counteract cellular intrinsic antiviral resistance. All PML isoforms are highly SUMO modified, and all such modified forms are sensitive to ICP0-mediated degradation. However, in contrast to the situation with the other isoforms, ICP0 also targets PML.I that is not modified by SUMO, and PML in general is degraded more rapidly than the bulk of other SUMO-modified proteins. We report here that ICP0 interacts with PML.I in both yeast two-hybrid and coimmunoprecipitation assays. This interaction is dependent on PML.I isoform-specific sequences and the N-terminal half of ICP0 and is required for SUMO-modification-independent degradation of PML.I by ICP0. Degradation of the other PML isoforms by ICP0 was less efficient in cells specifically depleted of PML.I. Therefore, ICP0 has two distinct mechanisms of targeting PML: one dependent on SUMO modification and the other via SUMO-independent interaction with PML.I. We conclude that the ICP0-PML.I interaction reflects a countermeasure to PML-related antiviral restriction. PMID:22875967

  7. Increased dysbindin-1B isoform expression in schizophrenia and its propensity in aggresome formation

    PubMed Central

    Xu, Yiliang; Sun, Yuhui; Ye, Haihong; Zhu, Li; Liu, Jianghong; Wu, Xiaofeng; Wang, Le; He, Tingting; Shen, Yan; Wu, Jane Y; Xu, Qi

    2015-01-01

    Genetic variations in the human dysbindin-1 gene (DTNBP1) have been associated with schizophrenia. As a result of alternative splicing, the human DTNBP1 gene generates at least three distinct protein isoforms, dysbindin-1A, -1B and -1C. Significant effort has focused on dysbindin-1A, an important player in multiple steps of neurodevelopment. However, the other isoforms, dysbindin-1B and dysbindin-1C have not been well characterized. Nor have been associated with human diseases. Here we report an increase in expression of DTNBP1b mRNA in patients with paranoid schizophrenia as compared with healthy controls. A single-nucleotide polymorphism located in intron 9, rs117610176, has been identified and associated with paranoid schizophrenia, and its C allele leads to an increase of DTNBP1b mRNA splicing. Our data show that different dysbindin splicing isoforms exhibit distinct subcellular distribution, suggesting their distinct functional activities. Dysbindin-1B forms aggresomes at the perinuclear region, whereas dysbindin-1A and -1C proteins exhibit diffused patterns in the cytoplasm. Dysbindin-1A interacts with dysbindin-1B, getting recruited to the aggresome structure when co-expressed with dysbindin-1B. Moreover, cortical neurons over-expressing dysbindin-1B show reduction in neurite outgrowth, suggesting that dysbindin-1B may interfere with dysbindin-1A function in a dominant-negative manner. Taken together, our study uncovers a previously unknown association of DTNBP1b expression with schizophrenia in addition to its distinct biochemical and functional properties.

  8. Analysis of knockout mutants reveals non-redundant functions of poly(ADP-ribose)polymerase isoforms in Arabidopsis.

    PubMed

    Pham, Phuong Anh; Wahl, Vanessa; Tohge, Takayuki; de Souza, Laise Rosado; Zhang, Youjun; Do, Phuc Thi; Olas, Justyna J; Stitt, Mark; Araújo, Wagner L; Fernie, Alisdair R

    2015-11-01

    The enzyme poly(ADP-ribose)polymerase (PARP) has a dual function being involved both in the poly(ADP-ribosyl)ation and being a constituent of the NAD(+) salvage pathway. To date most studies, both in plant and non-plant systems, have focused on the signaling role of PARP in poly(ADP-ribosyl)ation rather than any role that can be ascribed to its metabolic function. In order to address this question we here used a combination of expression, transcript and protein localization studies of all three PARP isoforms of Arabidopsis alongside physiological analysis of the corresponding mutants. Our analyses indicated that whilst all isoforms of PARP were localized to the nucleus they are also present in non-nuclear locations with parp1 and parp3 also localised in the cytosol, and parp2 also present in the mitochondria. We next isolated and characterized insertional knockout mutants of all three isoforms confirming a complete knockout in the full length transcript levels of the target genes as well as a reduced total leaf NAD hydrolase activity in the two isoforms (PARP1, PARP2) that are highly expressed in leaves. Physiological evaluation of the mutant lines revealed that they displayed distinctive metabolic and root growth characteristics albeit unaltered leaf morphology under optimal growth conditions. We therefore conclude that the PARP isoforms play non-redundant non-nuclear metabolic roles and that their function is highly important in rapidly growing tissues such as the shoot apical meristem, roots and seeds. PMID:26428915

  9. Tumorigenic properties of alternative osteopontin isoforms in mesothelioma

    SciTech Connect

    Ivanov, Sergey V.; Ivanova, Alla V.; Goparaju, Chandra M.V.; Chen, Yuanbin; Beck, Amanda; Pass, Harvey I.

    2009-05-08

    Osteopontin (SPP1) is an inflammatory cytokine that we previously characterized as a diagnostic marker in patients with asbestos-induced malignant mesothelioma (MM). While SPP1 shows both pro- and anti-tumorigenic biological effects, little is known about the molecular basis of these activities. In this study, we demonstrate that while healthy pleura possesses all three differentially spliced SPP1 isoforms (A-C), in clinical MM specimens isoform A is markedly up-regulated and predominant. To provide a clue to possible functions of the SPP1 isoforms we next performed their functional evaluation via transient expression in MM cell lines. As a result, we report that isoforms A-C demonstrate different activities in cell proliferation, wound closure, and invasion assays. These findings suggest different functions for SPP1 isoforms and underline pro-tumorigenic properties of isoforms A and B.

  10. Expression of Contractile Protein Isoforms in Microgravity

    NASA Technical Reports Server (NTRS)

    Anderson, Page A. W.

    1996-01-01

    The general objective of this experiment is to determine the effect of space flight parameters, including microgravity, on ontogenesis and embryogenesis of Japanese quail. Nine U.S. and two Russian investigators are cooperating in this study. Specific objectives of the participating scientists include assessing the gross and microscopic morphological and histological development of the embryo, as well as the temporal and spacial development of specific cells, tissues, and organs. Temporally regulated production of specific proteins is also being investigated. Our objective is to determine the effects of microgravity on developmentally programmed expression of Troponin T and I isoforms known to regulate cardiac and skeletal muscle contraction.

  11. Decoding RAS isoform and codon-specific signalling

    PubMed Central

    Newlaczyl, Anna U.; Hood, Fiona E.; Coulson, Judy M.; Prior, Ian A.

    2014-01-01

    RAS proteins are key signalling hubs that are oncogenically mutated in 30% of all cancer cases. Three genes encode almost identical isoforms that are ubiquitously expressed, but are not functionally redundant. The network responses associated with each isoform and individual oncogenic mutations remain to be fully characterized. In the present article, we review recent data defining the differences between the RAS isoforms and their most commonly mutated codons and discuss the underlying mechanisms. PMID:25109951

  12. Separation of plasmid DNA isoforms using centrifugal ultrafiltration.

    PubMed

    Borujeni, Ehsan Espah; Zydney, Andrew L

    2012-07-01

    Centrifugal ultrafiltration is a well-established method for concentrating and purifying DNA. Here, we describe the use of centrifugal ultrafiltration for the separation of plasmid DNA isoforms based on differences in elongational flexibility of the supercoiled, open-circular, and linear plasmids. Transmission of each isoform is minimal below a critical value of the filtration velocity, which is directly related to the magnitude of the centrifugal speed and the system geometry. A discontinuous diafiltration process was used to enrich the desired isoform, as determined by agarose gel electrophoresis. The simplicity and efficacy of this membrane-based separation are attractive for multiple applications requiring the use of separated DNA isoforms. PMID:22780319

  13. Basal activity of GIRK5 isoforms.

    PubMed

    Salvador, Carolina; Mora, Silvia I; Ordaz, Benito; Antaramian, Anaid; Vaca, Luis; Escobar, Laura I

    2003-02-14

    G protein-coupled inwardly rectifying K(+) channels (GIRK or Kir3) form functional heterotetramers gated by Gbetagamma subunits. GIRK channels are critical for functions as diverse as heart rate modulation and neuronal post-synaptic inhibition. GIRK5 (Kir3.5) is the oocyte homologue of the mammalian GIRK subunits that conform the K(ACh) channel. It has been claimed that even when the oocytes express GIRK5 proteins they do not form functional channels. However, the GIRK5 gene shows three initiation sites that suggest the existence of three isoforms. In a previous work we demonstrated the functionality of homomultimers of the shortest isoform overexpressed in the own oocytes. Remarkably, the basal GIRK5-Delta25 inward currents were not coupled to the activation of a G-protein receptor in the oocytes. These results encouraged us to study this channel in another expression system. In this work we show that Sf21 insect cells can be successfully transfected with this channel. GIRK5-Delta25 homomultimers produce time-dependent inward currents only with GTPgammaS in the recording pipette. Therefore, alternative modes of stimulus input to heterotrimeric G-proteins should be present in the oocytes to account for these results. PMID:12535718

  14. Immunochemical characterization and transacting properties of upstream stimulatory factor isoforms.

    PubMed

    Viollet, B; Lefrançois-Martinez, A M; Henrion, A; Kahn, A; Raymondjean, M; Martinez, A

    1996-01-19

    The ubiquitous upstream stimulatory factor (USF) transcription factors encoded by two distinct genes (USF1 and USF2) exist under the form of various dimers able to bind E-boxes. We report the molecular cloning and functional characterization of USF2 isoforms, corresponding to a 44-kDa subunit, USF2a, and a new 38-kDa subunit, USF2b, generated by differential splicing. Using specific anti-USF antibodies, we define the different binding complexes in various nuclear extracts. In vivo, the USF1/USF2a heterodimer represents over 66% of the USF binding activity whereas the USF1 and USF2a homodimers represent less than 10%, which strongly suggests an in vivo preferential association in heterodimers. In particular, an USF1/USF2b heterodimer accounted for almost 15% of the USF species in some cells. The preferential heterodimerization of USF subunits was reproduced ex vivo, while the in vitro association of cotranslated subunits, or recombinant USF proteins, appeared to be random. In transiently transfected HeLa or hepatoma cells, USF2a and USF1 homodimers transactivated a minimal promoter with similar efficiency, whereas USF2b, which lacks an internal 67-amino acid domain, was a poor transactivator. Additionally, USF2b was an efficient as USF1 and USF2a homodimers in transactivating the liver-specific pyruvate kinase gene promoter. PMID:8576131

  15. Prion neuropathology follows the accumulation of alternate prion protein isoforms after infective titre has peaked

    PubMed Central

    Sandberg, Malin K.; Al-Doujaily, Huda; Sharps, Bernadette; De Oliveira, Michael Wiggins; Schmidt, Christian; Richard-Londt, Angela; Lyall, Sarah; Linehan, Jacqueline M.; Brandner, Sebastian; Wadsworth, Jonathan D. F.; Clarke, Anthony R.; Collinge, John

    2014-01-01

    Prions are lethal infectious agents thought to consist of multi-chain forms (PrPSc) of misfolded cellular prion protein (PrPC). Prion propagation proceeds in two distinct mechanistic phases: an exponential phase 1, which rapidly reaches a fixed level of infectivity irrespective of PrPC expression level, and a plateau (phase 2), which continues until clinical onset with duration inversely proportional to PrPC expression level. We hypothesized that neurotoxicity relates to distinct neurotoxic species produced following a pathway switch when prion levels saturate. Here we show a linear increase of proteinase K-sensitive PrP isoforms distinct from classical PrPSc at a rate proportional to PrPC concentration, commencing at the phase transition and rising until clinical onset. The unaltered level of total PrP during phase 1, when prion infectivity increases a million-fold, indicates that prions comprise a small minority of total PrP. This is consistent with PrPC concentration not being rate limiting to exponential prion propagation and neurotoxicity relating to critical concentrations of alternate PrP isoforms whose production is PrPC concentration dependent. PMID:25005024

  16. Tunable protein synthesis by transcript isoforms in human cells

    PubMed Central

    Floor, Stephen N; Doudna, Jennifer A

    2016-01-01

    Eukaryotic genes generate multiple RNA transcript isoforms though alternative transcription, splicing, and polyadenylation. However, the relationship between human transcript diversity and protein production is complex as each isoform can be translated differently. We fractionated a polysome profile and reconstructed transcript isoforms from each fraction, which we term Transcript Isoforms in Polysomes sequencing (TrIP-seq). Analysis of these data revealed regulatory features that control ribosome occupancy and translational output of each transcript isoform. We extracted a panel of 5′ and 3′ untranslated regions that control protein production from an unrelated gene in cells over a 100-fold range. Select 5′ untranslated regions exert robust translational control between cell lines, while 3′ untranslated regions can confer cell type-specific expression. These results expose the large dynamic range of transcript-isoform-specific translational control, identify isoform-specific sequences that control protein output in human cells, and demonstrate that transcript isoform diversity must be considered when relating RNA and protein levels. DOI: http://dx.doi.org/10.7554/eLife.10921.001 PMID:26735365

  17. Spinach pyruvate kinase isoforms: partial purification and regulatory properties

    SciTech Connect

    Baysdorfer, C.; Bassham, J.A.

    1984-02-01

    Pyruvate kinase from spinach (Spinacea oleracea L.) leaves consists of two isoforms, separable by blue agarose chromatography. Both isoforms share similar pH profiles and substrate and alternate nucleotide K/sub m/ values. In addition, both isoforms are inhibited by oxalate and ATP and activated by AMP. The isoforms differ in their response to three key metabolites; citrate, aspartate, and glutamate. The first isoform is similar to previously reported plant pyruvate kinases in its sensitivity to citrate inhibition. The K/sub i/ for this inhibition is 1.2 millimolar citrate. The second isoform is not affected by citrate but is regulated by aspartate and glutamate. Aspartate is an activator with a K/sub a/ of 0.05 millimolar, and glutamate is an inhibitor with a K/sub i/ of 0.68 millimolar. A pyruvate kinase with these properties has not been previously reported. Based on these considerations, the authors suggest that the activity of the first isoform is regulated by respiratory metabolism. The second isoform, in contrast, may be regulated by the demand for carbon skeletons for use in ammonia assimilation.

  18. CD45 Isoform Profile Identifies Natural Killer (NK) Subsets with Differential Activity

    PubMed Central

    Krzywinska, Ewelina; Cornillon, Amelie; Allende-Vega, Nerea; Vo, Dang-Nghiem; Rene, Celine; Lu, Zhao-Yang; Pasero, Christine; Olive, Daniel; Fegueux, Nathalie; Ceballos, Patrick; Hicheri, Yosr; Sobecki, Michal; Rossi, Jean-François; Cartron, Guillaume; Villalba, Martin

    2016-01-01

    The leucocyte-specific phosphatase CD45 is present in two main isoforms: the large CD45RA and the short CD45RO. We have recently shown that distinctive expression of these isoforms distinguishes natural killer (NK) populations. For example, co-expression of both isoforms identifies in vivo the anti tumor NK cells in hematological cancer patients. Here we show that low CD45 expression associates with less mature, CD56bright, NK cells. Most NK cells in healthy human donors are CD45RA+CD45RO-. The CD45RA-RO+ phenotype, CD45RO cells, is extremely uncommon in B or NK cells, in contrast to T cells. However, healthy donors possess CD45RAdimRO- (CD45RAdim cells), which show immature markers and are largely expanded in hematopoietic stem cell transplant patients. Blood borne cancer patients also have more CD45RAdim cells that carry several features of immature NK cells. However, and in opposition to their association to NK cell progenitors, they do not proliferate and show low expression of the transferrin receptor protein 1/CD71, suggesting low metabolic activity. Moreover, CD45RAdim cells properly respond to in vitro encounter with target cells by degranulating or gaining CD69 expression. In summary, they are quiescent NK cells, with low metabolic status that can, however, respond after encounter with target cells. PMID:27100180

  19. PI3K in cancer: divergent roles of isoforms, modes of activation, and therapeutic targeting

    PubMed Central

    Thorpe, Lauren M.; Yuzugullu, Haluk; Zhao, Jean J.

    2015-01-01

    Preface Phosphatidylinositol 3-Kinases (PI3Ks) are critical coordinators of intracellular signaling in response to extracellular stimuli. Hyperactivation of PI3K signaling cascades is one of the most common events in human cancers. In this Review, we discuss recent advances in our knowledge of the roles of distinct PI3K isoforms in normal and oncogenic signaling, the different ways in which PI3K can be upregulated, and the current state and future potential of targeting this pathway in the clinic. PMID:25533673

  20. IL-33 isoforms: their future as vaccine adjuvants?

    PubMed Central

    Villarreal, Daniel O; Weiner, David B

    2015-01-01

    The identification and characterization of cytokine isoforms is likely to provide critical important new insight into immunobiology. Cytokine isoforms can provide additional diversity to their complex biological effects that participate in control and protection against different foreign pathogens. Recently, IL-33 has been identified as a proinflammatory cytokine having several different biologically active isoform products. Originally associated with Th2 immunity, new evidence now supports the role of two IL-33 isoforms to facilitate the generation of protective Th1 and CD8 T cell immunity against specific pathogens. Therefore, a better understanding of the IL-33 isoforms will inform us on how to utilize them to facilitate their development as tools as vaccine adjuvants for immune therapy. PMID:25656504

  1. Direct force measurements reveal that protein Tau confers short-range attractions and isoform-dependent steric stabilization to microtubules

    PubMed Central

    Chung, Peter J.; Choi, Myung Chul; Miller, Herbert P.; Feinstein, H. Eric; Raviv, Uri; Li, Youli; Wilson, Leslie; Feinstein, Stuart C.; Safinya, Cyrus R.

    2015-01-01

    Microtubules (MTs) are hollow cytoskeletal filaments assembled from αβ-tubulin heterodimers. Tau, an unstructured protein found in neuronal axons, binds to MTs and regulates their dynamics. Aberrant Tau behavior is associated with neurodegenerative dementias, including Alzheimer’s. Here, we report on a direct force measurement between paclitaxel-stabilized MTs coated with distinct Tau isoforms by synchrotron small-angle X-ray scattering (SAXS) of MT-Tau mixtures under osmotic pressure (P). In going from bare MTs to MTs with Tau coverage near the physiological submonolayer regime (Tau/tubulin-dimer molar ratio; ΦTau = 1/10), isoforms with longer N-terminal tails (NTTs) sterically stabilized MTs, preventing bundling up to PB ∼ 10,000–20,000 Pa, an order of magnitude larger than bare MTs. Tau with short NTTs showed little additional effect in suppressing the bundling pressure (PB ∼ 1,000–2,000 Pa) over the same range. Remarkably, the abrupt increase in PB observed for longer isoforms suggests a mushroom to brush transition occurring at 1/13 < ΦTau < 1/10, which corresponds to MT-bound Tau with NTTs that are considerably more extended than SAXS data for Tau in solution indicate. Modeling of Tau-mediated MT–MT interactions supports the hypothesis that longer NTTs transition to a polyelectrolyte brush at higher coverages. Higher pressures resulted in isoform-independent irreversible bundling because the polyampholytic nature of Tau leads to short-range attractions. These findings suggest an isoform-dependent biological role for regulation by Tau, with longer isoforms conferring MT steric stabilization against aggregation either with other biomacromolecules or into tight bundles, preventing loss of function in the crowded axon environment. PMID:26542680

  2. Cell Elasticity Is Regulated by the Tropomyosin Isoform Composition of the Actin Cytoskeleton

    PubMed Central

    Jalilian, Iman; Heu, Celine; Cheng, Hong; Freittag, Hannah; Desouza, Melissa; Stehn, Justine R.; Bryce, Nicole S.; Whan, Renee M.; Hardeman, Edna C.

    2015-01-01

    The actin cytoskeleton is the primary polymer system within cells responsible for regulating cellular stiffness. While various actin binding proteins regulate the organization and dynamics of the actin cytoskeleton, the proteins responsible for regulating the mechanical properties of cells are still not fully understood. In the present study, we have addressed the significance of the actin associated protein, tropomyosin (Tpm), in influencing the mechanical properties of cells. Tpms belong to a multi-gene family that form a co-polymer with actin filaments and differentially regulate actin filament stability, function and organization. Tpm isoform expression is highly regulated and together with the ability to sort to specific intracellular sites, result in the generation of distinct Tpm isoform-containing actin filament populations. Nanomechanical measurements conducted with an Atomic Force Microscope using indentation in Peak Force Tapping in indentation/ramping mode, demonstrated that Tpm impacts on cell stiffness and the observed effect occurred in a Tpm isoform-specific manner. Quantitative analysis of the cellular filamentous actin (F-actin) pool conducted both biochemically and with the use of a linear detection algorithm to evaluate actin structures revealed that an altered F-actin pool does not absolutely predict changes in cell stiffness. Inhibition of non-muscle myosin II revealed that intracellular tension generated by myosin II is required for the observed increase in cell stiffness. Lastly, we show that the observed increase in cell stiffness is partially recapitulated in vivo as detected in epididymal fat pads isolated from a Tpm3.1 transgenic mouse line. Together these data are consistent with a role for Tpm in regulating cell stiffness via the generation of specific populations of Tpm isoform-containing actin filaments. PMID:25978408

  3. A differential association of Apolipoprotein E isoforms with the Aβ oligomer in solution

    PubMed Central

    Petrlova, Jitka; Hong, Hyun-Seok; Bricarello, Daniel; Harishchandra, Ghimire; Lorigan, Gary; Jin, Lee-Way; Voss, John C.

    2010-01-01

    The molecular pathogenesis of disorders arising from protein mis-folding and aggregation is difficult to elucidate, involving a complex ensemble of intermediates whose toxicity depends upon their state of progression along distinct processing pathways. To address the complex mis-folding and aggregation that initiates the toxic cascade resulting in Alzheimer's disease, we have developed a TOAC spin-labeled Aβ peptide to observe its isoform-dependent interaction with the apoE protein. While most individuals carry the E3 isoform of apoE, approximately 15% of humans carry the E4 isoform, which is recognized as the most significant genetic determinant for Alzheimer's. ApoE is consistently associated with the amyloid plaque marker for Alzheimer's disease. A vital question centers on the influence of the two predominant isoforms, E3 and E4, on Aβ peptide processing and hence Aβ toxicity. We employed EPR spectroscopy of incorporated spin labels to investigate the interaction of apoE with the toxic oligomeric species of Aβ in solution. EPR spectra of the spin labeled side chain report on side chain and backbone dynamics, as well as the spatial proximity of spins in an assembly. Our results indicate oligomer binding involves the C-terminal domain of apoE, with apoE3 reporting a much greater response through this conformational marker. Coupled with SPR binding measurements, apoE3 displays a higher affinity and capacity for the toxic Aβ oligomer. These findings support the hypothesis that apoE polymorphism and Alzheimer's risk can largely be attributed to the reduced ability of apoE4 to function as a clearance vehicle for the toxic form of Aβ. PMID:21069870

  4. New Phosphospecific Antibody Reveals Isoform-Specific Phosphorylation of CPEB3 Protein

    PubMed Central

    Sehgal, Kapil; Sylvester, Marc; Skubal, Magdalena; Josten, Michele; Steinhäuser, Christian; De Koninck, Paul; Theis, Martin

    2016-01-01

    Cytoplasmic Polyadenylation Element Binding proteins (CPEBs) are a family of polyadenylation factors interacting with 3’UTRs of mRNA and thereby regulating gene expression. Various functions of CPEBs in development, synaptic plasticity, and cellular senescence have been reported. Four CPEB family members of partially overlapping functions have been described to date, each containing a distinct alternatively spliced region. This region is highly conserved between CPEBs-2-4 and contains a putative phosphorylation consensus, overlapping with the exon seven of CPEB3. We previously found CPEBs-2-4 splice isoforms containing exon seven to be predominantly present in neurons, and the isoform expression pattern to be cell type-specific. Here, focusing on the alternatively spliced region of CPEB3, we determined that putative neuronal isoforms of CPEB3 are phosphorylated. Using a new phosphospecific antibody directed to the phosphorylation consensus we found Protein Kinase A and Calcium/Calmodulin-dependent Protein Kinase II to robustly phosphorylate CPEB3 in vitro and in primary hippocampal neurons. Interestingly, status epilepticus induced by systemic kainate injection in mice led to specific upregulation of the CPEB3 isoforms containing exon seven. Extensive analysis of CPEB3 phosphorylation in vitro revealed two other phosphorylation sites. In addition, we found plethora of potential kinases that might be targeting the alternatively spliced kinase consensus site of CPEB3. As this site is highly conserved between the CPEB family members, we suggest the existence of a splicing-based regulatory mechanism of CPEB function, and describe a robust phosphospecific antibody to study it in future. PMID:26915047

  5. Optical Tools To Study the Isoform-Specific Roles of Small GTPases in Immune Cells.

    PubMed

    Miskolci, Veronika; Wu, Bin; Moshfegh, Yasmin; Cox, Dianne; Hodgson, Louis

    2016-04-15

    Despite the 92% homology of the hematopoietic cell-specific Rac2 to the canonical isoform Rac1, these isoforms have been shown to play nonredundant roles in immune cells. To study isoform-specific dynamics of Rac in live cells, we developed a genetically encoded, single-chain FRET-based biosensor for Rac2. We also made significant improvements to our existing single-chain Rac1 biosensor. We optimized the biosensor constructs for facile expression in hematopoietic cells and performed functional validations in murine macrophage sublines of RAW264.7 cells. Rac2, Rac1, and Cdc42 have been implicated in the formation of actin-rich protrusions by macrophages, but their individual activation dynamics have not been previously characterized. We found that both Rac1 and Rac2 had similar activation kinetics, yet they had distinct spatial distributions in response to the exogenous stimulus, fMLF. Active Rac1 was mainly localized to the cell periphery, whereas active Rac2 was distributed throughout the cell, with an apparent higher concentration in the perinuclear region. We also performed an extensive morphodynamic analysis of Rac1, Rac2, and Cdc42 activities during the extension of random protrusions. We found that Rac2 appears to play a leading role in the generation of random protrusions, as we observed an initial strong activation of Rac2 in regions distal from the leading edge, followed by the activation of Rac1, a second burst of Rac2 and then Cdc42 immediately behind the leading edge. Overall, isoform-specific biosensors that have been optimized for expression should be valuable for interrogating the coordination of Rho family GTPase activities in living cells. PMID:26951800

  6. New Phosphospecific Antibody Reveals Isoform-Specific Phosphorylation of CPEB3 Protein.

    PubMed

    Kaczmarczyk, Lech; Labrie-Dion, Étienne; Sehgal, Kapil; Sylvester, Marc; Skubal, Magdalena; Josten, Michele; Steinhäuser, Christian; De Koninck, Paul; Theis, Martin

    2016-01-01

    Cytoplasmic Polyadenylation Element Binding proteins (CPEBs) are a family of polyadenylation factors interacting with 3'UTRs of mRNA and thereby regulating gene expression. Various functions of CPEBs in development, synaptic plasticity, and cellular senescence have been reported. Four CPEB family members of partially overlapping functions have been described to date, each containing a distinct alternatively spliced region. This region is highly conserved between CPEBs-2-4 and contains a putative phosphorylation consensus, overlapping with the exon seven of CPEB3. We previously found CPEBs-2-4 splice isoforms containing exon seven to be predominantly present in neurons, and the isoform expression pattern to be cell type-specific. Here, focusing on the alternatively spliced region of CPEB3, we determined that putative neuronal isoforms of CPEB3 are phosphorylated. Using a new phosphospecific antibody directed to the phosphorylation consensus we found Protein Kinase A and Calcium/Calmodulin-dependent Protein Kinase II to robustly phosphorylate CPEB3 in vitro and in primary hippocampal neurons. Interestingly, status epilepticus induced by systemic kainate injection in mice led to specific upregulation of the CPEB3 isoforms containing exon seven. Extensive analysis of CPEB3 phosphorylation in vitro revealed two other phosphorylation sites. In addition, we found plethora of potential kinases that might be targeting the alternatively spliced kinase consensus site of CPEB3. As this site is highly conserved between the CPEB family members, we suggest the existence of a splicing-based regulatory mechanism of CPEB function, and describe a robust phosphospecific antibody to study it in future. PMID:26915047

  7. Cyclin D1 splice variants: polymorphism, risk, and isoform specific regulation in prostate cancer

    PubMed Central

    Comstock, Clay E.S.; Augello, Michael A.; Benito, Ruth Pe; Karch, Jason; Tran, Thai H.; Utama, Fransiscus E.; Tindall, Elizabeth A.; Wang, Ying; Burd, Craig J.; Groh, Eric M.; Hoang, Hoa N.; Giles, Graham G.; Severi, Gianluca; Hayes, Vanessa M.; Henderson, Brian E.; Marchand, Loic Le; Kolonel, Laurence N.; Haiman, Christopher A.; Baffa, Raffaele; Gomella, Leonard G.; Knudsen, Erik S.; Rui, Hallgeir; Henshall, Susan M.; Sutherland, Robert L.; Knudsen, Karen E.

    2010-01-01

    Purpose Alternative CCND1 splicing results in cyclin D1b, which has specialized, pro-tumorigenic functions in prostate not shared by the cyclin D1a (full-length) isoform. Here, the frequency, tumor relevance, and mechanisms controlling cyclin D1b were challenged. Experimental Design First, relative expression of both cyclin D1 isoforms was determined in prostate adenocarcinomas. Second, relevance of the androgen axis was determined. Third, minigenes were created to interrogate the role of the G/A870 polymorphism (within the splice site), and findings validated in primary tissue. Fourth, impact of G/A870 on cancer risk was assessed in two large case-control studies. Results Cyclin D1b is induced in tumors, and a significant subset expressed this isoform in the absence of detectable cyclin D1a. Accordingly, the isoforms showed non-correlated expression patterns, and hormone status did not alter splicing. While G/A870 was not independently predictive of cancer risk, A870 predisposed for transcript-b production in cells and in normal prostate. The influence of A870 on overall transcript-b levels was relieved in tumors, indicating that aberrations in tumorigenesis likely alter the influence of the polymorphism. Conclusions These studies reveal that cyclin D1b is specifically elevated in prostate tumorigenesis. Cyclin D1b expression patterns are distinct from that observed with cyclin D1a. The A870 allele predisposes for transcript-b production in a context-specific manner. While A870 does not independently predict cancer risk, tumor cells can bypass the influence of the polymorphism. These findings have major implications for the analyses of D-cyclin function in the prostate, and provide the foundation for future studies directed at identifying potential modifiers of the G/A870 polymorphism. PMID:19706803

  8. Oleosin Isoforms of High and Low Molecular Weights Are Present in the Oil Bodies of Diverse Seed Species 1

    PubMed Central

    Tzen, Jason T. C.; Lai, Yiu-Kay; Chan, Kwai-Lan; Huang, Anthony H. C.

    1990-01-01

    Oleosins are unique and major proteins localized on the surface of oil bodies in diverse seed species. We purified five different oleosins (maize [Zea mays L.] KD 16 and KD 18, soybean [Glycine max L.] KD 18 and KD 24, and rapeseed [Brassica campestris L.] KD 20), and raised chicken antibodies against them. These antibodies were used to test for immunological cross-reactivity among oleosins from diverse seed species. Within the same seed species, antibodies raised against one oleosin isoform did not cross-react with the other oleosin isoform (i.e. between maize oleosins KD 16 and KD 18, and between soybean oleosins KD 18 and KD 24). However, the respective antibodies were able to recognize oleosins from other seed species. Where interspecies cross-reactivity occurred, the results suggest that there are at least two immunologically distinct isoforms of oleosins present in diverse seed species, one of lower Mr, and another one of higher Mr. This suggestion is also supported by the relative similarities between the amino acid sequence of a small portion of rapeseed oleosin KD 20 and those of maize oleosins KD 16 and KD 18. In maize kernel, there was a tissue-specific differential presentation of the three oleosins, KD 16, KD 18, and KD 19, in the oil-storing scutellum, embryonic axis, and aleurone layer. The phylogenetic relationship between the high and low Mr isoforms within the same, and among diverse, seed species is discussed. Images Figure 1 Figure 2 Figure 4 PMID:16667830

  9. A second isoform of the ferredoxin:NADP oxidoreductase generated by an in-frame initiation of translation

    PubMed Central

    Thomas, Jean-Claude; Ughy, Bettina; Lagoutte, Bernard; Ajlani, Ghada

    2006-01-01

    Ferredoxin:NADP oxidoreductases (FNRs) constitute a family of flavoenzymes that catalyze the exchange of reducing equivalents between one-electron carriers and the two-electron-carrying NADP(H). The main role of FNRs in cyanobacteria and leaf plastids is to provide the NADPH for photoautotrophic metabolism. In root plastids, a distinct FNR isoform is found that has been postulated to function in the opposite direction, providing electrons for nitrogen assimilation at the expense of NADPH generated by heterotrophic metabolism. A multiple gene family encodes FNR isoenzymes in plants, whereas there is only one FNR gene (petH) in cyanobacteria. Nevertheless, we detected two FNR isoforms in the cyanobacterium Synechocystis sp. strain PCC6803. One of them (FNRS ≈34 kDa) is similar in size to the plastid FNR and specifically accumulates under heterotrophic conditions, whereas the other one (FNRL ≈46 kDa) contains an extra N-terminal domain that allows its association with the phycobilisome. Site-directed mutants allowed us to conclude that the smaller isoform, FNRS, is produced from an internal ribosome entry site within the petH ORF. Thus we have uncovered a mechanism by which two isoforms are produced from a single gene, which is, to our knowledge, novel in photosynthetic bacteria. Our results strongly suggest that FNRL is an NADP+ reductase, whereas FNRS is an NADPH oxidase. PMID:17116880

  10. SPINK5, the defective gene in netherton syndrome, encodes multiple LEKTI isoforms derived from alternative pre-mRNA processing.

    PubMed

    Tartaglia-Polcini, Alessandro; Bonnart, Chrystelle; Micheloni, Alessia; Cianfarani, Francesca; Andrè, Alessandra; Zambruno, Giovanna; Hovnanian, Alain; D'Alessio, Marina

    2006-02-01

    The multidomain serine protease inhibitor lymphoepithelial Kazal-type related inhibitor (LEKTI) represents a key regulator of the proteolytic events occurring during epidermal barrier formation and hair development, as attested by the severe autosomal recessive ichthyosiform skin condition Netherton syndrome (NS) caused by mutations in its encoding gene, serine protease inhibitor Kazal-type 5 (SPINK5). Synthesized as a proprotein, LEKTI is rapidly cleaved intracellularly, thus generating a number of potentially bioactive fragments that are secreted. Here, we show that SPINK5 generates three classes of transcripts encoding three different LEKTI isoforms, which differ in their C-terminal portion. In addition to the previously described 15 domain isoform, SPINK5 encodes a shorter LEKTI isoform composed of only the first 13 domains, as well as a longer isoform carrying a 30-amino-acid residue insertion between the 13th and 14th inhibitory domains. We demonstrate that variable amounts of SPINK5 alternative transcripts are detected in all SPINK5 transcriptionally active tissues. Finally, we show that in differentiated cultured human keratinocytes all SPINK5 alternative transcripts are translated into protein and that the LEKTI precursors generate distinct secreted C-terminal proteolytic fragments from a similar cleavage site. Since several data indicate a biological role for the pro-LEKTI-cleaved polypeptides, we hypothesize that the alternative processing of the SPINK5 pre-messenger RNA represents an additional mechanism to further increase the structural and functional diversity of the LEKTI bioactive fragments. PMID:16374478

  11. Opposing roles of the two isoforms of ErbB3 binding protein 1 in human cancer cells.

    PubMed

    Ko, Hyo Rim; Chang, Yun Sil; Park, Won Soon; Ahn, Jee-Yin

    2016-09-15

    The different functions of the two isoforms of ErbB3 binding protein 1 (Ebp1), p48 and p42, have recently become the focus of interest as they reveal contradictory roles in cell growth promoting ability. The conformational change that crystal structure of p42 was shown to lack α helices at the amino-terminus present in p48 represents the differential binding partners and protein modifications of two Ebp1 isoforms. N-terminal specific phosphorylation by CDK2 and deregulation of the p53 tumor suppressor through specific interaction with HDM2 and Akt activation is postulated to contribute to p48-mediated tumorigenesis. The short isoform p42 Ebp1, which is actual binding partner of ErbB3 has been implicated as a tumor suppressor with many binding partners such as Rb, HDAC2, Sin3A and the p85 subunit of PI3K with HSP70/CHIP, inhibiting its own antiproliferative activity or inhibiting PI3K activity. The aim of the current review is to provide a summary on distinctive cellular functions of two Ebp1 proteins and their molecular partners that might be responsible for the unique functions of each isoform of Ebp1. PMID:27130196

  12. Two new isoforms of the human hepatoma-derived growth factor interact with components of the cytoskeleton.

    PubMed

    Nüße, Jessica; Mirastschijski, Ursula; Waespy, Mario; Oetjen, Janina; Brandes, Nadine; Rebello, Osmond; Paroni, Federico; Kelm, Sørge; Dietz, Frank

    2016-05-01

    Hepatoma-derived growth factor (HDGF) is involved in diverse, apparently unrelated processes, such as cell proliferation, apoptosis, DNA-repair, transcriptional control, ribosome biogenesis and cell migration. Most of the interactions of HDGF with diverse molecules has been assigned to the hath region of HDGF. In this study we describe two previously unknown HDGF isoforms, HDGF-B and HDGF-C, generated via alternative splicing with structurally unrelated N-terminal regions of their hath region, which is clearly different from the well described isoform, HDGF-A. In silico modeling revealed striking differences near the PHWP motif, an essential part of the binding site for glycosaminoglycans and DNA/RNA. This observation prompted the hypothesis that these isoforms would have distinct interaction patterns with correspondingly diverse roles on cellular processes. Indeed, we discovered specific associations of HDGF-B and HDGF-C with cytoskeleton elements, such as tubulin and dynein, suggesting previously unknown functions of HDGF in retrograde transport, site directed localization and/or cytoskeleton organization. In contrast, the main isoform HDGF-A does not interact directly with the cytoskeleton, but via RNA with messenger ribonucleoprotein (mRNP) complexes. In summary, the discovery of HDGF splice variants with their discrete binding activities and subcellular distributions opened new avenues for understanding its biological function and importance. PMID:26845719

  13. Isoform-Specific Up-Regulation of Plasma Membrane Ca2+ATPase Expression During Colon and Gastric Cancer Cell Differentiation

    PubMed Central

    Ribiczey, Polett; Tordai, Attila; Andrikovics, Hajnalka; Filoteo, Adelaida G.; Penniston, John T.; Enouf, Jocelyne; Enyedi, Ágnes; Papp, Béla; Kovács, Tünde

    2007-01-01

    Summary In this work we demonstrate a differentiation-induced up-regulation of the expression of plasma membrane Ca2+ATPase (PMCA) isoforms being present in various gastric/colon cancer cell types. We found PMCA1b as the major isoform in non-differentiated cancer cell lines, whereas the expression level of PMCA4b was significantly lower. Cell differentiation initiated with short chain fatty acids (SCFAs) and trichostatin A, or spontaneous differentiation of post-confluent cell cultures resulted in a marked induction of PMCA4b expression, while only moderately increased PMCA1b levels. Up-regulation of PMCA4b expression was demonstrated both at the protein and mRNA levels, and closely correlated with the induction of established differentiation markers. In contrast, the expression level of the Na+/K+-ATPase or that of the sarco/endoplasmic reticulum Ca2+ATPase 2 protein did not change significantly under these conditions. In membrane vesicles obtained from SCFA-treated gastric/colon cancer cells a marked increase in the PMCA-dependent Ca2+ transport activity was observed, indicating a general increase of PMCA function during the differentiation of these cancer cells. Because various PMCA isoforms display distinct functional characteristics, we suggest that up-regulated PMCA expression, together with a major switch in PMCA isoform pattern may significantly contribute to the differentiation of gastric/colon cancer cells. The analysis of PMCA expression may provide a new diagnostic tool for monitoring the tumor phenotype. PMID:17433436

  14. Molecular cloning of a putative tetrodotoxin-resistant rat heart Na+ channel isoform.

    PubMed Central

    Rogart, R B; Cribbs, L L; Muglia, L K; Kephart, D D; Kaiser, M W

    1989-01-01

    Voltage-gated Na+ channels in mammalian heart differ from those in nerve and skeletal muscle. One major difference is that tetrodotoxin (TTX)-resistant cardiac Na+ channels are blocked by 1-10 microM TTX, whereas TTX-sensitive nerve Na+ channels are blocked by nanomolar TTX concentrations. We constructed a cDNA library from 6-day-old rat hearts, where only low-affinity [3H]saxitoxin receptors, corresponding to TTX-resistant Na+ channels, were detected. We isolated several overlapping cDNA clones encompassing 7542 nucleotides and encoding the entire alpha subunit of a cardiac-specific Na+ channel isoform (designated rat heart I) as well as several rat brain I Na+ channel cDNA clones. The derived amino acid sequence of rat heart I was highly homologous to, but distinct from, previous Na+ channel clones. RNase protection studies showed that the corresponding mRNA species is abundant in newborn and adult rat hearts, but not detectable in brain or innervated skeletal muscle. The same mRNA species appears upon denervation of skeletal muscle, likely accounting for expression of new TTX-resistant Na+ channels. Thus, this cardiac-specific Na+ channel clone appears to encode a distinct TTX-resistant isoform and is another member of the mammalian Na+ channel multigene family, found in newborn heart and denervated skeletal muscles. Images PMID:2554302

  15. Versican isoform V1 regulates proliferation and migration in high-grade gliomas.

    PubMed

    Onken, Julia; Moeckel, Sylvia; Leukel, Petra; Leidgens, Verena; Baumann, Fusun; Bogdahn, Ulrich; Vollmann-Zwerenz, Arabel; Hau, Peter

    2014-10-01

    Versican is a large chondroitin sulphate proteoglycan produced by several tumor cell types, including high-grade gliomas. Increased expression of distinct versican isoforms in the extracellular matrix plays a role in tumor cell growth, adhesion and migration. We have recently shown that transforming growth factor (TGF-beta)2, an important modulator of glioma invasion, interacts with versican isoforms V0/V1 during malignant progression of glioma in vitro. However, the distinct subtype of versican that modulates these effects could not be specified. Here, we show that transient down-regulation of V1 by siRNA leads to a significant reduction of proliferation and migration in glioblastoma cell lines and glioblastoma progenitor cells, whereas tumor cell attachment stays unaffected. We conclude that V1 plays a predominant role in modulating central pathophysiological mechanisms as proliferation and migration in glioblastoma. Considering that TGF-beta is a master regulator of glioma pathophysiology, and that V0/1 is induced by TGF-beta2, therapeutic regulation of V1 may induce meaningful effects on glioma cell migration not only in vitro, but also in vivo. PMID:25064688

  16. Diverse functions of myosin VI elucidated by an isoform-specific α-helix domain.

    PubMed

    Wollscheid, Hans-Peter; Biancospino, Matteo; He, Fahu; Magistrati, Elisa; Molteni, Erika; Lupia, Michela; Soffientini, Paolo; Rottner, Klemens; Cavallaro, Ugo; Pozzoli, Uberto; Mapelli, Marina; Walters, Kylie J; Polo, Simona

    2016-04-01

    Myosin VI functions in endocytosis and cell motility. Alternative splicing of myosin VI mRNA generates two distinct isoform types, myosin VIshort and myosin VIlong, which differ in the C-terminal region. Their physiological and pathological roles remain unknown. Here we identified an isoform-specific regulatory helix, named the α2-linker, that defines specific conformations and hence determines the target selectivity of human myosin VI. The presence of the α2-linker structurally defines a new clathrin-binding domain that is unique to myosin VIlong and masks the known RRL interaction motif. This finding is relevant to ovarian cancer, in which alternative myosin VI splicing is aberrantly regulated, and exon skipping dictates cell addiction to myosin VIshort in tumor-cell migration. The RRL interactor optineurin contributes to this process by selectively binding myosin VIshort. Thus, the α2-linker acts like a molecular switch that assigns myosin VI to distinct endocytic (myosin VIlong) or migratory (myosin VIshort) functional roles. PMID:26950368

  17. Neutralization of vascular endothelial growth factor antiangiogenic isoforms or administration of proangiogenic isoforms stimulates vascular development in the rat testis.

    PubMed

    Baltes-Breitwisch, Michelle M; Artac, Robin A; Bott, Rebecca C; McFee, Renee M; Kerl, Jill G; Clopton, Debra T; Cupp, Andrea S

    2010-08-01

    Vascular endothelial growth factor A (VEGFA) plays a role in both angiogenesis and seminiferous cord formation, and alternative splicing of the Vegfa gene produces both proangiogenic isoforms and antiangiogenic isoforms (B-isoforms). The objectives of this study were to evaluate the expression of pro- and antiangiogenic isoforms during testis development and to determine the role of VEGFA isoforms in testis morphogenesis. Quantitative RT-PCR determined that Vegfa_165b mRNA was most abundant between embryonic days 13.5 and 16 (E13.5 and 16; P<0.05). Compared with ovarian mRNA levels, Vegfa_120 was more abundant at E13-14 (P<0.05), Vegfa_164 was less abundant at E13 (P<0.05), and Vegfa_165b tended to be less abundant at E13 (P<0.09) in testes. Immunohistochemical staining localized antiangiogenic isoforms to subsets of germ cells at E14-16, and western blot analysis revealed similar protein levels for VEGFA_165B, VEGFA_189B, and VEGFA_206B at this time point. Treatment of E13 organ culture testes with VEGFA_120, VEGFA_164, and an antibody to antiangiogenic isoforms (anti-VEGFAxxxB) resulted in less organized and defined seminiferous cords compared with paired controls. In addition, 50 ng/ml VEGFA_120 and VEGFA_164 treatments increased vascular density in cultured testes by 60 and 48% respectively, and treatment with VEGFAxxxB antibody increased vascular density by 76% in testes (0.5 ng/ml) and 81% in ovaries (5 ng/ml) compared with controls (P<0.05). In conclusion, both pro- and antiangiogenic VEGFA isoforms are involved in the development of vasculature and seminiferous cords in rat testes, and differential expression of these isoforms may be important for normal gonadal development. PMID:20457593

  18. EASI--enrichment of alternatively spliced isoforms.

    PubMed

    Venables, Julian P; Burn, John

    2006-01-01

    Alternative splicing produces more than one protein from the majority of genes and the rarer forms can have dominant functions. Instability of alternative transcripts can also hinder the study of regulation of gene expression by alternative splicing. To investigate the true extent of alternative splicing we have developed a simple method of enriching alternatively spliced isoforms (EASI) from PCRs using beads charged with Thermus aquaticus single-stranded DNA-binding protein (T.Aq ssb). This directly purifies the single-stranded regions of heteroduplexes between alternative splices formed in the PCR, enabling direct sequencing of all the rare alternative splice forms of any gene. As a proof of principle the alternative transcripts of three tumour suppressor genes, TP53, MLH1 and MSH2, were isolated from testis cDNA. These contain missing exons, cryptic splice sites or include completely novel exons. EASI beads are stable for months in the fridge and can be easily combined with standard protocols to speed the cloning of novel transcripts. PMID:16951290

  19. p53 Isoforms: Key Regulators of the Cell Fate Decision.

    PubMed

    Joruiz, Sebastien M; Bourdon, Jean-Christophe

    2016-01-01

    It is poorly understood how a single protein, p53, can be responsive to so many stress signals and orchestrates very diverse cell responses to maintain/restore cell/tissue functions. The uncovering that TP53 gene physiologically expresses, in a tissue-dependent manner, several p53 splice variants (isoforms) provides an explanation to its pleiotropic biological activities. Here, we summarize a decade of research on p53 isoforms. The clinical studies and the diverse cellular and animal models of p53 isoforms (zebrafish, Drosophila, and mouse) lead us to realize that a p53-mediated cell response is, in fact, the sum of the intrinsic activities of the coexpressed p53 isoforms and that unbalancing expression of different p53 isoforms leads to cancer, premature aging, (neuro)degenerative diseases, inflammation, embryo malformations, or defects in tissue regeneration. Cracking the p53 isoforms' code is, thus, a necessary step to improve cancer treatment. It also opens new exciting perspectives in tissue regeneration. PMID:26801896

  20. Frac-seq reveals isoform-specific recruitment to polyribosomes

    PubMed Central

    Sterne-Weiler, Timothy; Martinez-Nunez, Rocio Teresa; Howard, Jonathan M.; Cvitovik, Ivan; Katzman, Sol; Tariq, Muhammad A.; Pourmand, Nader; Sanford, Jeremy R.

    2013-01-01

    Pre-mRNA splicing is required for the accurate expression of virtually all human protein coding genes. However, splicing also plays important roles in coordinating subsequent steps of pre-mRNA processing such as polyadenylation and mRNA export. Here, we test the hypothesis that nuclear pre-mRNA processing influences the polyribosome association of alternative mRNA isoforms. By comparing isoform ratios in cytoplasmic and polyribosomal extracts, we determined that the alternative products of ∼30% (597/1954) of mRNA processing events are differentially partitioned between these subcellular fractions. Many of the events exhibiting isoform-specific polyribosome association are highly conserved across mammalian genomes, underscoring their possible biological importance. We find that differences in polyribosome association may be explained, at least in part by the observation that alternative splicing alters the cis-regulatory landscape of mRNAs isoforms. For example, inclusion or exclusion of upstream open reading frames (uORFs) in the 5′UTR as well as Alu-elements and microRNA target sites in the 3′UTR have a strong influence on polyribosome association of alternative mRNA isoforms. Taken together, our data demonstrate for the first time the potential link between alternative splicing and translational control of the resultant mRNA isoforms. PMID:23783272

  1. A Network of Splice Isoforms for the Mouse.

    PubMed

    Li, Hong-Dong; Menon, Rajasree; Eksi, Ridvan; Guerler, Aysam; Zhang, Yang; Omenn, Gilbert S; Guan, Yuanfang

    2016-01-01

    The laboratory mouse is the primary mammalian species used for studying alternative splicing events. Recent studies have generated computational models to predict functions for splice isoforms in the mouse. However, the functional relationship network, describing the probability of splice isoforms participating in the same biological process or pathway, has not yet been studied in the mouse. Here we describe a rich genome-wide resource of mouse networks at the isoform level, which was generated using a unique framework that was originally developed to infer isoform functions. This network was built through integrating heterogeneous genomic and protein data, including RNA-seq, exon array, protein docking and pseudo-amino acid composition. Through simulation and cross-validation studies, we demonstrated the accuracy of the algorithm in predicting isoform-level functional relationships. We showed that this network enables the users to reveal functional differences of the isoforms of the same gene, as illustrated by literature evidence with Anxa6 (annexin a6) as an example. We expect this work will become a useful resource for the mouse genetics community to understand gene functions. The network is publicly available at: http://guanlab.ccmb.med.umich.edu/isoformnetwork. PMID:27079421

  2. Isoform dependent regulation of human HCN channels by cholesterol

    PubMed Central

    Fürst, Oliver; D’Avanzo, Nazzareno

    2015-01-01

    Cholesterol has been shown to regulate numerous ion channels. HCN channels represent the molecular correlate of If or Ih in sinoatrial node (SAN) and neuronal cells. Previous studies have implicated a role for cholesterol in the regulation of rabbit HCN4 channels with effects on pacing in the rabbit SAN. Using electrophysiological and biochemical approaches, we examined the effect of cholesterol modulation on human HCN1, HCN2 and HCN4 isoforms. Patch-clamp experiments uncovered isoform specific differences in the effect of cholesterol on gating kinetics upon depletion by MβCD or mevastatin or enrichment using MβCD/cholesterol. Most dramatically cholesterol had isoform specific effects on mode-shifting, which has been suggested to play a key role in stabilizing firing rate and preventing arrhythmic firing in SAN cells and neurons. Mode-shifting in HCN1 channels was insensitive to cholesterol manipulation, while HCN2 and HCN4 were strongly affected. Trafficking of each isoform to the plasma membrane was also affected by cholesterol modulation differentially between isoforms, however, each isoform remained localized in lipid raft domains after cholesterol depletion. These effects may contribute to the side effects of cholesterol reducing therapies including disrupted heart rhythm and neuropathic pain, as well as the susceptibility of sinus dysfunction in patients with elevated cholesterol. PMID:26404789

  3. A Network of Splice Isoforms for the Mouse

    PubMed Central

    Li, Hong-Dong; Menon, Rajasree; Eksi, Ridvan; Guerler, Aysam; Zhang, Yang; Omenn, Gilbert S.; Guan, Yuanfang

    2016-01-01

    The laboratory mouse is the primary mammalian species used for studying alternative splicing events. Recent studies have generated computational models to predict functions for splice isoforms in the mouse. However, the functional relationship network, describing the probability of splice isoforms participating in the same biological process or pathway, has not yet been studied in the mouse. Here we describe a rich genome-wide resource of mouse networks at the isoform level, which was generated using a unique framework that was originally developed to infer isoform functions. This network was built through integrating heterogeneous genomic and protein data, including RNA-seq, exon array, protein docking and pseudo-amino acid composition. Through simulation and cross-validation studies, we demonstrated the accuracy of the algorithm in predicting isoform-level functional relationships. We showed that this network enables the users to reveal functional differences of the isoforms of the same gene, as illustrated by literature evidence with Anxa6 (annexin a6) as an example. We expect this work will become a useful resource for the mouse genetics community to understand gene functions. The network is publicly available at: http://guanlab.ccmb.med.umich.edu/isoformnetwork. PMID:27079421

  4. Asf1b, the necessary Asf1 isoform for proliferation, is predictive of outcome in breast cancer

    PubMed Central

    Corpet, Armelle; De Koning, Leanne; Toedling, Joern; Savignoni, Alexia; Berger, Frédérique; Lemaître, Charlène; O'Sullivan, Roderick J; Karlseder, Jan; Barillot, Emmanuel; Asselain, Bernard; Sastre-Garau, Xavier; Almouzni, Geneviève

    2011-01-01

    Mammalian cells possess two isoforms of the histone H3–H4 chaperone anti-silencing function 1 (Asf1), Asf1a and Asf1b. However to date, whether they have individual physiological roles has remained elusive. Here, we aim to elucidate the functional importance of Asf1 isoforms concerning both basic and applied aspects. First, we reveal a specific proliferation-dependent expression of human Asf1b unparalleled by Asf1a. Strikingly, in cultured cells, both mRNA and protein corresponding to Asf1b decrease upon cell cycle exit. Depletion of Asf1b severely compromises proliferation, leads to aberrant nuclear structures and a distinct transcriptional signature. Second, a major physiological implication is found in the applied context of tissue samples derived from early stage breast tumours in which we examined Asf1a/b levels. We reveal that overexpression of Asf1b mRNA correlate with clinical data and disease outcome. Together, our results highlight a distribution of tasks between the distinct Asf1 isoforms, which emphasizes a specialized function of Asf1b required for proliferation capacity. We discuss the implications of these results for breast cancer diagnosis and prognosis. PMID:21179005

  5. Asf1b, the necessary Asf1 isoform for proliferation, is predictive of outcome in breast cancer.

    PubMed

    Corpet, Armelle; De Koning, Leanne; Toedling, Joern; Savignoni, Alexia; Berger, Frédérique; Lemaître, Charlène; O'Sullivan, Roderick J; Karlseder, Jan; Barillot, Emmanuel; Asselain, Bernard; Sastre-Garau, Xavier; Almouzni, Geneviève

    2011-02-01

    Mammalian cells possess two isoforms of the histone H3-H4 chaperone anti-silencing function 1 (Asf1), Asf1a and Asf1b. However to date, whether they have individual physiological roles has remained elusive. Here, we aim to elucidate the functional importance of Asf1 isoforms concerning both basic and applied aspects. First, we reveal a specific proliferation-dependent expression of human Asf1b unparalleled by Asf1a. Strikingly, in cultured cells, both mRNA and protein corresponding to Asf1b decrease upon cell cycle exit. Depletion of Asf1b severely compromises proliferation, leads to aberrant nuclear structures and a distinct transcriptional signature. Second, a major physiological implication is found in the applied context of tissue samples derived from early stage breast tumours in which we examined Asf1a/b levels. We reveal that overexpression of Asf1b mRNA correlate with clinical data and disease outcome. Together, our results highlight a distribution of tasks between the distinct Asf1 isoforms, which emphasizes a specialized function of Asf1b required for proliferation capacity. We discuss the implications of these results for breast cancer diagnosis and prognosis. PMID:21179005

  6. Multiple isoform recovery (MIR)-PCR: a simple method for the isolation of related mRNA isoforms.

    PubMed Central

    Fagotti, A; Gabbiani, G; Pascolini, R; Neuville, P

    1998-01-01

    We present a rapid and efficient method for the detection of related transcripts with different expression levels. This approach combines the rapid amplification of cDNA ends (RACE) method with a cDNA subtractive technique. The strategy is based on successive subtractions of prevalent isoforms resulting in enrichment of less expressed transcripts. For each subtraction, a biotinylated primer specific for the prevalent isoform is hybridized on the total cDNA and the hybrid is retained on a streptavidin affinity column. The unbound cDNA serves as a template for subsequent isoform identification. To illustrate its application we describe the isolation of three new actin cDNA isoforms in the freshwater planarian Dugesia (S) polychroa. PMID:9518500

  7. Non-Muscle Myosin II Isoforms Have Different Functions in Matrix Rearrangement by MDA-MB-231 Cells

    PubMed Central

    Hindman, Bridget; Goeckeler, Zoe; Sierros, Kostas; Wysolmerski, Robert

    2015-01-01

    The role of a stiffening extra-cellular matrix (ECM) in cancer progression is documented but poorly understood. Here we use a conditioning protocol to test the role of nonmuscle myosin II isoforms in cell mediated ECM arrangement using collagen constructs seeded with breast cancer cells expressing shRNA targeted to either the IIA or IIB heavy chain isoform. While there are several methods available to measure changes in the biophysical characteristics of the ECM, we wanted to use a method which allows for the measurement of global stiffness changes as well as a dynamic response from the sample over time. The conditioning protocol used allows the direct measurement of ECM stiffness. Using various treatments, it is possible to determine the contribution of various construct and cellular components to the overall construct stiffness. Using this assay, we show that both the IIA and IIB isoforms are necessary for efficient matrix remodeling by MDA-MB-231 breast cancer cells, as loss of either isoform changes the stiffness of the collagen constructs as measured using our conditioning protocol. Constructs containing only collagen had an elastic modulus of 0.40 Pascals (Pa), parental MDA-MB-231 constructs had an elastic modulus of 9.22 Pa, while IIA and IIB KD constructs had moduli of 3.42 and 7.20 Pa, respectively. We also calculated the cell and matrix contributions to the overall sample elastic modulus. Loss of either myosin isoform resulted in decreased cell stiffness, as well as a decrease in the stiffness of the cell-altered collagen matrices. While the total construct modulus for the IIB KD cells was lower than that of the parental cells, the IIB KD cell-altered matrices actually had a higher elastic modulus than the parental cell-altered matrices (4.73 versus 4.38 Pa). These results indicate that the IIA and IIB heavy chains play distinct and non-redundant roles in matrix remodeling. PMID:26136073

  8. Managing Brain Extracellular K+ during Neuronal Activity: The Physiological Role of the Na+/K+-ATPase Subunit Isoforms

    PubMed Central

    Larsen, Brian Roland; Stoica, Anca; MacAulay, Nanna

    2016-01-01

    During neuronal activity in the brain, extracellular K+ rises and is subsequently removed to prevent a widespread depolarization. One of the key players in regulating extracellular K+ is the Na+/K+-ATPase, although the relative involvement and physiological impact of the different subunit isoform compositions of the Na+/K+-ATPase remain unresolved. The various cell types in the brain serve a certain temporal contribution in the face of network activity; astrocytes respond directly to the immediate release of K+ from neurons, whereas the neurons themselves become the primary K+ absorbers as activity ends. The kinetic characteristics of the catalytic α subunit isoforms of the Na+/K+-ATPase are, partly, determined by the accessory β subunit with which they combine. The isoform combinations expressed by astrocytes and neurons, respectively, appear to be in line with the kinetic characteristics required to fulfill their distinct physiological roles in clearance of K+ from the extracellular space in the face of neuronal activity. Understanding the nature, impact and effects of the various Na+/K+-ATPase isoform combinations in K+ management in the central nervous system might reveal insights into pathological conditions such as epilepsy, migraine, and spreading depolarization following cerebral ischemia. In addition, particular neurological diseases occur as a result of mutations in the α2- (familial hemiplegic migraine type 2) and α3 isoforms (rapid-onset dystonia parkinsonism/alternating hemiplegia of childhood). This review addresses aspects of the Na+/K+-ATPase in the regulation of extracellular K+ in the central nervous system as well as the related pathophysiology. Understanding the physiological setting in non-pathological tissue would provide a better understanding of the pathological events occurring during disease. PMID:27148079

  9. Managing Brain Extracellular K(+) during Neuronal Activity: The Physiological Role of the Na(+)/K(+)-ATPase Subunit Isoforms.

    PubMed

    Larsen, Brian Roland; Stoica, Anca; MacAulay, Nanna

    2016-01-01

    During neuronal activity in the brain, extracellular K(+) rises and is subsequently removed to prevent a widespread depolarization. One of the key players in regulating extracellular K(+) is the Na(+)/K(+)-ATPase, although the relative involvement and physiological impact of the different subunit isoform compositions of the Na(+)/K(+)-ATPase remain unresolved. The various cell types in the brain serve a certain temporal contribution in the face of network activity; astrocytes respond directly to the immediate release of K(+) from neurons, whereas the neurons themselves become the primary K(+) absorbers as activity ends. The kinetic characteristics of the catalytic α subunit isoforms of the Na(+)/K(+)-ATPase are, partly, determined by the accessory β subunit with which they combine. The isoform combinations expressed by astrocytes and neurons, respectively, appear to be in line with the kinetic characteristics required to fulfill their distinct physiological roles in clearance of K(+) from the extracellular space in the face of neuronal activity. Understanding the nature, impact and effects of the various Na(+)/K(+)-ATPase isoform combinations in K(+) management in the central nervous system might reveal insights into pathological conditions such as epilepsy, migraine, and spreading depolarization following cerebral ischemia. In addition, particular neurological diseases occur as a result of mutations in the α2- (familial hemiplegic migraine type 2) and α3 isoforms (rapid-onset dystonia parkinsonism/alternating hemiplegia of childhood). This review addresses aspects of the Na(+)/K(+)-ATPase in the regulation of extracellular K(+) in the central nervous system as well as the related pathophysiology. Understanding the physiological setting in non-pathological tissue would provide a better understanding of the pathological events occurring during disease. PMID:27148079

  10. All Akt Isoforms (Akt1, Akt2, Akt3) Are Involved in Normal Hearing, but Only Akt2 and Akt3 Are Involved in Auditory Hair Cell Survival in the Mammalian Inner Ear

    PubMed Central

    Brand, Yves; Levano, Soledad; Radojevic, Vesna; Naldi, Arianne Monge; Setz, Cristian; Ryan, Allen F.; Pak, Kwang; Hemmings, Brian A.; Bodmer, Daniel

    2015-01-01

    The kinase Akt is a key downstream mediator of the phosphoinositide-3-kinase signaling pathway and participates in a variety of cellular processes. Akt comprises three isoforms each encoded by a separate gene. There is evidence to indicate that Akt is involved in the survival and protection of auditory hair cells in vitro. However, little is known about the physiological role of Akt in the inner ear—especially in the intact animal. To elucidate this issue, we first analyzed the mRNA expression of the three Akt isoforms in the inner ear of C57/BL6 mice by real-time PCR. Next, we tested the susceptibility to gentamicin-induced auditory hair cell loss in isoform-specific Akt knockout mice compared to wild-types (C57/BL6) in vitro. To analyze the effect of gene deletion in vivo, hearing and cochlear microanatomy were evaluated in Akt isoform knockout animals. In this study, we found that all three Akt isoforms are expressed in the cochlea. Our results further indicate that Akt2 and Akt3 enhance hair cell resistance to ototoxicity, while Akt1 does not. Finally, we determined that untreated Akt1 and Akt2/Akt3 double knockout mice display significant hearing loss, indicating a role for these isoforms in normal hearing. Taken together, our results indicate that each of the Akt isoforms plays a distinct role in the mammalian inner ear. PMID:25811375

  11. The SAP motif and C-terminal RS- and RD/E-rich region influences the sub-nuclear localization of Acinus isoforms.

    PubMed

    Wang, Fang; Wendling, Karen S; Soprano, Kenneth J; Soprano, Dianne Robert

    2014-12-01

    Acinus has been reported to function in apoptosis, RNA processing and regulation of gene transcription including RA-dependent transcription. There are three different isoforms of Acinus termed Acinus-L, Acinus-S', and Acinus-S. The isoforms of Acinus differ in their N-terminus while the C-terminus is consistent in all isoforms. The sub-nuclear localization of Acinus-L and Acinus-S' was determined using fluorescence microscopy. Acinus-S' colocalizes with SC35 in nuclear speckles while Acinus-L localizes diffusely throughout the nucleoplasm. RA treatment has little effect on the sub-nuclear localization of Acinus-L and Acinus-S'. The domains/regions necessary for the distinct sub-nuclear localization of Acinus-L and Acinus-S' were identified. The speckled sub-nuclear localization of Acinus-S' is dependent on its C-terminal RS- and RD/E-rich region but is independent of the phosphorylation status of Ser-453 and Ser-604 within this region. The unique N-terminal SAP motif of Acinus-L is responsible for its diffuse localization in the nucleus. Moreover, the sub-nuclear localization of Acinus isoforms is affected by each other, which is determined by the combinatorial effect of the more potent SAP motif of Acinus-L and the C-terminal RS- and RD/E-rich region in all Acinus isoforms. The C-terminal RS- and RD/E-rich region of Acinus mediates the colocalization of Acinus isoforms as well as with its interacting protein RNPS1. In conclusion, the SAP motif is responsible for the difference in the nuclear localization between Acinus-L and Acinus-S'. This difference in the nuclear localization of Acinus-S' and Acinus-L may suggest that these two isoforms have different functional roles. PMID:25079509

  12. Wnt isoform-specific interactions with coreceptor specify inhibition or potentiation of signaling by LRP6 antibodies.

    PubMed

    Gong, Yan; Bourhis, Eric; Chiu, Cecilia; Stawicki, Scott; DeAlmeida, Venita I; Liu, Bob Y; Phamluong, Khanhky; Cao, Tim C; Carano, Richard A D; Ernst, James A; Solloway, Mark; Rubinfeld, Bonnee; Hannoush, Rami N; Wu, Yan; Polakis, Paul; Costa, Mike

    2010-01-01

    β-Catenin-dependent Wnt signaling is initiated as Wnt binds to both the receptor FZD and coreceptor LRP5/6, which then assembles a multimeric complex at the cytoplasmic membrane face to recruit and inactivate the kinase GSK3. The large number and sequence diversity of Wnt isoforms suggest the possibility of domain-specific ligand-coreceptor interactions, and distinct binding sites on LRP6 for Wnt3a and Wnt9b have recently been identified in vitro. Whether mechanistically different interactions between Wnts and coreceptors might mediate signaling remains to be determined. It is also not clear whether coreceptor homodimerization induced extracellularly can activate Wnt signaling, as is the case for receptor tyrosine kinases. We generated monoclonal antibodies against LRP6 with the unexpected ability to inhibit signaling by some Wnt isoforms and potentiate signaling by other isoforms. In cell culture, two antibodies characterized further show reciprocal activities on most Wnts, with one antibody antagonizing and the other potentiating. We demonstrate that these antibodies bind to different regions of LRP6 protein, and inhibition of signaling results from blocking Wnt binding. Antibody-mediated dimerization of LRP6 can potentiate signaling only when a Wnt isoform is also able to bind the complex, presumably recruiting FZD. Endogenous autocrine Wnt signaling in different tumor cell lines can be either antagonized or enhanced by the LRP6 antibodies, indicating expression of different Wnt isoforms. As anticipated from the roles of Wnt signaling in cancer and bone development, antibody activities can also be observed in mice for inhibition of tumor growth and in organ culture for enhancement of bone mineral density. Collectively, our results indicate that separate binding sites for different subsets of Wnt isoforms determine the inhibition or potentiation of signaling conferred by LRP6 antibodies. This complexity of coreceptor-ligand interactions may allow for

  13. Rice PROTEIN l-ISOASPARTYL METHYLTRANSFERASE isoforms differentially accumulate during seed maturation to restrict deleterious isoAsp and reactive oxygen species accumulation and are implicated in seed vigor and longevity.

    PubMed

    Petla, Bhanu Prakash; Kamble, Nitin Uttam; Kumar, Meenu; Verma, Pooja; Ghosh, Shraboni; Singh, Ajeet; Rao, Venkateswara; Salvi, Prafull; Kaur, Harmeet; Saxena, Saurabh Chandra; Majee, Manoj

    2016-07-01

    PROTEIN l-ISOASPARTYL O-METHYLTRANSFERASE (PIMT) is a protein-repairing enzyme involved in seed vigor and longevity. However, the regulation of PIMT isoforms during seed development and the mechanism of PIMT-mediated improvement of seed vigor and longevity are largely unknown. In this study in rice (Oryza sativa), we demonstrate the dynamics and correlation of isoaspartyl (isoAsp)-repairing demands and PIMT activity, and their implications, during seed development, germination and aging, through biochemical, molecular and genetic studies. Molecular and biochemical analyses revealed that rice possesses various biochemically active and inactive PIMT isoforms. Transcript and western blot analyses clearly showed the seed development stage and tissue-specific accumulation of active isoforms. Immunolocalization studies revealed distinct isoform expression in embryo and aleurone layers. Further analyses of transgenic lines for each OsPIMT isoform revealed a clear role in the restriction of deleterious isoAsp and age-induced reactive oxygen species (ROS) accumulation to improve seed vigor and longevity. Collectively, our data suggest that a PIMT-mediated, protein repair mechanism is initiated during seed development in rice, with each isoform playing a distinct, yet coordinated, role. Our results also raise the intriguing possibility that PIMT repairs antioxidative enzymes and proteins which restrict ROS accumulation, lipid peroxidation, etc. in seed, particularly during aging, thus contributing to seed vigor and longevity. PMID:26987457

  14. Isoforms of endoplasmic reticulum Ca(2+)-ATPase are differentially expressed in normal and diabetic islets of Langerhans.

    PubMed Central

    Váradi, A; Molnár, E; Ostenson, C G; Ashcroft, S J

    1996-01-01

    Glucose-dependent sequestration of Ca2+ into endoplasmic reticulum and its subsequent release play an important role in the control of intracellular Ca2+ concentration, which regulates insulin secretion in pancreatic beta-cells. The active uptake of cytosolic Ca2+ into endoplasmic reticulum is mediated by sarco-(endo)plasmic reticulum Ca(2+)-ATPases (SERCAs). We found, using RT-PCR with isoform-specific primers, that SERCA 2 and SERCA 3 mRNAs are co-expressed in human and rat islets of Langerhans and in the RINm5F beta-cell line. Immunochemical analysis also revealed the existence of two SERCA proteins with molecular masses of 110 and 115 kDa in beta-cell membranes. The 115 kDa protein was identified as SERCA 2b by its reaction with an isoform-specific antibody and the 110 kDa protein most probably corresponds to SERCA 3. The presence of two functionally different SERCA isoforms raises the possibility that they are located in distinct Ca2+ stores. There is evidence that altered Ca2+ handling in the beta-cell may contribute to the decreased insulin secretion seen in non-insulin dependent diabetes mellitus (NIDDM). We therefore investigated SERCA 2 and SERCA 3 mRNA expression by quantitative RT-PCR in islets prepared from Goto-Kakizaki (GK) rats, a non-obese spontaneous model of NIDDM. We found a significant reduction (about 68%) in SERCA 3 isoform expression. Since SERCA 2 expression was not significantly reduced, these genes are independently regulated and probably play distinct roles in islets of Langerhans. The marked decrease of SERCA 3 expression may constitute a defect in Ca2+ signalling in GK rat islets which could be a component of NIDDM. PMID:8912690

  15. Microgravity modifies protein kinase C isoform translocation in the human monocytic cell line U937 and human peripheral blood T-cells

    NASA Technical Reports Server (NTRS)

    Hatton, Jason P.; Gaubert, Francois; Cazenave, Jean-Pierre; Schmitt, Didier; Hashemi, B. B. (Principal Investigator); Hughes-Fulford, M. (Principal Investigator)

    2002-01-01

    Individual protein kinase C (PKC) isoforms fulfill distinct roles in the regulation of the commitment to differentiation, cell cycle arrest, and apoptosis in both monocytes and T-cells. The human monocyte like cell line U937 and T-cells were exposed to microgravity, during spaceflight and the translocation (a critical step in PKC signaling) of individual isoforms to cell particulate fraction examined. PKC activating phorbol esters induced a rapid translocation of several PKC isoforms to the particulate fraction of U937 monocytes under terrestrial gravity (1 g) conditions in the laboratory. In microgravity, the translocation of PKC beta II, delta, and epsilon in response to phorbol esters was reduced in microgravity compared to 1 g, but was enhanced in weak hypergravity (1.4 g). All isoforms showed a net increase in particulate PKC following phorbol ester stimulation, except PKC delta which showed a net decrease in microgravity. In T-cells, phorbol ester induced translocation of PKC delta was reduced in microgravity, compared to 1 g, while PKC beta II translocation was not significantly different at the two g-levels. These data show that microgravity differentially alters the translocation of individual PKC isoforms in monocytes and T-cells, thus providing a partial explanation for the modifications previously observed in the activation of these cell types under microgravity.

  16. Differential induction of FosB isoforms throughout the brain by fluoxetine and chronic stress.

    PubMed

    Vialou, Vincent; Thibault, Mackenzie; Kaska, Sophia; Cooper, Sarah; Gajewski, Paula; Eagle, Andrew; Mazei-Robison, Michelle; Nestler, Eric J; Robison, A J

    2015-12-01

    Major depressive disorder is thought to arise in part from dysfunction of the brain's "reward circuitry", consisting of the mesolimbic dopamine system and the glutamatergic and neuromodulatory inputs onto this system. Both chronic stress and antidepressant treatment regulate gene transcription in many of the brain regions that make up these circuits, but the exact nature of the transcription factors and target genes involved in these processes remain unclear. Here, we demonstrate induction of the FosB family of transcription factors in ∼25 distinct regions of adult mouse brain, including many parts of the reward circuitry, by chronic exposure to the antidepressant fluoxetine. We further uncover specific patterns of FosB gene product expression (i.e., differential expression of full-length FosB, ΔFosB, and Δ2ΔFosB) in brain regions associated with depression--the nucleus accumbens (NAc), prefrontal cortex (PFC), and hippocampus--in response to chronic fluoxetine treatment, and contrast these patterns with differential induction of FosB isoforms in the chronic social defeat stress model of depression with and without fluoxetine treatment. We find that chronic fluoxetine, in contrast to stress, causes induction of the unstable full-length FosB isoform in the NAc, PFC, and hippocampus even 24 h following the final injection, indicating that these brain regions may undergo chronic activation when fluoxetine is on board, even in the absence of stress. We also find that only the stable ΔFosB isoform correlates with behavioral responses to stress. These data suggest that NAc, PFC, and hippocampus may present useful targets for directed intervention in mood disorders (ie, brain stimulation or gene therapy), and that determining the gene targets of FosB-mediated transcription in these brain regions in response to fluoxetine may yield novel inroads for pharmaceutical intervention in depressive disorders. PMID:26164345

  17. Cell-specific activity of neprilysin 2 isoforms and enzymic specificity compared with neprilysin.

    PubMed Central

    Rose, Christiane; Voisin, Stéphanie; Gros, Claude; Schwartz, Jean-Charles; Ouimet, Tanja

    2002-01-01

    Neprilysin (NEP) 2 is a recently cloned glycoprotein displaying a high degree of sequence identity with neprilysin (EC 3.4.24.11), the prototypical member of the M13 subfamily of metalloproteases. Whereas NEP is involved in the metabolism of several bioactive peptides by plasma membranes of various cells, the enzymic properties and physiological functions of NEP2 are unknown. Here we characterize the cell-expression modalities and enzymic specificity of two alternatively spliced isoforms of NEP2 in Chinese hamster ovary and AtT20 cells. In the two cell lines, both isoforms are type II glycoproteins inserted in the endoplasmic reticulum as inactive precursors. Maturation detected by Western-blot analysis of glycosidase digests was cell-specific and more efficient in the endocrine cell line. The enzymic activity of both isoforms semi-purified from AtT20 cells reveals comparable specificities in terms of model substrates, pH optima and inhibitory patterns. NEP2 activity was compared with that of NEP regarding potencies of transition-state inhibitors, modes of hydrolysis, maximal hydrolysis rates and apparent affinities of bioactive peptides. Although all transition-state inhibitors of NEP inhibited NEP2 activity, albeit with different potencies, and many peptides were cleaved at the same amide bond by both peptidases, differences could be observed, i.e. in the hydrolysis of gonadotropin-releasing hormone and cholecystokinin, which occurred at different sites and more efficiently in the case of NEP2. Differences in cleavage of bioactive peptides, in cell-trafficking patterns and in tissue distribution indicate that NEP and NEP2 play distinct physiological roles in spite of their high degree of sequence identity. PMID:11964170

  18. Isoforms Confer Characteristic Force Generation and Mechanosensation by Myosin II Filaments

    PubMed Central

    Stam, Samantha; Alberts, Jon; Gardel, Margaret L.; Munro, Edwin

    2015-01-01

    Myosin II isoforms with varying mechanochemistry and filament size interact with filamentous actin (F-actin) arrays to generate contractile forces in muscle and nonmuscle cells. How myosin II force production is shaped by isoform-specific motor properties and environmental stiffness remains poorly understood. Here, we used computer simulations to analyze force production by an ensemble of myosin motors against an elastically tethered actin filament. We found that force output depends on two timescales: the duration of F-actin attachment, which varies sharply with the ensemble size, motor duty ratio, and external load; and the time to build force, which scales with the ensemble stall force, gliding speed, and environmental stiffness. Although force-dependent kinetics were not required to sense changes in stiffness, the myosin catch bond produced positive feedback between the attachment time and force to trigger switch-like transitions from transient attachments, generating small forces, to high-force-generating runs. Using parameters representative of skeletal muscle myosin, nonmuscle myosin IIB, and nonmuscle myosin IIA revealed three distinct regimes of behavior, respectively: 1) large assemblies of fast, low-duty ratio motors rapidly build stable forces over a large range of environmental stiffness; 2) ensembles of slow, high-duty ratio motors serve as high-affinity cross-links with force buildup times that exceed physiological timescales; and 3) small assemblies of low-duty ratio motors operating at intermediate speeds are poised to respond sharply to changes in mechanical context—at low force or stiffness, they serve as low-affinity cross-links, but they can transition to force production via the positive-feedback mechanism described above. Together, these results reveal how myosin isoform properties may be tuned to produce force and respond to mechanical cues in their environment. PMID:25902439

  19. Heterogeneity of presynaptic proteins: do not forget isoforms

    PubMed Central

    Bragina, Luca; Fattorini, Giorgia; Giovedì, Silvia; Bosco, Federica; Benfenati, Fabio; Conti, Fiorenzo

    2013-01-01

    Analysis of presynaptic protein expression in glutamatergic and GABAergic central synapses performed in several laboratories and with different techniques is unveiling a complex scenario, largely because each presynaptic protein exists in several isoforms. The interpretation of these findings is generally based on the notion that each synapse and each synaptic vesicle contains one of the isoforms of each family of presynaptic proteins. We verified whether this interpretation is tenable by performing triple labeling and immunoisolation studies with the aim of detecting two isoforms of a given presynaptic protein in glutamatergic or GABAergic axon terminals and/or synaptic vesicles (SVs). Here, we show that: (1) the possibility that not all families of presynaptic proteins are expressed in all terminals must be taken into serious account; (2) the expression of a given protein isoform in a terminal does not exclude the expression of other isoforms of the same protein in the same terminal and in the same vesicle. These conclusions open new and interesting problems; their experimental analysis might improve our understanding of the physiology and pathophysiology of central synapses. PMID:23382710

  20. SURVIV for survival analysis of mRNA isoform variation.

    PubMed

    Shen, Shihao; Wang, Yuanyuan; Wang, Chengyang; Wu, Ying Nian; Xing, Yi

    2016-01-01

    The rapid accumulation of clinical RNA-seq data sets has provided the opportunity to associate mRNA isoform variations to clinical outcomes. Here we report a statistical method SURVIV (Survival analysis of mRNA Isoform Variation), designed for identifying mRNA isoform variation associated with patient survival time. A unique feature and major strength of SURVIV is that it models the measurement uncertainty of mRNA isoform ratio in RNA-seq data. Simulation studies suggest that SURVIV outperforms the conventional Cox regression survival analysis, especially for data sets with modest sequencing depth. We applied SURVIV to TCGA RNA-seq data of invasive ductal carcinoma as well as five additional cancer types. Alternative splicing-based survival predictors consistently outperform gene expression-based survival predictors, and the integration of clinical, gene expression and alternative splicing profiles leads to the best survival prediction. We anticipate that SURVIV will have broad utilities for analysing diverse types of mRNA isoform variation in large-scale clinical RNA-seq projects. PMID:27279334

  1. p53 isoform profiling in glioblastoma and injured brain

    PubMed Central

    Takahashi, Rie; Giannini, Caterina; Sarkaria, Jann N.; Schroeder, Mark; Rogers, Joseph; Mastroeni, Diego; Scrable, Heidi

    2014-01-01

    The tumor suppressor p53 has been found to be the most commonly mutated gene in human cancers; however, the frequency of p53 mutations varies from 10–70% across different cancer types. This variability can partly be explained by inactivating mechanisms aside from direct genomic polymorphisms. The p53 gene encodes 12 isoforms, which have been shown to modulate full-length p53 activity in cancer. In this study, we characterized p53 isoform expression patterns in glioblastoma, gliosis, non-tumor brain, and neural progenitor cells by SDS-PAGE, immunoblot, mass spectrometry, and RT-PCR. At the protein level, we found that the most consistently expressed isoform in glioblastoma, Δ40p53, was uniquely expressed in regenerative processes, such as those involving neural progenitor cells and gliosis compared to tumor samples. Isoform profiling of glioblastoma tissues revealed the presence of both Δ40p53 and full-length p53, neither of which were detected in non-tumor cerebral cortex. Upon xenograft propagation of tumors, p53 levels increased. The variability of overall p53 expression and relative levels of isoforms suggest fluctuations in subpopulations of cells with greater or lesser capacity for proliferation, which can change as the tumor evolves under different growth conditions. PMID:22824800

  2. SURVIV for survival analysis of mRNA isoform variation

    PubMed Central

    Shen, Shihao; Wang, Yuanyuan; Wang, Chengyang; Wu, Ying Nian; Xing, Yi

    2016-01-01

    The rapid accumulation of clinical RNA-seq data sets has provided the opportunity to associate mRNA isoform variations to clinical outcomes. Here we report a statistical method SURVIV (Survival analysis of mRNA Isoform Variation), designed for identifying mRNA isoform variation associated with patient survival time. A unique feature and major strength of SURVIV is that it models the measurement uncertainty of mRNA isoform ratio in RNA-seq data. Simulation studies suggest that SURVIV outperforms the conventional Cox regression survival analysis, especially for data sets with modest sequencing depth. We applied SURVIV to TCGA RNA-seq data of invasive ductal carcinoma as well as five additional cancer types. Alternative splicing-based survival predictors consistently outperform gene expression-based survival predictors, and the integration of clinical, gene expression and alternative splicing profiles leads to the best survival prediction. We anticipate that SURVIV will have broad utilities for analysing diverse types of mRNA isoform variation in large-scale clinical RNA-seq projects. PMID:27279334

  3. Differential regulation of renal phospholipase C isoforms by catecholamines.

    PubMed

    Yu, P Y; Asico, L D; Eisner, G M; Jose, P A

    1995-01-01

    Dopamine and D1 agonists and NE all increase phosphatidyl inositol-specific phospholipase C (PLC) activity, but whereas dopamine produces a natriuresis, NE has an antinatriuretic effect. To determine if catecholamines differentially regulate the expression of PLC isoforms, we infused fenoldopam, a D1 agonist, or pramipexole, a D1/D2 agonist, intravenously or infused fenoldopam or NE into the renal artery of anesthetized rats. After 3-4 h of infusion, when the expected natriuresis (fenoldopam or pramipexole) or antinatriuresis (NE) occurred, the kidneys were removed for analysis of PLC isoform protein expression activity. Western blot analysis revealed that in renal cortical membranes, fenoldopam and pramipexole increased expression of PLC beta 1 and decreased expression of PLC gamma 1; PLC delta was unchanged. In the cytosol, pramipexole and fenoldopam increased expression of both PLC beta 1 and PLC gamma 1. No effects were noted in the medulla. A preferential D1 antagonist, SKF 83742, which by itself had no effect, blocked the effects of pramipexole, thus confirming the involvement of the D1 receptor. In contrast, NE also increased PLC beta 1 but did not affect PLC gamma 1 protein expression in membranes. The changes in PLC isoform expression were accompanied by similar changes in PLC isoform activity. These studies demonstrate for the first time differential regulation of PLC isoforms by catecholamines. PMID:7814630

  4. Roles and post-translational regulation of cardiac class IIa histone deacetylase isoforms.

    PubMed

    Weeks, Kate L; Avkiran, Metin

    2015-04-15

    Cardiomyocyte hypertrophy is an integral component of pathological cardiac remodelling in response to mechanical and chemical stresses in settings such as chronic hypertension or myocardial infarction. For hypertrophy to ensue, the pertinent mechanical and chemical signals need to be transmitted from membrane sensors (such as receptors for neurohormonal mediators) to the cardiomyocyte nucleus, leading to altered transcription of the genes that regulate cell growth. In recent years, nuclear histone deacetylases (HDACs) have attracted considerable attention as signal-responsive, distal regulators of the transcriptional reprogramming that in turn precipitates cardiomyocyte hypertrophy, with particular focus on the role of members of the class IIa family, such as HDAC4 and HDAC5. These histone deacetylase isoforms appear to repress cardiomyocyte hypertrophy through mechanisms that involve protein interactions in the cardiomyocyte nucleus, particularly with pro-hypertrophic transcription factors, rather than via histone deacetylation. In contrast, evidence indicates that class I HDACs promote cardiomyocyte hypertrophy through mechanisms that are dependent on their enzymatic activity and thus sensitive to pharmacological HDAC inhibitors. Although considerable progress has been made in understanding the roles of post-translational modifications (PTMs) such as phosphorylation, oxidation and proteolytic cleavage in regulating class IIa HDAC localisation and function, more work is required to explore the contributions of other PTMs, such as ubiquitination and sumoylation, as well as potential cross-regulatory interactions between distinct PTMs and between class IIa and class I HDAC isoforms. PMID:25362149

  5. Cloning and primary structure of a human islet isoform of glutamic acid decarboxylase from chromosome 10

    SciTech Connect

    Karlsen, A.E.; Hagopian, W.A.; Grubin, C.E.; Dube, S.; Disteche, C.M.; Adler, D.A.; Baermeier, H.; Lernmark, A. ); Mathewes, S.; Grant, F.J.; Foster, D. )

    1991-10-01

    Glutamic acid decarboxylase which catalyzes formation of {gamma}-aminobutyric acid from L-glutamic acid, is detectable in different isoforms with distinct electrophoretic and kinetic characteristics. GAD has also been implicated as an autoantigen in the vastly differing autoimmune disease stiff-man syndrome and insulin-dependent diabetes mellitus. Despite the differing GAD isoforms, only one type of GAD cDNA (GAD-1), localized to a syntenic region of chromosome 2, has been isolated from rat, mouse, and cat. Using sequence information from GAD-1 to screen a human pancreatic islet cDNA library, the authors describe the isolation of an additional GAD cDNA (GAD-2), which was mapped to the short arm of human chromosome 10. Genomic Southern blotting with GAD-2 demonstrated a hybridization pattern different form that detected by GAD-1. GAD-2 recognizes a 5.6-kilobase transcript in both islets and brain, in contrast to GAD-1, which detects a 3.7-kilobase transcript in brain only. The deduced 585-amino acid sequence coded for by GAD-2 shows < 65% identify to previously published, highly conserved GAD-1 brain sequences, which show > 96% deduced amino acid sequence homology among the three species.

  6. Identification of caleosin and two oleosin isoforms in oil bodies of pine megagametophytes.

    PubMed

    Pasaribu, Buntora; Chung, Tse-Yu; Chen, Chii-Shiarng; Wang, Song-Liang; Jiang, Pei-Luen; Tzen, Jason T C

    2014-09-01

    Numerous oil bodies of 0.2-2 μm occupied approximately 80% of intracellular space in mature pine (Pinus massoniana) megagametophytes. They were stably isolated and found to comprise mostly triacylglycerols as examined by thin layer chromatography analysis and confirmed by both Nile red and BODIPY stainings. Fatty acids released from the triacylglycerols of pine oil bodies were mainly unsaturated, including linoleic acid (60%), adrenic acid (12.3%) and vaccenic acid (9.7%). Proteins extracted from pine oil bodies were subjected to immunological cross-recognition, and the results showed that three proteins of 28, 16 and 14 kDa were detected by antibodies against sesame seed caleosin, sesame oleosin-L and lily pollen oleosin-P, respectively. Complete cDNA fragments encoding these three pine oil-body proteins, tentatively named caleosin, oleosin-L and oleosin-G, were obtained by PCR cloning and further confirmed by mass spectrometric analysis. Consistently, phylogenetic tree analyses showed that pine caleosin was closely-related to the caleosin of cycad megagametophyte among known caleosin sequences. While pine oleosin-L was found clustered with seed oleosin isoforms of angiosperm species, oleosin-G was distinctively grouped with the oleosin-P of lily pollen. The oleosin-G identified in pine megagametophytes seems to represent a new class of seed oleosin isoform evolutionarily close to the pollen oleosin-P. PMID:24954070

  7. Discontinuous thoracic venous cardiomyocytes and heart exhibit synchronized developmental switch of troponin isoforms

    PubMed Central

    Kracklauer, Martin P.; Feng, Han-Zhong; Jiang, Wenrui; Lin, Jenny L.-C.; Lin, Jim J.-C.; Jin, J.-P.

    2012-01-01

    Cardiomyocyte-like cells have been reported in thoracic veins of rodents and other mammals, but their differentiation state and relationship to the muscle mass in the heart remain to be characterized. Here we investigated the distribution, ultrastructure, and the expression and developmental regulation of myofilament proteins in mouse and rat pulmonary and azygos venous cardiomyocytes. Tracing cardiomyocytes in transgenic mouse tissues with a lacZ reporter gene driven by cloned rat cardiac troponin T promoter demonstrated scattered distribution of cardiomyocytes discontinuous from the atrial sleeves. The longitudinal axis of venous cardiomyocytes is perpendicular to that of the vessel. These cells contain typical sarcomere structures and intercalated discs as shown in electron microscopic images and express cardiac isoforms of troponin T, troponin I and myosin. The expression of troponin I isoform genes and the alternative splicing of cardiac troponin T in thoracic venous cardiomyocytes are regulated during postnatal development in a precise synchrony with that in the heart. Nonetheless, the patterns of cardiac troponin T splicing in adult rat thoracic venous cardiomyocytes are slightly but clearly distinct from those in the atrial and ventricular muscles. The data indicate that mouse and rat thoracic venous cardiomyocytes residing in extra-cardiac tissue possess a physiologically differentiated state and an intrinsically preset developmental clock, which are apparently independent of the very different hemodynamic environments and functional features of the vessels and heart. PMID:23176202

  8. Molecular and Enzymatic Characterization of Three Phosphoinositide-Specific Phospholipase C Isoforms from Potato1

    PubMed Central

    Kopka, Joachim; Pical, Christophe; Gray, Julie E.; Müller-Röber, Bernd

    1998-01-01

    Many cellular responses to stimulation of cell-surface receptors by extracellular signals are transmitted across the plasma membrane by hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2), which is cleaved into diacylglycerol and inositol-1,4,5-tris-phosphate by phosphoinositide-specific phospholipase C (PI-PLC). We present structural, biochemical, and RNA expression data for three distinct PI-PLC isoforms, StPLC1, StPLC2, and StPLC3, which were cloned from a guard cell-enriched tissue preparation of potato (Solanum tuberosum) leaves. All three enzymes contain the catalytic X and Y domains, as well as C2-like domains also present in all PI-PLCs. Analysis of the reaction products obtained from PIP2 hydrolysis unequivocally identified these enzymes as genuine PI-PLC isoforms. Recombinant StPLCs showed an optimal PIP2-hydrolyzing activity at 10 μm Ca2+ and were inhibited by Al3+ in equimolar amounts. In contrast to PI-PLC activity in plant plasma membranes, however, recombinant enzymes could not be activated by Mg2+. All three stplc genes are expressed in various tissues of potato, including leaves, flowers, tubers, and roots, and are affected by drought stress in a gene-specific manner. PMID:9449844

  9. Proteolytic Isoforms of SPARC Induce Adipose Stromal Cell Mobilization in Obesity.

    PubMed

    Tseng, Chieh; Kolonin, Mikhail G

    2016-01-01

    Adipose stromal cells (ASC) are mesenchymal adipocyte progenitors that reside in the peri-endothelium of fat tissue. ASC mobilization and migration accompany white adipose tissue (WAT) remodeling and pathological conditions. Mechanisms regulating ASC trafficking are largely unknown. We previously reported that binding of the matricellular protein secreted protein acidic and rich in cysteine (SPARC) to β1 integrin on ASC surface induces their motility. Here, we show that SPARC is required for ASC mobilization. We report two SPARC proteolytic isoforms, C-SPARC (lacking the N terminus) and N-SPARC (lacking the C terminus), generated in mesenteric WAT of obese mice. C-SPARC, but not N-SPARC, binds to β1 integrin on ASC, while N-SPARC preferentially binds to the extracellular matrix (ECM) and blocks ECM/integrin interaction. Interestingly, both C-SPARC and N-SPARC induce ASC deadhesion from the ECM, which is associated with modulation of integrin-dependent FAK-ERK signaling and integrin-independent ILK-Akt signaling. We show that these SPARC isoforms, acting on ASC through distinct mechanisms, have an additive effect in inducing ASC migration. PMID:26381424

  10. Differential Effects of Tra2ß Isoforms on HIV-1 RNA Processing and Expression

    PubMed Central

    Platt, Craig; Calimano, Maria; Nemet, Josip; Bubenik, Jodi; Cochrane, Alan

    2015-01-01

    Balanced processing of HIV-1 RNA is critical to virus replication and is regulated by host factors. In this report, we demonstrate that overexpression of either Tra2α or Tra2β results in a marked reduction in HIV-1 Gag/ Env expression, an effect associated with changes in HIV-1 RNA accumulation, altered viral splice site usage, and a block to export of HIV-1 genomic RNA. A natural isoform of Tra2β (Tra2ß3), lacking the N-terminal RS domain, also suppressed HIV-1 expression but had different effects on viral RNA processing. The functional differences between the Tra2β isoforms were also observed in the context of another RNA substrate indicating that these factors have distinct functions within the cell. Finally, we demonstrate that Tra2ß depletion results in a selective reduction in HIV-1 Env expression as well as an increase in multiply spliced viral RNA. Together, the findings indicate that Tra2α/β can play important roles in regulating HIV-1 RNA metabolism and expression. PMID:25970345

  11. Neurotoxin-induced selective ubiquitination and regulation of MEF2A isoform in neuronal stress response

    PubMed Central

    She, Hua; Yang, Qian; Mao, Zixu

    2014-01-01

    The myocyte enhancer factor 2A-D (MEF2) proteins are members of the MCM1-agamous-deficiens-serum (MADS) response factor family of transcription factors. Various MEF2 isoform proteins are enriched in neurons and exhibit distinct patterns of expression in different regions of the brain. In neurons, MEF2 functions as a converging factor to regulate many neuronal functions including survival. MEF2 activities are tightly controlled in neurons in response to stress. Whether stress signal may differentially regulate MEF2s remains largely unknown. In this work, we showed that MEF2A but not MEF2C or MEF2D was modified by ubiquitination in dopaminergic neuronal cell line SN4741 cells. MEF2A was ubiquitinated at its N’-terminus, and the ubiquitination of MEF2A was first detectable in the nuclear compartment and later in the cytoplasm. Ubiquitination of MEF2A correlated with reduced DNA-binding activity and transcriptional activity. More importantly, disturbing the degradation of ubiquitinated MEF2A through proteasome pathway by neurotoxins known to induce Parkinson’s disease (PD) features in model animals caused accumulation of ubiquitinated MEF2A, reduced MEF2 activity, and impaired cellular viability. Our work thus provides the first evidence to demonstrate an isoforms specific regulation of MEF2s by ubiquitination-proteasome pathway in dopaminergic neuronal cell by neurotoxins, suggesting that stress signal and cellular context dependent dysregulation of MEF2s may underlie the loss of neuronal viability. PMID:22764880

  12. Polarizing the Neuron through Sustained Co-expression of Alternatively Spliced Isoforms.

    PubMed

    Yap, Karen; Xiao, Yixin; Friedman, Brad A; Je, H Shawn; Makeyev, Eugene V

    2016-05-10

    Alternative splicing (AS) is an important source of proteome diversity in eukaryotes. However, how this affects protein repertoires at a single-cell level remains an open question. Here, we show that many 3'-terminal exons are persistently co-expressed with their alternatives in mammalian neurons. In an important example of this scenario, cell polarity gene Cdc42, a combination of polypyrimidine tract-binding, protein-dependent, and constitutive splicing mechanisms ensures a halfway switch from the general (E7) to the neuron-specific (E6) alternative 3'-terminal exon during neuronal differentiation. Perturbing the nearly equimolar E6/E7 ratio in neurons results in defects in both axonal and dendritic compartments and suggests that Cdc42E7 is involved in axonogenesis, whereas Cdc42E6 is required for normal development of dendritic spines. Thus, co-expression of a precise blend of functionally distinct splice isoforms rather than a complete switch from one isoform to another underlies proper structural and functional polarization of neurons. PMID:27134173

  13. Roles and post-translational regulation of cardiac class IIa histone deacetylase isoforms

    PubMed Central

    Weeks, Kate L; Avkiran, Metin

    2015-01-01

    Cardiomyocyte hypertrophy is an integral component of pathological cardiac remodelling in response to mechanical and chemical stresses in settings such as chronic hypertension or myocardial infarction. For hypertrophy to ensue, the pertinent mechanical and chemical signals need to be transmitted from membrane sensors (such as receptors for neurohormonal mediators) to the cardiomyocyte nucleus, leading to altered transcription of the genes that regulate cell growth. In recent years, nuclear histone deacetylases (HDACs) have attracted considerable attention as signal-responsive, distal regulators of the transcriptional reprogramming that in turn precipitates cardiomyocyte hypertrophy, with particular focus on the role of members of the class IIa family, such as HDAC4 and HDAC5. These histone deacetylase isoforms appear to repress cardiomyocyte hypertrophy through mechanisms that involve protein interactions in the cardiomyocyte nucleus, particularly with pro-hypertrophic transcription factors, rather than via histone deacetylation. In contrast, evidence indicates that class I HDACs promote cardiomyocyte hypertrophy through mechanisms that are dependent on their enzymatic activity and thus sensitive to pharmacological HDAC inhibitors. Although considerable progress has been made in understanding the roles of post-translational modifications (PTMs) such as phosphorylation, oxidation and proteolytic cleavage in regulating class IIa HDAC localisation and function, more work is required to explore the contributions of other PTMs, such as ubiquitination and sumoylation, as well as potential cross-regulatory interactions between distinct PTMs and between class IIa and class I HDAC isoforms. PMID:25362149

  14. Chimeric calcium/calmodulin-dependent protein kinase in tobacco: differential regulation by calmodulin isoforms

    NASA Technical Reports Server (NTRS)

    Liu, Z.; Xia, M.; Poovaiah, B. W.

    1998-01-01

    cDNA clones of chimeric Ca2+/calmodulin-dependent protein kinase (CCaMK) from tobacco (TCCaMK-1 and TCCaMK-2) were isolated and characterized. The polypeptides encoded by TCCaMK-1 and TCCaMK-2 have 15 different amino acid substitutions, yet they both contain a total of 517 amino acids. Northern analysis revealed that CCaMK is expressed in a stage-specific manner during anther development. Messenger RNA was detected when tobacco bud sizes were between 0.5 cm and 1.0 cm. The appearance of mRNA coincided with meiosis and became undetectable at later stages of anther development. The reverse polymerase chain reaction (RT-PCR) amplification assay using isoform-specific primers showed that both of the CCaMK mRNAs were expressed in anther with similar expression patterns. The CCaMK protein expressed in Escherichia coli showed Ca2+-dependent autophosphorylation and Ca2+/calmodulin-dependent substrate phosphorylation. Calmodulin isoforms (PCM1 and PCM6) had differential effects on the regulation of autophosphorylation and substrate phosphorylation of tobacco CCaMK, but not lily CCaMK. The evolutionary tree of plant serine/threonine protein kinases revealed that calmodulin-dependent kinases form one subgroup that is distinctly different from Ca2+-dependent protein kinases (CDPKs) and other serine/threonine kinases in plants.

  15. PPARγ isoforms differentially regulate metabolic networks to mediate mouse prostatic epithelial differentiation

    PubMed Central

    Strand, D W; Jiang, M; Murphy, T A; Yi, Y; Konvinse, K C; Franco, O E; Wang, Y; Young, J D; Hayward, S W

    2012-01-01

    Recent observations indicate prostatic diseases are comorbidities of systemic metabolic dysfunction. These discoveries revealed fundamental questions regarding the nature of prostate metabolism. We previously showed that prostate-specific ablation of PPARγ in mice resulted in tumorigenesis and active autophagy. Here, we demonstrate control of overlapping and distinct aspects of prostate epithelial metabolism by ectopic expression of individual PPARγ isoforms in PPARγ knockout prostate epithelial cells. Expression and activation of either PPARγ 1 or 2 reduced de novo lipogenesis and oxidative stress and mediated a switch from glucose to fatty acid oxidation through regulation of genes including Pdk4, Fabp4, Lpl, Acot1 and Cd36. Differential effects of PPARγ isoforms included decreased basal cell differentiation, Scd1 expression and triglyceride fatty acid desaturation and increased tumorigenicity by PPARγ1. In contrast, PPARγ2 expression significantly increased basal cell differentiation, Scd1 expression and AR expression and responsiveness. Finally, in confirmation of in vitro data, a PPARγ agonist versus high-fat diet (HFD) regimen in vivo confirmed that PPARγ agonization increased prostatic differentiation markers, whereas HFD downregulated PPARγ-regulated genes and decreased prostate differentiation. These data provide a rationale for pursuing a fundamental metabolic understanding of changes to glucose and fatty acid metabolism in benign and malignant prostatic diseases associated with systemic metabolic stress. PMID:22874998

  16. Laminin isoforms in endothelial and perivascular basement membranes

    PubMed Central

    Yousif, Lema F.; Di Russo, Jacopo; Sorokin, Lydia

    2013-01-01

    Laminins, one of the major functional components of basement membranes, are found underlying endothelium, and encasing pericytes and smooth muscle cells in the vessel wall. Depending on the type of blood vessel (capillary, venule, postcapillary venule, vein or artery) and their maturation state, both the endothelial and mural cell phenotype vary, with associated changes in laminin isoform expression. Laminins containing the α4 and α5 chains are the major isoforms found in the vessel wall, with the added contribution of laminin α2 in larger vessels. We here summarize current data on the precise localization of these laminin isoforms and their receptors in the different layers of the vessel wall, and their potential contribution to vascular homeostasis. PMID:23263631

  17. A penalized likelihood approach for robust estimation of isoform expression

    PubMed Central

    2016-01-01

    Ultra high-throughput sequencing of transcriptomes (RNA-Seq) has enabled the accurate estimation of gene expression at individual isoform level. However, systematic biases introduced during the sequencing and mapping processes as well as incompleteness of the transcript annotation databases may cause the estimates of isoform abundances to be unreliable, and in some cases, highly inaccurate. This paper introduces a penalized likelihood approach to detect and correct for such biases in a robust manner. Our model extends those previously proposed by introducing bias parameters for reads. An L1 penalty is used for the selection of non-zero bias parameters. We introduce an efficient algorithm for model fitting and analyze the statistical properties of the proposed model. Our experimental studies on both simulated and real datasets suggest that the model has the potential to improve isoform-specific gene expression estimates and identify incompletely annotated gene models.

  18. Oxygenation properties and isoform diversity of snake hemoglobins.

    PubMed

    Storz, Jay F; Natarajan, Chandrasekhar; Moriyama, Hideaki; Hoffmann, Federico G; Wang, Tobias; Fago, Angela; Malte, Hans; Overgaard, Johannes; Weber, Roy E

    2015-11-01

    Available data suggest that snake hemoglobins (Hbs) are characterized by a combination of unusual structural and functional properties relative to the Hbs of other amniote vertebrates, including oxygenation-linked tetramer-dimer dissociation. However, standardized comparative data are lacking for snake Hbs, and the Hb isoform composition of snake red blood cells has not been systematically characterized. Here we present the results of an integrated analysis of snake Hbs and the underlying α- and β-type globin genes to characterize 1) Hb isoform composition of definitive erythrocytes, and 2) the oxygenation properties of isolated isoforms as well as composite hemolysates. We used species from three families as subjects for experimental studies of Hb function: South American rattlesnake, Crotalus durissus (Viperidae); Indian python, Python molurus (Pythonidae); and yellow-bellied sea snake, Pelamis platura (Elapidae). We analyzed allosteric properties of snake Hbs in terms of the Monod-Wyman-Changeux model and Adair four-step thermodynamic model. Hbs from each of the three species exhibited high intrinsic O2 affinities, low cooperativities, small Bohr factors in the absence of phosphates, and high sensitivities to ATP. Oxygenation properties of the snake Hbs could be explained entirely by allosteric transitions in the quaternary structure of intact tetramers, suggesting that ligation-dependent dissociation of Hb tetramers into αβ-dimers is not a universal feature of snake Hbs. Surprisingly, the major Hb isoform of the South American rattlesnake is homologous to the minor HbD of other amniotes and, contrary to the pattern of Hb isoform differentiation in birds and turtles, exhibits a lower O2 affinity than the HbA isoform. PMID:26354849

  19. Apolipoprotein E isoform-specific effects on lipoprotein receptor processing

    PubMed Central

    Bachmeier, Corbin; Shackleton, Ben; Ojo, Joseph; Paris, Daniel; Mullan, Michael; Crawford, Fiona

    2014-01-01

    Recent findings indicate an isoform-specific role for apolipoprotein E (apoE) in the elimination of beta-amyloid (Aβ) from the brain. ApoE is closely associated with various lipoprotein receptors, which contribute to Aβ brain removal via metabolic clearance or transit across the blood-brain barrier (BBB). These receptors are subject to ectodomain shedding at the cell surface, which alters endocytic transport and mitigates Aβ elimination. To further understand the manner in which apoE influences Aβ brain clearance, these studies investigated the effect of apoE on lipoprotein receptor shedding. Consistent with prior reports, we observed an increased shedding of the low density lipoprotein receptor (LDLR) and the LDLR-related protein 1 (LRP1) following Aβ exposure in human brain endothelial cells. When Aβ was co-treated with each apoE isoform, there was a reduction in Aβ-induced shedding with apoE2 and apoE3, while lipoprotein receptor shedding in the presence of apoE4 remained elevated. Likewise, intracranial administration of Aβ to apoE targeted replacement mice (expressing the human apoE isoforms) resulted in an isoform-dependent effect on lipoprotein receptor shedding in the brain (apoE4>apoE3>apoE2). Moreover, these results show a strong inverse correlation with our prior work in apoE transgenic mice in which apoE4 animals showed reduced Aβ clearance across the BBB compared to apoE3 animals. Based on these results, apoE4 appears less efficient than other apoE isoforms in regulating lipoprotein receptor shedding, which may explain the differential effects of these isoforms in removing Aβ from the brain. PMID:25015123

  20. Modulation of neuronal differentiation by CD40 isoforms

    SciTech Connect

    Hou Huayu; Obregon, Demian; Lou, Deyan; Ehrhart, Jared; Fernandez, Frank; Silver, Archie; Tan Jun

    2008-05-02

    Neuron differentiation is a complex process involving various cell-cell interactions, and multiple signaling pathways. We showed previously that CD40 is expressed and functional on mouse and human neurons. In neurons, ligation of CD40 protects against serum withdrawal-induced injury and plays a role in survival and differentiation. CD40 deficient mice display neuron dysfunction, aberrant neuron morphologic changes, and associated gross brain abnormalities. Previous studies by Tone and colleagues suggested that five isoforms of CD40 exist with two predominant isoforms expressed in humans: signal-transducible CD40 type I and a C-terminal truncated, non-signal-transducible CD40 type II. We hypothesized that differential expression of CD40 isoform type I and type II in neurons may modulate neuron differentiation. Results show that adult wild-type, and CD40{sup -/-} deficient mice predominantly express CD40 type I and II isoforms. Whereas adult wild-type mice express mostly CD40 type I in cerebral tissues at relatively high levels, in age and gender-matched CD40{sup -/-} mice CD40 type I expression was almost completely absent; suggesting a predominance of the non-signal-transducible CD40 type II isoform. Younger, 1 day old wild-type mice displayed less CD40 type I, and more CD40 type II, as well as, greater expression of soluble CD40 (CD40L/CD40 signal inhibitor), compared with 1 month old mice. Neuron-like N2a cells express CD40 type I and type II isoforms while in an undifferentiated state, however once induced to differentiate, CD40 type I predominates. Further, differentiated N2a cells treated with CD40 ligand express high levels of neuron specific nuclear protein (NeuN); an effect reduced by anti-CD40 type I siRNA, but not by control (non-targeting) siRNA. Altogether these data suggest that CD40 isoforms may act in a temporal fashion to modulate neuron differentiation during brain development. Thus, modulation of neuronal CD40 isoforms and CD40 signaling may

  1. Systematically Differentiating Functions for Alternatively Spliced Isoforms through Integrating RNA-seq Data

    PubMed Central

    Menon, Rajasree; Wen, Yuchen; Omenn, Gilbert S.; Kretzler, Matthias; Guan, Yuanfang

    2013-01-01

    Integrating large-scale functional genomic data has significantly accelerated our understanding of gene functions. However, no algorithm has been developed to differentiate functions for isoforms of the same gene using high-throughput genomic data. This is because standard supervised learning requires ‘ground-truth’ functional annotations, which are lacking at the isoform level. To address this challenge, we developed a generic framework that interrogates public RNA-seq data at the transcript level to differentiate functions for alternatively spliced isoforms. For a specific function, our algorithm identifies the ‘responsible’ isoform(s) of a gene and generates classifying models at the isoform level instead of at the gene level. Through cross-validation, we demonstrated that our algorithm is effective in assigning functions to genes, especially the ones with multiple isoforms, and robust to gene expression levels and removal of homologous gene pairs. We identified genes in the mouse whose isoforms are predicted to have disparate functionalities and experimentally validated the ‘responsible’ isoforms using data from mammary tissue. With protein structure modeling and experimental evidence, we further validated the predicted isoform functional differences for the genes Cdkn2a and Anxa6. Our generic framework is the first to predict and differentiate functions for alternatively spliced isoforms, instead of genes, using genomic data. It is extendable to any base machine learner and other species with alternatively spliced isoforms, and shifts the current gene-centered function prediction to isoform-level predictions. PMID:24244129

  2. Induction of the ChREBPβ Isoform Is Essential for Glucose-Stimulated β-Cell Proliferation.

    PubMed

    Zhang, Pili; Kumar, Anil; Katz, Liora S; Li, Lucy; Paulynice, Martine; Herman, Mark A; Scott, Donald K

    2015-12-01

    Carbohydrate-responsive element-binding protein (ChREBP) is a glucose-sensing transcription factor required for glucose-stimulated proliferation of pancreatic β-cells in rodents and humans. The full-length isoform (ChREBPα) has a low glucose inhibitory domain (LID) that restrains the transactivation domain when glucose catabolism is minimal. A novel isoform of ChREBP (ChREBPβ) was recently described that lacks the LID domain and is therefore constitutively and more potently active. ChREBPβ has not been described in β-cells nor has its role in glucose-stimulated proliferation been determined. We found that ChREBPβ is highly expressed in response to glucose, particularly with prolonged culture in hyperglycemic conditions. In addition, small interfering RNAs that knocked down ChREBPβ transcripts without affecting ChREBPα expression or activity decreased glucose-stimulated expression of carbohydrate response element-containing genes and glucose-stimulated proliferation in INS-1 cells and in isolated rat islets. Quantitative chromatin immunoprecipitation, electrophoretic mobility shift assays, and luciferase reporter assays were used to demonstrate that ChREBP binds to a newly identified powerful carbohydrate response element in β-cells and hepatocytes, distinct from that in differentiated 3T3-L1 adipocytes. We conclude that ChREBPβ contributes to glucose-stimulated gene expression and proliferation in β-cells, with recruitment of ChREBPα to tissue-specific elements of the ChREBPβ isoform promoter. PMID:26384380

  3. Toward Understanding the Molecular Bases of Stretch Activation: A STRUCTURAL COMPARISON OF THE TWO TROPONIN C ISOFORMS OF LETHOCERUS.

    PubMed

    Sanfelice, Domenico; Sanz-Hernández, Máximo; de Simone, Alfonso; Bullard, Belinda; Pastore, Annalisa

    2016-07-29

    Muscles are usually activated by calcium binding to the calcium sensory protein troponin-C, which is one of the three components of the troponin complex. However, in cardiac and insect flight muscle activation is also produced by mechanical stress. Little is known about the molecular bases of this calcium-independent activation. In Lethocerus, a giant water bug often used as a model system because of its large muscle fibers, there are two troponin-C isoforms, called F1 and F2, that have distinct roles in activating the muscle. It has been suggested that this can be explained either by differences in structural features or by differences in the interactions with other proteins. Here we have compared the structural and dynamic properties of the two proteins and shown how they differ. We have also mapped the interactions of the F2 isoform with peptides spanning the sequence of its natural partner, troponin-I. Our data have allowed us to build a model of the troponin complex and may eventually help in understanding the specialized function of the F1 and F2 isoforms and the molecular mechanism of stretch activation. PMID:27226601

  4. Actin stress fiber disruption and tropomysin isoform switching in normal thyroid epithelial cells stimulated by thyrotropin and phorbol esters

    SciTech Connect

    Roger, P.P.; Rickaert, F.; Lamy, F.; Authelet, M.; Dumont, J.E. )

    1989-05-01

    Thyrotropin (TSH), through cyclic AMP, promotes both proliferation and differentiation expression in dog thyroid epithelial cells in primary culture, whereas the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) also stimulates proliferation but antagonizes differentiating effects of TSH. In this study, within 20 min both factors triggered the disruption of actin-containing stress fibers. This process preceded distinct morphological changes: cytoplasmic retraction and arborization in response to TSH and cyclic AMP, cell shape distortion, and increased motility in response to TPA and diacylglycerol. TSH and TPA also induced a marked decrease in the synthesis of three high M{sub r} tropomyosin isoforms, which were not present in dog thyroid tissue but appeared in culture during cell spreading and stress fiber formation. The tropomyosin isoform switching observed here closely resembled similar processes in various cells transformed by oncogenic viruses. However, it did not correlate with differentiation or mitogenic activation. Contrasting with current hypothesis on this process in transformed cells, tropomyosin isoform switching in normal thyroid cells was preceded and thus might be caused by early disruption of stress fibers.

  5. Pinpointing retrovirus entry sites in cells expressing alternatively spliced receptor isoforms by single virus imaging

    PubMed Central

    2014-01-01

    Background The majority of viruses enter host cells via endocytosis. Current knowledge of viral entry pathways is largely based upon infectivity measurements following genetic and/or pharmacological interventions that disrupt vesicular trafficking and maturation. Imaging of single virus entry in living cells provides a powerful means to delineate viral trafficking pathways and entry sites under physiological conditions. Results Here, we visualized single avian retrovirus co-trafficking with markers for early (Rab5) and late (Rab7) endosomes, acidification of endosomal lumen and the resulting viral fusion measured by the viral content release into the cytoplasm. Virus-carrying vesicles either merged with the existing Rab5-positive early endosomes or slowly accumulated Rab5. The Rab5 recruitment to virus-carrying endosomes correlated with acidification of their lumen. Viral fusion occurred either in early (Rab5-positive) or intermediate (Rab5- and Rab7-positive) compartments. Interestingly, different isoforms of the cognate receptor directed virus entry from distinct endosomes. In cells expressing the transmembrane receptor, viruses preferentially entered and fused with slowly maturing early endosomes prior to accumulation of Rab7. By comparison, in cells expressing the GPI-anchored receptor, viruses entered both slowly and quickly maturing endosomes and fused with early (Rab5-positive) and intermediate (Rab5- and Rab7-positive) compartments. Conclusions Since the rate of low pH-triggered fusion was independent of the receptor isoform, we concluded that the sites of virus entry are determined by the kinetic competition between endosome maturation and viral fusion. Our findings demonstrate the ability of this retrovirus to enter cells via alternative endocytic pathways and establish infection by releasing its content from distinct endosomal compartments. PMID:24935247

  6. Diversified Expression of NG2/CSPG4 Isoforms in Glioblastoma and Human Foetal Brain Identifies Pericyte Subsets

    PubMed Central

    Rizzi, Marco; Errede, Mariella; Wälchli, Thomas; Mucignat, Maria Teresa; Frei, Karl; Roncali, Luisa; Perris, Roberto; Virgintino, Daniela

    2013-01-01

    NG2/CSPG4 is a complex surface-associated proteoglycan (PG) recognized to be a widely expressed membrane component of glioblastoma (WHO grade IV) cells and angiogenic pericytes. To determine the precise expression pattern of NG2/CSPG4 on glioblastoma cells and pericytes, we generated a panel of >60 mouse monoclonal antibodies (mAbs) directed against the ectodomain of human NG2/CSPG4, partially characterized the mAbs, and performed a high-resolution distributional mapping of the PG in human foetal, adult and glioblastoma-affected brains. The reactivity pattern initially observed on reference tumour cell lines indicated that the mAbs recognized 48 immunologically distinct NG2/CSPG4 isoforms, and a total of 14 mAbs was found to identify NG2/CSPG4 isoforms in foetal and neoplastic cerebral sections. These were consistently absent in the adult brain, but exhibited a complementary expression pattern in angiogenic vessels of both tumour and foetal tissues. Considering the extreme pleomorphism of tumour areas, and with the aim of subsequently analysing the distributional pattern of the NG2/CSPG4 isoforms on similar histological vessel typologies, a preliminary study was carried out with endothelial cell and pericyte markers, and with selected vascular basement membrane (VBM) components. On both tumour areas characterized by 'glomeruloid' and 'garland vessels', which showed a remarkably similar cellular and molecular organization, and on developing brain vessels, spatially separated, phenotypically diversified pericyte subsets with a polarized expression of key surface components, including NG2/CSPG4, were disclosed. Interestingly, the majority of the immunolocalized NG2/CSPG4 isoforms present in glioblastoma tissue were present in foetal brain, except for one isoform that seemed to be exclusive of tumour cells, being absent in foetal brain. The results highlight an unprecedented, complex pattern of NG2/CSPG4 isoform expression in foetal and neoplastic CNS, discriminating

  7. Circulating non-22 kDa growth hormone isoforms after a repeated GHRH stimulus in normal subjects.

    PubMed

    Coya, R; Algorta, J; Boguszewski, C L; Vela, A; Carlsson, L M S; Aniel-Quiroga, A; Busturia, M A; Martul, P

    2005-04-01

    peak among subjects might be due to distinct recovery patterns of somatrotrophic function and/or differences in metabolic clearance of GH isoforms. PMID:15809015

  8. Role of p53 isoforms and aggregations in cancer.

    PubMed

    Kim, SeJin; An, Seong Soo A

    2016-06-01

    p53 is a master regulatory protein that is involved in diverse cellular metabolic processes such as apoptosis, DNA repair, and cell cycle arrest. The protective function of p53 (in its homotetrameric form) as a tumor suppressor is lost in more than 50% of human cancers.Despite considerable experimental evidence suggesting the presence of multiple p53 states, it has been difficult to correlate the status of p53 with cancer response to treatments and clinical outcomes, which suggest the importance of complex but essential p53 regulatory pathways.Recent studies have indicated that the expression pattern of p53 isoforms may play a crucial role in regulating normal and cancer cell fates in response to diverse stresses. The human TP53 gene encodes at least 12 p53 isoforms, which are produced in normal tissue through alternative initiation of translation, usage of alternative promoters, and alternative splicing. Furthermore, some researchers have suggested that the formation of mutant p53 aggregates may be associated with cancer pathogenesis due to loss-of function (LoF), dominant-negative (DN), and gain-of function (GoF) effects.As different isoforms or the aggregation state of p53 may influence tumorigenesis, this review aims to examine the correlation of p53 isoforms and aggregation with cancer. PMID:27368003

  9. Differential isoform expression and selective muscle involvement in muscular dystrophies.

    PubMed

    Huovinen, Sanna; Penttilä, Sini; Somervuo, Panu; Keto, Joni; Auvinen, Petri; Vihola, Anna; Huovinen, Sami; Pelin, Katarina; Raheem, Olayinka; Salenius, Juha; Suominen, Tiina; Hackman, Peter; Udd, Bjarne

    2015-10-01

    Despite the expression of the mutated gene in all muscles, selective muscles are involved in genetic muscular dystrophies. Different muscular dystrophies show characteristic patterns of fatty degenerative changes by muscle imaging, even to the extent that the patterns have been used for diagnostic purposes. However, the underlying molecular mechanisms explaining the selective involvement of muscles are not known. To test the hypothesis that different muscles may express variable amounts of different isoforms of muscle genes, we applied a custom-designed exon microarray containing probes for 57 muscle-specific genes to assay the transcriptional profiles in sets of human adult lower limb skeletal muscles. Quantitative real-time PCR and whole transcriptome sequencing were used to further analyze the results. Our results demonstrate significant variations in isoform and gene expression levels in anatomically different muscles. Comparison of the known patterns of selective involvement of certain muscles in two autosomal dominant titinopathies and one autosomal dominant myosinopathy, with the isoform and gene expression results, shows a correlation between the specific muscles involved and significant differences in the level of expression of the affected gene and exons in these same muscles compared with some other selected muscles. Our results suggest that differential expression levels of muscle genes and isoforms are one determinant in the selectivity of muscle involvement in muscular dystrophies. PMID:26269091

  10. Role of p53 isoforms and aggregations in cancer

    PubMed Central

    Kim, SeJin; An, Seong Soo A.

    2016-01-01

    Abstract p53 is a master regulatory protein that is involved in diverse cellular metabolic processes such as apoptosis, DNA repair, and cell cycle arrest. The protective function of p53 (in its homotetrameric form) as a tumor suppressor is lost in more than 50% of human cancers. Despite considerable experimental evidence suggesting the presence of multiple p53 states, it has been difficult to correlate the status of p53 with cancer response to treatments and clinical outcomes, which suggest the importance of complex but essential p53 regulatory pathways. Recent studies have indicated that the expression pattern of p53 isoforms may play a crucial role in regulating normal and cancer cell fates in response to diverse stresses. The human TP53 gene encodes at least 12 p53 isoforms, which are produced in normal tissue through alternative initiation of translation, usage of alternative promoters, and alternative splicing. Furthermore, some researchers have suggested that the formation of mutant p53 aggregates may be associated with cancer pathogenesis due to loss-of function (LoF), dominant-negative (DN), and gain-of function (GoF) effects. As different isoforms or the aggregation state of p53 may influence tumorigenesis, this review aims to examine the correlation of p53 isoforms and aggregation with cancer. PMID:27368003

  11. Regulatory Divergence of Transcript Isoforms in a Mammalian Model System

    PubMed Central

    Thybert, David; Stefflova, Klara; Watt, Stephen; Flicek, Paul; Brazma, Alvis; Marioni, John C.; Odom, Duncan T.

    2015-01-01

    Phenotypic differences between species are driven by changes in gene expression and, by extension, by modifications in the regulation of the transcriptome. Investigation of mammalian transcriptome divergence has been restricted to analysis of bulk gene expression levels and gene-internal splicing. Using allele-specific expression analysis in inter-strain hybrids of Mus musculus, we determined the contribution of multiple cellular regulatory systems to transcriptome divergence, including: alternative promoter usage, transcription start site selection, cassette exon usage, alternative last exon usage, and alternative polyadenylation site choice. Between mouse strains, a fifth of genes have variations in isoform usage that contribute to transcriptomic changes, half of which alter encoded amino acid sequence. Virtually all divergence in isoform usage altered the post-transcriptional regulatory instructions in gene UTRs. Furthermore, most genes with isoform differences between strains contain changes originating from multiple regulatory systems. This result indicates widespread cross-talk and coordination exists among different regulatory systems. Overall, isoform usage diverges in parallel with and independently to gene expression evolution, and the cis and trans regulatory contribution to each differs significantly. PMID:26339903

  12. Arabidopsis UDP-sugar pyrophosphorylase: evidence for two isoforms

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Arabidopsis UDP-sugar pyrophosphorylase (AtUSP, EC 2.7.7.64) is a broad substrate pyrophosphorylase that exhibits activity with GlcA-1-P, Gal-1-P, and Glc-1-P. Immunoblots using polyclonal antibodies raised to recombinant AtUSP demonstrated the presence of two USP isoforms of approximately 70 kDa (U...

  13. APPRIS: annotation of principal and alternative splice isoforms.

    PubMed

    Rodriguez, Jose Manuel; Maietta, Paolo; Ezkurdia, Iakes; Pietrelli, Alessandro; Wesselink, Jan-Jaap; Lopez, Gonzalo; Valencia, Alfonso; Tress, Michael L

    2013-01-01

    Here, we present APPRIS (http://appris.bioinfo.cnio.es), a database that houses annotations of human splice isoforms. APPRIS has been designed to provide value to manual annotations of the human genome by adding reliable protein structural and functional data and information from cross-species conservation. The visual representation of the annotations provided by APPRIS for each gene allows annotators and researchers alike to easily identify functional changes brought about by splicing events. In addition to collecting, integrating and analyzing reliable predictions of the effect of splicing events, APPRIS also selects a single reference sequence for each gene, here termed the principal isoform, based on the annotations of structure, function and conservation for each transcript. APPRIS identifies a principal isoform for 85% of the protein-coding genes in the GENCODE 7 release for ENSEMBL. Analysis of the APPRIS data shows that at least 70% of the alternative (non-principal) variants would lose important functional or structural information relative to the principal isoform. PMID:23161672

  14. A human Polycomb isoform lacking the Pc box does not participate to PRC1 complexes but forms protein assemblies and represses transcription.

    PubMed

    Völkel, Pamela; Le Faou, Perrine; Vandamme, Julien; Pira, Dorcas; Angrand, Pierre-Olivier

    2012-05-01

    Polycomb repression controls the expression of hundreds of genes involved in development and is mediated by essentially two classes of chromatin-associated protein complexes. The Polycomb repressive complex 2 (PRC2) trimethylates histone H3 at lysine 27, an epigenetic mark that serves as a docking site for the PRC1 protein complex. Drosophila core PRC1 is composed of four subunits: Polycomb (Pc), Posterior sex combs (Psc), Polyhomeotic (Ph) and Sex combs extra (Sce). Each of these proteins has multiple orthologs in vertebrates, thus generating an enormous scope for potential combinatorial diversity. In particular, mammalian genomes encode five Pc family members: CBX2, CBX4, CBX6, CBX7 and CBX8. To complicate matters further, distinct isoforms might arise from single genes. Here, we address the functional role of the two human CBX2 isoforms. Owing to different polyadenylation sites and alternative splicing events, the human CBX2 locus produces two transcripts: a 5-exon transcript that encodes the 532-amino acid CBX2-1 isoform that contains the conserved chromodomain and Pc box and a 4-exon transcript encoding a shorter isoform, CBX2-2, lacking the Pc box but still possessing a chromodomain. Using biochemical approaches and a novel in vivo imaging assay, we show that the short CBX2-2 isoform lacking the Pc box, does not participate in PRC1 protein complexes, but self-associates in vivo and forms complexes of high molecular weight. Furthermore, the CBX2 short isoform is still able to repress transcription, suggesting that Polycomb repression might occur in the absence of PRC1 formation. PMID:22419124

  15. Isoform-specific Binding of Selenoprotein P to the β-Propeller Domain of Apolipoprotein E Receptor 2 Mediates Selenium Supply*

    PubMed Central

    Kurokawa, Suguru; Bellinger, Frederick P.; Hill, Kristina E.; Burk, Raymond F.; Berry, Marla J.

    2014-01-01

    Sepp1 supplies selenium to tissues via receptor-mediated endocytosis. Mice, rats, and humans have 10 selenocysteines in Sepp1, which are incorporated via recoding of the stop codon, UGA. Four isoforms of rat Sepp1 have been identified, including full-length Sepp1 and three others, which terminate at the second, third, and seventh UGA codons. Previous studies have shown that the longer Sepp1 isoforms bind to the low density lipoprotein receptor apoER2, but the mechanism remains unclear. To identify the essential residues for apoER2 binding, an in vitro Sepp1 binding assay was developed using different Sec to Cys substituted variants of Sepp1 produced in HEK293T cells. ApoER2 was found to bind the two longest isoforms. These results suggest that Sepp1 isoforms with six or more selenocysteines are taken up by apoER2. Furthermore, the C-terminal domain of Sepp1 alone can bind to apoER2. These results indicate that apoER2 binds to the Sepp1 C-terminal domain and does not require the heparin-binding site, which is located in the N-terminal domain. Site-directed mutagenesis identified three residues of Sepp1 that are necessary for apoER2 binding. Sequential deletion of extracellular domains of apoER2 surprisingly identified the YWTD β-propeller domain as the Sepp1 binding site. Finally, we show that apoER2 missing the ligand-binding repeat region, which can result from cleavage at a furin cleavage site present in some apoER2 isoforms, can act as a receptor for Sepp1. Thus, longer isoforms of Sepp1 with high selenium content interact with a binding site distinct from the ligand-binding domain of apoER2 for selenium delivery. PMID:24532792

  16. A nonerythroid isoform of protein 4.1R interacts with components of the contractile apparatus in skeletal myofibers.

    PubMed

    Kontrogianni-Konstantopoulos, A; Huang, S C; Benz, E J

    2000-11-01

    The approximately 80-kDa erythroid 4.1R protein is a major component of the erythrocyte cytoskeleton, where it links transmembrane proteins to the underlying spectrin/actin complexes. A diverse collection of 4.1R isoforms has been described in nonerythroid cells, ranging from approximately 30 to approximately 210 kDa. In the current study, we identified the number and primary structure of 4.1R isoforms expressed in adult skeletal muscle and characterized the localization patterns of 4.1R message and protein. Skeletal muscle 4.1R appears to originate solely from the upstream translation initiation codon (AUG-1) residing in exon 2'. Combinations of alternatively spliced downstream exons generate an array of distinct 4.1R spliceoforms. Two major isoform classes of approximately 105/110 and approximately 135 kDa are present in muscle homogenates. 4.1R transcripts are distributed in highly ordered signal stripes, whereas 4.1R protein(s) decorate the sarcoplasm in transverse striations that are in register with A-bands. An approximately 105/110-kDa 4.1R isoform appears to occur in vivo in a supramolecular complex with major sarcomeric proteins, including myosin, alpha-actin, and alpha-tropomyosin. In vitro binding assays showed that 4.1R may interact directly with the aforementioned contractile proteins through its 10-kDa domain. All of these observations suggest a topological model whereby 4.1R may play a scaffolding role by anchoring the actomyosin myofilaments and possibly modulating their displacements during contraction/relaxation. PMID:11071908

  17. A Nonerythroid Isoform of Protein 4.1R Interacts with Components of the Contractile Apparatus in Skeletal Myofibers

    PubMed Central

    Kontrogianni-Konstantopoulos, Aikaterini; Huang, Shu-Ching; Benz, Edward J.

    2000-01-01

    The ∼80-kDa erythroid 4.1R protein is a major component of the erythrocyte cytoskeleton, where it links transmembrane proteins to the underlying spectrin/actin complexes. A diverse collection of 4.1R isoforms has been described in nonerythroid cells, ranging from ∼30 to ∼210 kDa. In the current study, we identified the number and primary structure of 4.1R isoforms expressed in adult skeletal muscle and characterized the localization patterns of 4.1R message and protein. Skeletal muscle 4.1R appears to originate solely from the upstream translation initiation codon (AUG-1) residing in exon 2′. Combinations of alternatively spliced downstream exons generate an array of distinct 4.1R spliceoforms. Two major isoform classes of ∼105/110 and ∼135 kDa are present in muscle homogenates. 4.1R transcripts are distributed in highly ordered signal stripes, whereas 4.1R protein(s) decorate the sarcoplasm in transverse striations that are in register with A-bands. An ∼105/110-kDa 4.1R isoform appears to occur in vivo in a supramolecular complex with major sarcomeric proteins, including myosin, α-actin, and α-tropomyosin. In vitro binding assays showed that 4.1R may interact directly with the aforementioned contractile proteins through its 10-kDa domain. All of these observations suggest a topological model whereby 4.1R may play a scaffolding role by anchoring the actomyosin myofilaments and possibly modulating their displacements during contraction/relaxation. PMID:11071908

  18. Plectin isoform P1b and P1d deficiencies differentially affect mitochondrial morphology and function in skeletal muscle

    PubMed Central

    Winter, Lilli; Kuznetsov, Andrey V.; Grimm, Michael; Zeöld, Anikó; Fischer, Irmgard; Wiche, Gerhard

    2015-01-01

    Plectin, a versatile 500-kDa cytolinker protein, is essential for muscle fiber integrity and function. The most common disease caused by mutations in the human plectin gene, epidermolysis bullosa simplex with muscular dystrophy (EBS-MD), is characterized by severe skin blistering and progressive muscular dystrophy. Besides displaying pathological desmin-positive protein aggregates and degenerative changes in the myofibrillar apparatus, skeletal muscle specimens of EBS-MD patients and plectin-deficient mice are characterized by massive mitochondrial alterations. In this study, we demonstrate that structural and functional alterations of mitochondria are a primary aftermath of plectin deficiency in muscle, contributing to myofiber degeneration. We found that in skeletal muscle of conditional plectin knockout mice (MCK-Cre/cKO), mitochondrial content was reduced, and mitochondria were aggregated in sarcoplasmic and subsarcolemmal regions and were no longer associated with Z-disks. Additionally, decreased mitochondrial citrate synthase activity, respiratory function and altered adenosine diphosphate kinetics were characteristic of plectin-deficient muscles. To analyze a mechanistic link between plectin deficiency and mitochondrial alterations, we comparatively assessed mitochondrial morphology and function in whole muscle and teased muscle fibers of wild-type, MCK-Cre/cKO and plectin isoform-specific knockout mice that were lacking just one isoform (either P1b or P1d) while expressing all others. Monitoring morphological alterations of mitochondria, an isoform P1b-specific phenotype affecting the mitochondrial fusion–fission machinery and manifesting with upregulated mitochondrial fusion-associated protein mitofusin-2 could be identified. Our results show that the depletion of distinct plectin isoforms affects mitochondrial network organization and function in different ways. PMID:26019234

  19. Thermodynamic analysis of progesterone receptor-promoter interactions reveals a molecular model for isoform-specific function.

    PubMed

    Connaghan-Jones, Keith D; Heneghan, Aaron F; Miura, Michael T; Bain, David L

    2007-02-13

    Human progesterone receptors (PR) exist as two functionally distinct isoforms, PR-A and PR-B. The proteins are identical except for an additional 164 residues located at the N terminus of PR-B. To determine the mechanisms responsible for isoform-specific functional differences, we present here a thermodynamic dissection of PR-A-promoter interactions and compare the results to our previous work on PR-B. This analysis has generated a number of results inconsistent with the traditional, biochemically based model of receptor function. Specifically, statistical models invoking preformed PR-A dimers as the active binding species demonstrate that intrinsic binding energetics are over an order of magnitude greater than is apparent. High-affinity binding is opposed, however, by a large energetic penalty. The consequences of this penalty are 2-fold: Successive monomer binding to a palindromic response element is thermodynamically favored over preformed dimer binding, and DNA-induced dimerization of the monomers is largely abolished. Furthermore, PR-A binding to multiple PREs is only weakly cooperative, as judged by a 5-fold increase in overall stability. Comparison of these results to our work on PR-B demonstrates that whereas both isoforms appear to have similar DNA binding affinities, PR-B in fact has a greatly increased intrinsic binding affinity and cooperative binding ability relative to PR-A. These differences thus suggest that residues unique to PR-B allosterically regulate the energetics of cooperative promoter assembly. From a functional perspective, the differences in microscopic affinities predict receptor-promoter occupancies that accurately correlate with the transcriptional activation profiles seen for each isoform. PMID:17277083

  20. Plectin isoform P1b and P1d deficiencies differentially affect mitochondrial morphology and function in skeletal muscle.

    PubMed

    Winter, Lilli; Kuznetsov, Andrey V; Grimm, Michael; Zeöld, Anikó; Fischer, Irmgard; Wiche, Gerhard

    2015-08-15

    Plectin, a versatile 500-kDa cytolinker protein, is essential for muscle fiber integrity and function. The most common disease caused by mutations in the human plectin gene, epidermolysis bullosa simplex with muscular dystrophy (EBS-MD), is characterized by severe skin blistering and progressive muscular dystrophy. Besides displaying pathological desmin-positive protein aggregates and degenerative changes in the myofibrillar apparatus, skeletal muscle specimens of EBS-MD patients and plectin-deficient mice are characterized by massive mitochondrial alterations. In this study, we demonstrate that structural and functional alterations of mitochondria are a primary aftermath of plectin deficiency in muscle, contributing to myofiber degeneration. We found that in skeletal muscle of conditional plectin knockout mice (MCK-Cre/cKO), mitochondrial content was reduced, and mitochondria were aggregated in sarcoplasmic and subsarcolemmal regions and were no longer associated with Z-disks. Additionally, decreased mitochondrial citrate synthase activity, respiratory function and altered adenosine diphosphate kinetics were characteristic of plectin-deficient muscles. To analyze a mechanistic link between plectin deficiency and mitochondrial alterations, we comparatively assessed mitochondrial morphology and function in whole muscle and teased muscle fibers of wild-type, MCK-Cre/cKO and plectin isoform-specific knockout mice that were lacking just one isoform (either P1b or P1d) while expressing all others. Monitoring morphological alterations of mitochondria, an isoform P1b-specific phenotype affecting the mitochondrial fusion-fission machinery and manifesting with upregulated mitochondrial fusion-associated protein mitofusin-2 could be identified. Our results show that the depletion of distinct plectin isoforms affects mitochondrial network organization and function in different ways. PMID:26019234

  1. Cell, Isoform, and Environment Factors Shape Gradients and Modulate Chemotaxis

    PubMed Central

    Chang, S. Laura; Cavnar, Stephen P.; Takayama, Shuichi; Luker, Gary D.; Linderman, Jennifer J.

    2015-01-01

    Chemokine gradient formation requires multiple processes that include ligand secretion and diffusion, receptor binding and internalization, and immobilization of ligand to surfaces. To understand how these events dynamically shape gradients and influence ensuing cell chemotaxis, we built a multi-scale hybrid agent-based model linking gradient formation, cell responses, and receptor-level information. The CXCL12/CXCR4/CXCR7 signaling axis is highly implicated in metastasis of many cancers. We model CXCL12 gradient formation as it is impacted by CXCR4 and CXCR7, with particular focus on the three most highly expressed isoforms of CXCL12. We trained and validated our model using data from an in vitro microfluidic source-sink device. Our simulations demonstrate how isoform differences on the molecular level affect gradient formation and cell responses. We determine that ligand properties specific to CXCL12 isoforms (binding to the migration surface and to CXCR4) significantly impact migration and explain differences in in vitro chemotaxis data. We extend our model to analyze CXCL12 gradient formation in a tumor environment and find that short distance, steep gradients characteristic of the CXCL12-γ isoform are effective at driving chemotaxis. We highlight the importance of CXCL12-γ in cancer cell migration: its high effective affinity for both extracellular surface sites and CXCR4 strongly promote CXCR4+ cell migration. CXCL12-γ is also more difficult to inhibit, and we predict that co-inhibition of CXCR4 and CXCR7 is necessary to effectively hinder CXCL12-γ-induced migration. These findings support the growing importance of understanding differences in protein isoforms, and in particular their implications for cancer treatment. PMID:25909600

  2. SUGARBEET ROOT SUCROSE SYNTHASE ISOFORMS DIFFER IN DEVELOPMENTAL EXPRESSION, SUBUNIT COMPOSITION AND RESPONSE TO PH.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Two sucrose synthase isoforms have been identified by activity stained isoelectric focused polyacrylamide electrophoresis in developing sugarbeet (Beta vulgaris L.) root. Sucrose synthase isoform I (SuSyI) was present from the early stages of development to maturity. Sucrose synthase isoform II (S...

  3. The Three Maize Sucrose synthase Isoforms Differ in Distribution, Localization, and Phosphorylation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Although sucrose synthase (SUS) is widely appreciated for its role in plant metabolism and growth, very little is known about the contribution of each of the SUS isoforms to these processes. Using isoform-specific antibodies, we evaluated the three known isoforms individually at the protein level. ...

  4. Genetic variations of 14-3-3E1 isoform in rainbow trout (Oncorhynchus mykiss)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The highly conserved family of 14-3-3 proteins functions in the regulation of a wide variety of cellular processes. The presence of 14-3-3 isoforms and the diversity of cellular processes regulated by 14-3-3 isoforms suggest functional specificity of the isoforms. Several studies have observed diffe...

  5. Touch communicates distinct emotions.

    PubMed

    Hertenstein, Matthew J; Keltner, Dacher; App, Betsy; Bulleit, Brittany A; Jaskolka, Ariane R

    2006-08-01

    The study of emotional signaling has focused almost exclusively on the face and voice. In 2 studies, the authors investigated whether people can identify emotions from the experience of being touched by a stranger on the arm (without seeing the touch). In the 3rd study, they investigated whether observers can identify emotions from watching someone being touched on the arm. Two kinds of evidence suggest that humans can communicate numerous emotions with touch. First, participants in the United States (Study 1) and Spain (Study 2) could decode anger, fear, disgust, love, gratitude, and sympathy via touch at much-better-than-chance levels. Second, fine-grained coding documented specific touch behaviors associated with different emotions. In Study 3, the authors provide evidence that participants can accurately decode distinct emotions by merely watching others communicate via touch. The findings are discussed in terms of their contributions to affective science and the evolution of altruism and cooperation. PMID:16938094

  6. One isoform of Arg/Abl2 tyrosine kinase is nuclear and the other seven cytosolic isoforms differently modulate cell morphology, motility and the cytoskeleton

    SciTech Connect

    Bianchi, Cristina; Torsello, Barbara; Di Stefano, Vitalba; Zipeto, Maria A.; Facchetti, Rita; Bombelli, Silvia; Perego, Roberto A.

    2013-08-01

    The non-receptor tyrosine kinase Abelson related gene (Arg/Abl2) regulates cell migration and morphogenesis by modulating the cytoskeleton. Arg promotes actin-based cell protrusions and spreading, and inhibits cell migration by attenuating stress fiber formation and contractility via activation of the RhoA inhibitor, p190RhoGAP, and by regulating focal adhesion dynamics also via CrkII phosphorylation. Eight full-length Arg isoforms with different N- and C-termini are endogenously expressed in human cells. In this paper, the eight Arg isoforms, subcloned in the pFLAG-CMV2 vector, were transfected in COS-7 cells in order to study their subcellular distribution and role in cell morphology, migration and cytoskeletal modulation. The transfected 1BSCTS Arg isoform has a nuclear distribution and phosphorylates CrkII in the nucleus, whilst the other isoforms are detected in the cytoplasm. The 1BLCTL, 1BSCTL, 1ASCTS isoforms were able to significantly decrease stress fibers, induce cell shrinkage and filopodia-like protrusions with a significant increase in p190RhoGAP phosphorylation. In contrast, 1ALCTL, 1ALCTS, 1ASCTL and 1BLCTS isoforms do not significantly decrease stress fibers and induce the formation of retraction tail-like protrusions. The 1BLCTL and 1ALCTL isoforms have different effects on cell migration and focal adhesions. All these data may open new perspectives to study the mechanisms of cell invasiveness. -Highlights: • Each of the eight Arg isoforms was transfected in COS-7 cells. • Only the 1BSCTS Arg isoform has a nuclear distribution in transfected cells. • The cytoplasmic isoforms and F-actin colocalize cortically and in cell protrusions. • Arg isoforms differently phosphorylate p190RhoGAP and CrkII. • Arg isoforms differently modulate stress fibers, cell protrusions and motility.

  7. Cell-Surface and Secreted Isoforms of CSF-1 Exert Opposing Roles in Macrophage-Mediated Neural Damage in Cx32-Deficient Mice

    PubMed Central

    Basu, Ranu; Stanley, E. Richard

    2016-01-01

    Previous studies in myelin-mutant mouse models of the inherited and incurable nerve disorder, Charcot-Marie-Tooth (CMT) neuropathy, have demonstrated that low-grade secondary inflammation implicating phagocytosing macrophages amplifies demyelination, Schwann cell dedifferentiation and perturbation of axons. The cytokine colony stimulating factor-1 (CSF-1) acts as an important regulator of these macrophage-related disease mechanisms, as genetic and pharmacologic approaches to block the CSF-1/CSF-1R signaling result in a significant alleviation of pathological alterations in mutant peripheral nerves. In mouse models of CMT1A and CMT1X, as well as in human biopsies, CSF-1 is predominantly expressed by endoneurial fibroblasts, which are closely associated with macrophages, suggesting local stimulatory mechanisms. Here we investigated the impact of cell-surface and secreted isoforms of CSF-1 on macrophage-related disease in connexin32-deficient (Cx32def) mice, a mouse model of CMT1X. Our present observations suggest that the secreted proteoglycan isoform (spCSF-1) is predominantly expressed by fibroblasts, whereas the membrane-spanning cell-surface isoform (csCSF-1) is expressed by macrophages. Using crossbreeding approaches to selectively restore or overexpress distinct isoforms in CSF-1-deficient (osteopetrotic) Cx32def mice, we demonstrate that both isoforms equally regulate macrophage numbers dose-dependently. However, spCSF-1 mediates macrophage activation and macrophage-related neural damage, whereas csCSF-1 inhibits macrophage activation and attenuates neuropathy. These results further corroborate the important role of secondary inflammation in mouse models of CMT1 and might identify specific targets for therapeutic approaches to modulate innate immune reactions. SIGNIFICANCE STATEMENT Mouse models of Charcot-Marie-Tooth neuropathy have indicated that low-grade secondary inflammation involving phagocytosing macrophages amplifies demyelination, Schwann cell

  8. EGFR soluble isoforms and their transcripts are expressed in meningiomas.

    PubMed

    Guillaudeau, Angélique; Durand, Karine; Bessette, Barbara; Chaunavel, Alain; Pommepuy, Isabelle; Projetti, Fabrice; Robert, Sandrine; Caire, François; Rabinovitch-Chable, Hélène; Labrousse, François

    2012-01-01

    The EGFR (epidermal growth factor receptor) is involved in the oncogenesis of many tumors. In addition to the full-length EGFR (isoform a), normal and tumor cells produce soluble EGFR isoforms (sEGFR) that lack the intracellular domain. sEGFR isoforms b, c and d are encoded by EGFR variants 2 (v2), 3 (v3) and 4 (v4) mRNA resulting from gene alternative splicing. Accordingly, the results of EGFR protein expression analysis depend on the domain targeted by the antibodies. In meningiomas, EGFR expression investigations mainly focused on EGFR isoform a. sEGFR and EGFRvIII mutant, that encodes a constitutively active truncated receptor, have not been studied. In a 69 meningiomas series, protein expression was analyzed by immunohistochemistry using extracellular domain targeted antibody (ECD-Ab) and intracellular domain targeted antibody (ICD-Ab). EGFRv1 to v4 and EGFRvIII mRNAs were quantified by RT-PCR and EGFR amplification revealed by MLPA. Results were analyzed with respect to clinical data, tumor resection (Simpson grade), histological type, tumor grade, and patient outcome.Immunochemical staining was stronger with ECD-Ab than with ICD-Ab. Meningiomas expressed EGFRv1 to -v4 mRNAs but not EGFRvIII mutant. Intermediate or high ECD-Ab staining and high EGFRv1 to v4 mRNA levels were associated to a better progression free survival (PFS). PFS was also improved in women, when tumor resection was evaluated as Simpson 1 or 2, in grade I vs. grade II and III meningiomas and when Ki67 labeling index was lower than 10%. Our results suggest that, EGFR protein isoforms without ICD and their corresponding mRNA variants are expressed in meningiomas in addition to the whole isoform a. EGFRvIII was not expressed. High expression levels seem to be related to a better prognosis. These results indicate that the oncogenetic mechanisms involving the EGFR pathway in meningiomas could be different from other tumor types. PMID:22623992

  9. Alternative mRNA splicing of SMRT creates functional diversity by generating corepressor isoforms with different affinities for different nuclear receptors.

    PubMed

    Goodson, Michael L; Jonas, Brian A; Privalsky, Martin L

    2005-03-01

    Many eukaryotic transcription factors are bimodal in their regulatory properties and can both repress and activate expression of their target genes. These divergent transcriptional properties are conferred through recruitment of auxiliary proteins, denoted coactivators and corepressors. Repression plays a particularly critical role in the functions of the nuclear receptors, a large family of ligand-regulated transcription factors involved in metazoan development, differentiation, reproduction, and homeostasis. The SMRT corepressor interacts directly with nuclear receptors and serves, in turn, as a platform for the assembly of a larger corepressor complex. We report here that SMRT is expressed in cells by alternative mRNA splicing to yield two distinct variants or isoforms. We designate these isoforms SMRTalpha and SMRTtau and demonstrate that these isoforms have significantly different affinities for different nuclear receptors. These isoforms are evolutionarily conserved and are expressed in a tissue-specific manner. Our results suggest that differential mRNA splicing serves to customize corepressor function in different cells, allowing the transcriptional properties of nuclear receptors to be adapted to different contexts. PMID:15632172

  10. Quantitative rt-PCR analysis of uncoupling protein isoforms in mouse brain cortex: methodological optimization and comparison of expression with brown adipose tissue and skeletal muscle.

    PubMed

    Lengacher, Sylvain; Magistretti, Pierre J; Pellerin, Luc

    2004-07-01

    Uncoupling proteins (UCPs) present in the inner mitochondrial membrane are involved in uncoupling respiration from ATP synthesis. Five UCP isoforms have been identified but information about their presence and level of expression in the central nervous system remains incomplete. To determine the nature and proportion of UCP isoform mRNAs present in brain cortex, we developed and optimized a specific quantitative reverse-transcription polymerase chain reaction procedure. Optimal range of RNA concentrations to be used in the reverse-transcriptase reaction was determined. Primer design and concentration were optimized for each target gene while polymerase chain reaction efficiency was assessed for a range of reverse-transcriptase dilutions. Genomic contribution to the quantitative signal was evaluated for each isoform and minimized. Three reference genes were tested for normalization, and beta-actin was found to be the most stable among tissues. Results indicate that brain cortex contains significant amounts of all UCP mRNAs, with UCP5 and UCP4 being the most abundant, as opposed to brown adipose tissue and skeletal muscle, which predominantly express UCP1 and UCP3, respectively. These data provide a first quantitative assessment of UCP mRNA expression in mouse brain, showing the presence of all five isoforms with distinct proportions, thus suggesting specific roles in the central nervous system. PMID:15241186

  11. Effect of resistance exercise intensity on the expression of PGC-1α isoforms and the anabolic and catabolic signaling mediators, IGF-1 and myostatin, in human skeletal muscle.

    PubMed

    Schwarz, Neil A; McKinley-Barnard, Sarah K; Spillane, Mike B; Andre, Thomas L; Gann, Joshua J; Willoughby, Darryn S

    2016-08-01

    The purpose of this study was to investigate the acute messenger (mRNA) expression of the peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) isoforms, insulin-like growth factor-1Ea (IGF-1Ea), and myostatin in response to 2 resistance exercise intensities. In a uniform-balanced, crossover design, 10 participants performed 2 separate testing sessions involving a lower body resistance exercise component consisting of a lower intensity (50% of 1-repetition maximum; 1RM) protocol and a higher intensity (80% of 1RM) protocol of equal volumes. Muscle samples were obtained at before exercise, 45 min, 3 h, 24 h, and 48 h postexercise. Resistance exercise did not alter total PGC-1α mRNA expression; however, distinct responses of each PGC-1α isoform were observed. The response of each isoform was consistent between sessions, suggesting no effect of resistance exercise intensity on the complex transcriptional expression of the PGC-1α gene. IGF-1Ea mRNA expression significantly increased following the higher intensity session compared with pre-exercise and the lower intensity session. Myostatin mRNA expression was significantly reduced compared with pre-exercise values at all time points with no difference between exercise intensity. Further research is needed to determine the effects of the various isoforms of PGC-1α in human skeletal muscle on the translational level as well as their relation to the expression of IGF-1 and myostatin. PMID:27467217

  12. Haplotype and isoform specific expression estimation using multi-mapping RNA-seq reads

    PubMed Central

    2011-01-01

    We present a novel pipeline and methodology for simultaneously estimating isoform expression and allelic imbalance in diploid organisms using RNA-seq data. We achieve this by modeling the expression of haplotype-specific isoforms. If unknown, the two parental isoform sequences can be individually reconstructed. A new statistical method, MMSEQ, deconvolves the mapping of reads to multiple transcripts (isoforms or haplotype-specific isoforms). Our software can take into account non-uniform read generation and works with paired-end reads. PMID:21310039

  13. [PHF10 isoforms are phosphorylated in the PBAF mammalian chromatin remodeling complex].

    PubMed

    Brechalov, A V; Valieva, M E; Georgieva, S G; Soshnikova, N V

    2016-01-01

    Chromatin remodeling complex PBAF(SWI/SNF) alters the structure of chromatin and controls gene expression. PHF10 is a specific subunit of PBAF complex and is expressed as four isoforms in mammalian cells. We demonstrated that all isoforms are expressed in various human cell types of different histological origins. All four isoforms are extensively phosphorylated and their phosphorylation level is depended on the cell type. Phosphorylation of PHF10 isoforms occurs while they are incorporated as a subunit of the PBAF complex, and therefore phosphorylation of PHF10 isoforms may play an essential role in regulation of PBAF complex's function and mechanism of action. PMID:27239853

  14. Disease of distinction.

    PubMed

    Schwartz, Stephan A

    2006-01-01

    Gout is one of the rare diseases that defines its sufferers by class and culture. It is also one of the first chronic diseases to be clinically described. The Egyptians had identified gout as a distinct disorder by 2640 bce. This paper traces the history of gout from its earliest recorded period down to modern times, with a particular emphasis on the cultural, political, geopolitical, and social aspects. Included is a discussion of its role in the American Revolution. Today, gout is a well-understood clinically managed arthritic disease that excites little comment. This is an entirely modern perspective. For most of human history, gout was a disease of distinction that dominated much of medicine, playing the same role in Rome's third century bce aristocracy it would later play in the aristocracies of 17th and 18th century France and England, when each of these countries dominated the world. Because it was considered a disease of lifestyle until modern times, when genetics began to be understood, gout was associated with rich, high status Caucasian men and their excessive consumption of drink and rich foods. It was virtually unknown in Asia, until Western dietary practices became widespread there. From earliest history, gout has been linked with high IQ and sexual promiscuity, which made it grist for artists and writers, and their social commentary up to the time of Dickens; this is discussed, with examples. Because of its association with the rich, gout also developed a powerful moralistic aspect, particularly during the Christian era when the concept of sin was a cultural fundamental. The loose living and indulgence of the rich and the gout it produced made the disease a parable of Christian ethics. The Italian poet Francesco Petrarch (1304-1374) was one of the first to establish this nuance, and it influenced how gout was seen for centuries. Part of what gave gout its special character was that while it tortured, it rarely killed. Indeed, when death was a

  15. Differential Effects of Hepatocyte Nuclear Factor 4α Isoforms on Tumor Growth and T-Cell Factor 4/AP-1 Interactions in Human Colorectal Cancer Cells

    PubMed Central

    Vuong, Linh M.; Chellappa, Karthikeyani; Dhahbi, Joseph M.; Deans, Jonathan R.; Fang, Bin; Bolotin, Eugene; Titova, Nina V.; Hoverter, Nate P.; Spindler, Stephen R.; Waterman, Marian L.

    2015-01-01

    The nuclear receptor hepatocyte nuclear factor 4α (HNF4α) is tumor suppressive in the liver but amplified in colon cancer, suggesting that it also might be oncogenic. To investigate whether this discrepancy is due to different HNF4α isoforms derived from its two promoters (P1 and P2), we generated Tet-On-inducible human colon cancer (HCT116) cell lines that express either the P1-driven (HNF4α2) or P2-driven (HNF4α8) isoform and analyzed them for tumor growth and global changes in gene expression (transcriptome sequencing [RNA-seq] and chromatin immunoprecipitation sequencing [ChIP-seq]). The results show that while HNF4α2 acts as a tumor suppressor in the HCT116 tumor xenograft model, HNF4α8 does not. Each isoform regulates the expression of distinct sets of genes and recruits, colocalizes, and competes in a distinct fashion with the Wnt/β-catenin mediator T-cell factor 4 (TCF4) at CTTTG motifs as well as at AP-1 motifs (TGAXTCA). Protein binding microarrays (PBMs) show that HNF4α and TCF4 share some but not all binding motifs and that single nucleotide polymorphisms (SNPs) in sites bound by both HNF4α and TCF4 can alter binding affinity in vitro, suggesting that they could play a role in cancer susceptibility in vivo. Thus, the HNF4α isoforms play distinct roles in colon cancer, which could be due to differential interactions with the Wnt/β-catenin/TCF4 and AP-1 pathways. PMID:26240283

  16. PAX6 Isoforms, along with Reprogramming Factors, Differentially Regulate the Induction of Cornea-specific Genes

    PubMed Central

    Sasamoto, Yuzuru; Hayashi, Ryuhei; Park, Sung-Joon; Saito-Adachi, Mihoko; Suzuki, Yutaka; Kawasaki, Satoshi; Quantock, Andrew J.; Nakai, Kenta; Tsujikawa, Motokazu; Nishida, Kohji

    2016-01-01

    PAX6 is the key transcription factor involved in eye development in humans, but the differential functions of the two PAX6 isoforms, isoform-a and isoform-b, are largely unknown. To reveal their function in the corneal epithelium, PAX6 isoforms, along with reprogramming factors, were transduced into human non-ocular epithelial cells. Herein, we show that the two PAX6 isoforms differentially and cooperatively regulate the expression of genes specific to the structure and functions of the corneal epithelium, particularly keratin 3 (KRT3) and keratin 12 (KRT12). PAX6 isoform-a induced KRT3 expression by targeting its upstream region. KLF4 enhanced this induction. A combination of PAX6 isoform-b, KLF4, and OCT4 induced KRT12 expression. These new findings will contribute to furthering the understanding of the molecular basis of the corneal epithelium specific phenotype. PMID:26899008

  17. Functional impact of splice isoform diversity in individual cells

    PubMed Central

    Yap, Karen; Makeyev, Eugene V.

    2016-01-01

    Alternative pre-mRNA splicing provides an effective means for expanding coding capacity of eukaryotic genomes. Recent studies suggest that co-expression of different splice isoforms may increase diversity of RNAs and proteins at a single-cell level. A pertinent question in the field is whether such co-expression is biologically meaningful or, rather, represents insufficiently stringent splicing regulation. Here we argue that isoform co-expression may produce functional outcomes that are difficult and sometimes impossible to achieve using other regulation strategies. Far from being a ‘splicing noise’, co-expression is often established through co-ordinated activity of specific cis-elements and trans-acting factors. Further work in this area may uncover new biological functions of alternative splicing (AS) and generate important insights into mechanisms allowing different cell types to attain their unique molecular identities. PMID:27528755

  18. Foundations of Distinctive Feature Theory.

    ERIC Educational Resources Information Center

    Baltaxe, Christiane A. M.

    This treatise on the theoretical and historical foundations of distinctive feature theory traces the evolution of the distinctive features concept in the context of related notions current in linguistic theory, discusses the evolution of individual distinctive features, and criticizes certain acoustic and perceptual correlates attributed to these…

  19. Comparison of liver oncogenic potential among human RAS isoforms

    PubMed Central

    Chung, Sook In; Moon, Hyuk; Ju, Hye-Lim; Kim, Dae Yeong; Cho, Kyung Joo; Ribback, Silvia; Dombrowski, Frank; Calvisi, Diego F.; Ro, Simon Weonsang

    2016-01-01

    Mutation in one of three RAS genes (i.e., HRAS, KRAS, and NRAS) leading to constitutive activation of RAS signaling pathways is considered a key oncogenic event in human carcinogenesis. Whether activated RAS isoforms possess different oncogenic potentials remains an unresolved question. Here, we compared oncogenic properties among RAS isoforms using liver-specific transgenesis in mice. Hydrodynamic transfection was performed using transposons expressing short hairpin RNA downregulating p53 and an activated RAS isoform, and livers were harvested at 23 days after gene delivery. No differences were found in the hepatocarcinogenic potential among RAS isoforms, as determined by both gross examination of livers and liver weight per body weight ratio (LW/BW) of mice expressing HRASQ61L, KRAS4BG12V and NRASQ61K. However, the tumorigenic potential differed significantly between KRAS splicing variants. The LW/BW ratio in KRAS4AG12V mice was significantly lower than in KRAS4BG12V mice (p < 0.001), and KRAS4AG12V mice lived significantly longer than KRRAS4BG12V mice (p < 0.0001). Notably, tumors from KRAS4AG12V mice displayed higher expression of the p16INK4A tumor suppressor when compared with KRAS4BG12V tumors. Forced overexpression of p16INK4A significantly reduced tumor growth in KRAS4BG12V mice, suggesting that upregulation of p16INK4A by KRAS4AG12V presumably delays tumor development driven by the latter oncogene. PMID:26799184

  20. Structural differences between C-terminal regions of tropomyosin isoforms

    PubMed Central

    Śliwińska, Małgorzata

    2013-01-01

    Tropomyosins are actin-binding regulatory proteins which overlap end-to-end along the filament. High resolution structures of the overlap regions were determined for muscle and non-muscle tropomyosins in the absence of actin. Conformations of the junction regions bound to actin are unknown. In this work, orientation of the overlap on actin alone and on actin–myosin complex was evaluated by measuring FRET distances between a donor (AEDANS) attached to tropomyosin and an acceptor (DABMI) bound to actin’s Cys374. Donor was attached to the Cys residue introduced by site-directed mutagenesis near the C-terminal half of the overlap. The recombinant alpha-tropomyosin isoforms used in this study – skeletal muscle skTM, non-muscle TM2 and TM5a, and chimeric TM1b9a had various amino acid sequences of the N- and C-termini involved in the end-to-end overlap. The donor-acceptor distances calculated for each isoform varied between 36.4 Å and 48.1 Å. Rigor binding of myosin S1 increased the apparent FRET distances of skTM and TM2, but decreased the distances separating TM5a and TM1b9a from actin. The results show that isoform-specific sequences of the end-to-end overlaps determine orientations and dynamics of tropomyosin isoforms on actin. This can be important for specificity of tropomyosin in the regulation of actin filament diverse functions. PMID:24167776

  1. Different Protein Kinase C Isoforms Determine Growth Factor Specificity in Neuronal Cells

    PubMed Central

    Corbit, Kevin C.; Soh, Jae-Won; Yoshida, Keiko; Eves, Eva M.; Weinstein, I. Bernard; Rosner, Marsha Rich

    2000-01-01

    Although mitogenic and differentiating factors often activate a number of common signaling pathways, the mechanisms leading to their distinct cellular outcomes have not been elucidated. In a previous report, we demonstrated that mitogen-activated protein (MAP) kinase (ERK) activation by the neurogenic agents fibroblast growth factor (FGF) and nerve growth factor is dependent on protein kinase Cδ (PKCδ), whereas MAP kinase activation in response to the mitogen epidermal growth factor (EGF) is independent of PKCδ in rat hippocampal (H19-7) and pheochromocytoma (PC12) cells. We now show that EGF activates MAP kinase through a PKCζ-dependent pathway involving phosphatidylinositol 3-kinase and PDK1 in H19-7 cells. PKCζ, like PKCδ, acts upstream of MEK, and PKCζ can potentiate Raf-1 activation by EGF. Inhibition of PKCζ also blocks EGF-induced DNA synthesis as monitored by bromodeoxyuridine incorporation in H19-7 cells. Finally, in embryonic rat brain hippocampal cell cultures, inhibitors of PKCζ or PKCδ suppress MAP kinase activation by EGF or FGF, respectively, indicating that these factors activate distinct signaling pathways in primary as well as immortalized neural cells. Taken together, these results implicate different PKC isoforms as determinants of growth factor signaling specificity within the same cell. Furthermore, these data provide a mechanism whereby different growth factors can differentially activate a common signaling intermediate and thereby generate biological diversity. PMID:10891480

  2. Characterisation of CDKL5 Transcript Isoforms in Human and Mouse.

    PubMed

    Hector, Ralph D; Dando, Owen; Landsberger, Nicoletta; Kilstrup-Nielsen, Charlotte; Kind, Peter C; Bailey, Mark E S; Cobb, Stuart R

    2016-01-01

    Mutations in the X-linked Cyclin-Dependent Kinase-Like 5 gene (CDKL5) cause early onset infantile spasms and subsequent severe developmental delay in affected children. Deleterious mutations have been reported to occur throughout the CDKL5 coding region. Several studies point to a complex CDKL5 gene structure in terms of exon usage and transcript expression. Improvements in molecular diagnosis and more extensive research into the neurobiology of CDKL5 and pathophysiology of CDKL5 disorders necessitate an updated analysis of the gene. In this study, we have analysed human and mouse CDKL5 transcript patterns both bioinformatically and experimentally. We have characterised the predominant brain isoform of CDKL5, a 9.7 kb transcript comprised of 18 exons with a large 6.6 kb 3'-untranslated region (UTR), which we name hCDKL5_1. In addition we describe new exonic regions and a range of novel splice and UTR isoforms. This has enabled the description of an updated gene model in both species and a standardised nomenclature system for CDKL5 transcripts. Profiling revealed tissue- and brain development stage-specific differences in expression between transcript isoforms. These findings provide an essential backdrop for the diagnosis of CDKL5-related disorders, for investigations into the basic biology of this gene and its protein products, and for the rational design of gene-based and molecular therapies for these disorders. PMID:27315173

  3. Specific roles of GABAB(1) receptor isoforms in cognition

    PubMed Central

    Jacobson, Laura H.; Kelly, Peter H.; Bettler, Bernhard; Kaupmann, Klemens; Cryan, John F.

    2010-01-01

    The GABAB receptor is a heterodimer of GABAB(1) and GABAB(2) subunits. There are two isoforms of the GABAB(1) subunit: GABAB(1a) and GABAB(1b). Recent studies with mutant mice suggest a differential role for the two GABAB(1) isoforms in behavioural processes. As pharmacological and genetic studies have implicated GABAB receptors in cognition we investigated the behaviour of GABAB(1a) −/− and GABAB(1b) −/− mice in different types of cognitive paradigms. GABAB(1a) −/− and GABAB(1b) −/− mice were both impaired relative to wildtype controls in a continuous spontaneous alternation behaviour test of working spatial memory. In contrast to the reported phenotype of GABAB(1) −/− mice, however, neither GABAB(1a) −/− nor GABAB(1b) −/− mice were deficient in a passive avoidance task. On the other hand, GABAB(1a) −/− mice were impaired in familiar and novel object recognition. We conclude that GABAB(1) isoforms contribute differentially to GABAB receptor-mediated cognitive processes. PMID:17498817

  4. Isoform-specific targeting of ROCK proteins in immune cells

    PubMed Central

    Zanin-Zhorov, Alexandra; Flynn, Ryan; Waksal, Samuel D.; Blazar, Bruce R.

    2016-01-01

    ABSTRACT Rho-associated kinase 1 (ROCK1) and ROCK2 are activated by Rho GTPase and control cytoskeleton rearrangement through modulating the phosphorylation of their down-stream effector molecules. Although these 2 isoforms share more than 90% homology within their kinase domain the question of whether ROCK proteins function identically in different cell types is not clear. By using both pharmacological inhibition and genetic knockdown approaches recent studies suggest that the ROCK2 isoform plays an exclusive role in controlling of T-cell plasticity and macrophage polarization. Specifically, selective ROCK2 inhibition shifts the balance between pro-inflammatory and regulatory T-cell subsets via concurrent regulation of STAT3 and STAT5 phosphorylation, respectively. Furthermore, the administration of an orally available selective ROCK2 inhibitor effectively ameliorates clinical manifestations in experimental models of autoimmunity and chronic graft-vs.-host disease (cGVHD). Because ROCK2 inhibition results in the suppression of M2-type macrophages while favoring polarization of M1-type macrophages, ROCK2 inhibition can correct the macrophage imbalance seen during age-related macular degeneration (AMD). In summary, the exclusive role of ROCK2 in immune system modulation argues for the development and testing of isoform-specific ROCK2 inhibitors for the treatment of inflammatory disorders. PMID:27254302

  5. Cholesterol efflux is LXRα isoform-dependent in human macrophages

    PubMed Central

    2014-01-01

    Background The nuclear receptor liver X receptor (LXR) has two isoforms: LXRα and LXRβ. LXR activation promotes cholesterol efflux in macrophages, but the relative importance of each LXR isoform in mediating cholesterol efflux remains elusive. Methods We evaluated the ability of different doses of LXRs agonist T0901317 to affect cholesterol efflux in human macrophages and its relationship with mRNA and protein levels of several well-characterized proteins involved in cholesterol efflux, including ABCA1, ABCG1, SR-BI, LXRβ and LXRα, using quantitative real-time PCR, Western blotting, and siRNA techniques. Results Here we show that LXRα rather than LXRβ sustains baseline cholesterol efflux in human blood-derived macrophages. Treatment of human macrophages with a non-isoform-specific LXR agonist T0901317 substantially increased HDL- and apoA-I-mediated cholesterol efflux, which was associated with increased mRNA and protein expression levels of ABCA1, ABCG1, SR-BI, LXRα and LXRβ. The siRNA- mediated silencing of LXRα, but not LXRβ significantly reduced the protein levels of ABCA1,ABCG1, and SR-BI as wellas HDL- and ApoA1-mediated cholesterol in human macrophages. Conclusions These findings imply that LXRα- rather than LXRβ- specific agonists may promote reverse cholesterol transport in humans. PMID:24996838

  6. Characterisation of CDKL5 Transcript Isoforms in Human and Mouse

    PubMed Central

    Dando, Owen; Landsberger, Nicoletta; Kilstrup-Nielsen, Charlotte; Kind, Peter C.; Bailey, Mark E. S.; Cobb, Stuart R.

    2016-01-01

    Mutations in the X-linked Cyclin-Dependent Kinase-Like 5 gene (CDKL5) cause early onset infantile spasms and subsequent severe developmental delay in affected children. Deleterious mutations have been reported to occur throughout the CDKL5 coding region. Several studies point to a complex CDKL5 gene structure in terms of exon usage and transcript expression. Improvements in molecular diagnosis and more extensive research into the neurobiology of CDKL5 and pathophysiology of CDKL5 disorders necessitate an updated analysis of the gene. In this study, we have analysed human and mouse CDKL5 transcript patterns both bioinformatically and experimentally. We have characterised the predominant brain isoform of CDKL5, a 9.7 kb transcript comprised of 18 exons with a large 6.6 kb 3’-untranslated region (UTR), which we name hCDKL5_1. In addition we describe new exonic regions and a range of novel splice and UTR isoforms. This has enabled the description of an updated gene model in both species and a standardised nomenclature system for CDKL5 transcripts. Profiling revealed tissue- and brain development stage-specific differences in expression between transcript isoforms. These findings provide an essential backdrop for the diagnosis of CDKL5-related disorders, for investigations into the basic biology of this gene and its protein products, and for the rational design of gene-based and molecular therapies for these disorders. PMID:27315173

  7. Expression profile of parkin isoforms in human gliomas.

    PubMed

    Maugeri, Grazia; D'Amico, Agata Grazia; Magro, Gaetano; Salvatorelli, Lucia; Barbagallo, Giuseppe M V; Saccone, Salvatore; Drago, Filippo; Cavallaro, Sebastiano; D'Agata, Velia

    2015-10-01

    Mutations of parkin gene are not restricted to familial forms of Parkinsonism but they also occur in a wide variety of malignancies including gliomas. Parkin over-expression reduces glioma cells proliferation and analysis of its expression is predictive for the survival outcome of patients with glioma. To date have been identified 21 parkin alternative splice variants. However, most of the studies have focused their attention exclusively on full-length protein. In the present study, the expression profile of parkin isoforms in different grades of astrocytomas was analyzed for the first time, in order to evaluate their involvement in this malignancy. Furthermore, to investigate their role in cellular processes, their expression in three glioblastoma cell lines was analyzed following treatment with the proteasome inhibitor MG132, or induction of mitophagy with CCCP, or after serum deprivation. Results suggested that H20, H1 and H5 isoforms are always expressed in tumors both in vivo and in vitro models. Therefore, these isoforms might be used as specific biomarkers to develop a prognostic tool for brain tumors. PMID:26238155

  8. Inhibition of mammalian carbonic anhydrase isoforms I-XIV with a series of phenolic acid esters.

    PubMed

    Maresca, Alfonso; Akyuz, Gulay; Osman, Sameh M; AlOthman, Zeid; Supuran, Claudiu T

    2015-11-15

    A series of phenolic acid esters incorporating caffeic, ferulic, and p-coumaric acid, and benzyl, m/p-hydroxyphenethyl- as well as p-hydroxy-phenethoxy-phenethyl moieties were investigated for their inhibitory effects against the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1). Many of the mammalian isozymes of human (h) or murine (m) origin, hCA I-hCA XII, mCA XIII and hCA XIV, were inhibited in the submicromolar range by these derivatives (with KIs of 0.31-1.03 μM against hCA VA, VB, VI, VII, IX and XIV). The off-target, highly abundant isoforms hCA I and II, as well as hCA III, IV and XII were poorly inhibited by many of these esters, although the original phenolic acids were micromolar inhibitors. These phenols, like others investigated earlier, possess a CA inhibition mechanism distinct of the sulfonamides/sulfamates, clinically used drugs for the treatment of a multitude of pathologies, but with severe side effects due to hCA I/II inhibition. Unlike the sulfonamides, which bind to the catalytic zinc ion, phenols are anchored at the Zn(II)-coordinated water molecule, binding more externally within the active site cavity, and making contacts with amino acid residues at the entrance of the active site. As this is the region with the highest variability between the many CA isozymes found in mammals, this class of compounds shows isoform-selective inhibitory profiles, which may be exploited for obtaining pharmacological agents with less side effects compared to other classes of inhibitors. PMID:26498394

  9. Trangenic misexpression of the differentiation-specific desmocollin isoform 1 in basal keratinocytes.

    PubMed

    Henkler, F; Strom, M; Mathers, K; Cordingley, H; Sullivan, K; King, I

    2001-01-01

    Keratinocytes undergoing terminal differentiation are characterized by well-defined changes in protein expression, which contribute towards the transformation of cytoarchitecture and epithelial morphology. Characteristic patterns of desmosomal cadherins are tightly regulated and distinct isoforms are expressed during development and differentiation of epithelial tissues. Desmocollin-1 is strictly confined to suprabasal layers of epidermis, but it is absent in mitotically active, basal keratinocytes. This raises the question of whether basal desmocollin-1 could alter desmosomal functions and compromise keratinocyte proliferation, stratification, or early differentiation in skin. In this study, we misexpressed human desmocollin-1 in mouse epidermis, under control of the keratin-14 promoter. Transgenic animals were generated, which showed a specific expression of transgenic human desmocollin-1 in epidermal basal cells. High level transgenic expression, which was equal to or greater than endogenous protein levels, was observed in mice with multiple copy integration of the transgene. A punctate distribution of desmocollin-1 was demonstrated at the cell membrane by indirect immunofluorescence. Transgenic human desmocollin-1 colocalized with endogenous desmosomal marker proteins, indicating efficient incorporation into desmosomes. Transgenic mice did not display any obvious abnormalities, either in the histology of skin and hair follicles, or in the ultrastructure of desmosomes. These observations suggest that desmocollin-1 can function as a desmosomal cadherin both in basal and suprabasal cells. We propose that the differentiation-specific desmocollin isoforms desmocollin-1 and desmocollin-3 are functionally equivalent in basal epidermal cells and suggest that their changing expression patterns are markers, but not regulators, of the initial steps in keratinocyte differentiation. PMID:11168810

  10. Characterization and expression of human bifunctional 3'-phosphoadenosine 5'-phosphosulphate synthase isoforms.

    PubMed Central

    Fuda, Hirotoshi; Shimizu, Chikara; Lee, Young C; Akita, Harukuni; Strott, Charles A

    2002-01-01

    Sulphonation, a fundamental process essential for normal growth and development, requires the sulphonate donor molecule 3'-phosphoadenosine 5'-phosphosulphate (PAPS), which is produced from ATP and inorganic sulphate by the bifunctional enzyme PAPS synthase. In humans, two genes encode isoenzymes that are 77% identical at the amino acid level, and alternative splicing creates two subtypes of PAPS synthase 2. The question as to whether distinctions in amino acid composition are reflected in differences in activity has been examined. The specific activity of the PAPS synthase 2 subtypes is 10- to 15-fold higher than that for PAPS synthase 1. The greater catalytic efficiency of the PAPS synthase 2 subtypes is demonstrated further by the 3- to 6-fold higher k(cat)/K(m) ratios for ATP and inorganic sulphate as compared with the ratios for PAPS synthase 1. In humans, PAPS synthase 1 is expressed ubiquitously, and is the dominant isoform in most tissues, whereas expression of the PAPS synthase 2 subtypes is variable and tissue-specific. It is noteworthy that, similar to other human tissues, PAPS synthase 1 also appears to be the dominant isoform expressed in cartilage. The latter finding initially created a conundrum, since there is a specific human dwarfing disorder that is known to be caused by a mutation in the PAPS synthase 2 gene. This apparent enigma would seem to be resolved by examination of cartilage from guinea-pigs as an animal model. Similar to humans, cartilage from mature animals predominantly expresses PAPS synthase 1. In contrast, expression of PAPS synthase 1 is relatively low in the cartilage of immature guinea-pigs, including the growth plate of long bones, whereas PAPS synthase 2 is the highly expressed isoenzyme. PMID:11931637

  11. SP-R210 (Myo18A) Isoforms as Intrinsic Modulators of Macrophage Priming and Activation

    PubMed Central

    DiAngelo, Susan L.; Silveyra, Patricia; Umstead, Todd M.; Halstead, E. Scott; Davies, Michael L.; Hu, Sanmei; Floros, Joanna; McCormack, Francis X.; Christensen, Neil D.; Chroneos, Zissis C.

    2015-01-01

    The surfactant protein (SP-A) receptor SP-R210 has been shown to increase phagocytosis of SP-A-bound pathogens and to modulate cytokine secretion by immune cells. SP-A plays an important role in pulmonary immunity by enhancing opsonization and clearance of pathogens and by modulating macrophage inflammatory responses. Alternative splicing of the Myo18A gene results in two isoforms: SP-R210S and SP-R210L, with the latter predominantly expressed in alveolar macrophages. In this study we show that SP-A is required for optimal expression of SP-R210L on alveolar macrophages. Interestingly, pre-treatment with SP-A prepared by different methods either enhances or suppresses responsiveness to LPS, possibly due to differential co-isolation of SP-B or other proteins. We also report that dominant negative disruption of SP-R210L augments expression of receptors including SR-A, CD14, and CD36, and enhances macrophages’ inflammatory response to TLR stimulation. Finally, because SP-A is known to modulate CD14, we used a variety of techniques to investigate how SP-R210 mediates the effect of SP-A on CD14. These studies revealed a novel physical association between SP-R210S, CD14, and SR-A leading to an enhanced response to LPS, and found that SP-R210L and SP-R210S regulate internalization of CD14 via distinct macropinocytosis-like mechanisms. Together, our findings support a model in which SP-R210 isoforms differentially regulate trafficking, expression, and activation of innate immune receptors on macrophages. PMID:25965346

  12. Evolution of the plasma and tissue kallikreins, and their alternative splicing isoforms.

    PubMed

    Koumandou, Vassiliki Lila; Scorilas, Andreas

    2013-01-01

    Kallikreins are secreted serine proteases with important roles in human physiology. Human plasma kallikrein, encoded by the KLKB1 gene on locus 4q34-35, functions in the blood coagulation pathway, and in regulating blood pressure. The human tissue kallikrein and kallikrein-related peptidases (KLKs) have diverse expression patterns and physiological roles, including cancer-related processes such as cell growth regulation, angiogenesis, invasion, and metastasis. Prostate-specific antigen (PSA), the product of the KLK3 gene, is the most widely used biomarker in clinical practice today. A total of 15 KLKs are encoded by the largest contiguous cluster of protease genes in the human genome (19q13.3-13.4), which makes them ideal for evolutionary analysis of gene duplication events. Previous studies on the evolution of KLKs have traced mammalian homologs as well as a probable early origin of the family in aves, amphibia and reptilia. The aim of this study was to address the evolutionary and functional relationships between tissue KLKs and plasma kallikrein, and to examine the evolution of alternative splicing isoforms. Sequences of plasma and tissue kallikreins and their alternative transcripts were collected from the NCBI and Ensembl databases, and comprehensive phylogenetic analysis was performed by Bayesian as well as maximum likelihood methods. Plasma and tissue kallikreins exhibit high sequence similarity in the trypsin domain (>50%). Phylogenetic analysis indicates an early divergence of KLKB1, which groups closely with plasminogen, chymotrypsin, and complement factor D (CFD), in a monophyletic group distinct from trypsin and the tissue KLKs. Reconstruction of the earliest events leading to the diversification of the tissue KLKs is not well resolved, indicating rapid expansion in mammals. Alternative transcripts of each KLK gene show species-specific divergence, while examination of sequence conservation indicates that many annotated human KLK isoforms are missing

  13. Gene Turnover in the Avian Globin Gene Families and Evolutionary Changes in Hemoglobin Isoform Expression

    PubMed Central

    Opazo, Juan C.; Hoffmann, Federico G.; Natarajan, Chandrasekhar; Witt, Christopher C.; Berenbrink, Michael; Storz, Jay F.

    2015-01-01

    The apparent stasis in the evolution of avian chromosomes suggests that birds may have experienced relatively low rates of gene gain and loss in multigene families. To investigate this possibility and to explore the phenotypic consequences of variation in gene copy number, we examined evolutionary changes in the families of genes that encode the α- and β-type subunits of hemoglobin (Hb), the tetrameric α2β2 protein responsible for blood-O2 transport. A comparative genomic analysis of 52 bird species revealed that the size and membership composition of the α- and β-globin gene families have remained remarkably constant during approximately 100 My of avian evolution. Most interspecific variation in gene content is attributable to multiple independent inactivations of the αD-globin gene, which encodes the α-chain subunit of a functionally distinct Hb isoform (HbD) that is expressed in both embryonic and definitive erythrocytes. Due to consistent differences in O2-binding properties between HbD and the major adult-expressed Hb isoform, HbA (which incorporates products of the αA-globin gene), recurrent losses of αD-globin contribute to among-species variation in blood-O2 affinity. Analysis of HbA/HbD expression levels in the red blood cells of 122 bird species revealed high variability among lineages and strong phylogenetic signal. In comparison with the homologous gene clusters in mammals, the low retention rate for lineage-specific gene duplicates in the avian globin gene clusters suggests that the developmental regulation of Hb synthesis in birds may be more highly conserved, with orthologous genes having similar stage-specific expression profiles and similar functional properties in disparate taxa. PMID:25502940

  14. The solution structures of two soybean calmodulin isoforms provide a structural basis for their selective target activation properties.

    PubMed

    Ishida, Hiroaki; Huang, Hao; Yamniuk, Aaron P; Takaya, Yoshiaki; Vogel, Hans J

    2008-05-23

    The intracellular calcium ion is one of the most important secondary messengers in eukaryotic cells. Ca(2+) signals are translated into physiological responses by EF-hand calcium-binding proteins such as calmodulin (CaM). Multiple CaM isoforms occur in plant cells, whereas only a single CaM protein is found in animals. Soybean CaM isoform 1 (sCaM1) shares 90% amino acid sequence identity with animal CaM (aCaM), whereas sCaM4 is only 78% identical. These two sCaM isoforms have distinct target-enzyme activation properties and physiological functions. sCaM4 is highly expressed during the self-defense reaction of the plant and activates the enzyme nitric-oxide synthase (NOS), whereas sCaM1 is incapable of activating NOS. The mechanism of selective target activation by plant CaM isoforms is poorly understood. We have determined high resolution NMR solution structures of Ca(2+)-sCaM1 and -sCaM4. These were compared with previously determined Ca(2+)-aCaM structures. For the N-lobe of the protein, the solution structures of Ca(2+)-sCaM1, -sCaM4, and -aCaM all closely resemble each other. However, despite the high sequence identity with aCaM, the C-lobe of Ca(2+)-sCaM1 has a more open conformation and consequently a larger hydrophobic target-protein binding pocket than Ca(2+)-aCaM or -sCaM4, the presence of which was further confirmed through biophysical measurements. The single Val-144 --> Met substitution in the C-lobe of Ca(2+)-sCaM1, which restores its ability to activate NOS, alters the structure of the C-lobe to a more closed conformation resembling Ca(2+)-aCaM and -sCaM4. The relationships between the structural differences in the two Ca(2+)-sCaM isoforms and their selective target activation properties are discussed. PMID:18347016

  15. Molecular cloning and subcellular distribution of the novel PDE4B4 cAMP-specific phosphodiesterase isoform.

    PubMed Central

    Shepherd, Malcolm; McSorley, Theresa; Olsen, Aileen E; Johnston, Lee Ann; Thomson, Neil C; Baillie, George S; Houslay, Miles D; Bolger, Graeme B

    2003-01-01

    We have isolated cDNAs encoding PDE4B4, a new cAMP-specific phosphodiesterase (PDE4) isoform with novel properties. The amino acid sequence of PDE4B4 demonstrates that it is encoded by the PDE4B gene, but that it differs from the previously isolated PDE4B1, PDE4B2 and PDE4B3 isoforms by the presence of a novel N-terminal region of 17 amino acids. PDE4B4 contains both of the upstream conserved region 1 (UCR1) and UCR2 regulatory units that are characteristic of 'long' PDE4 isoforms. RNase protection demonstrated that PDE4B4 mRNA is expressed preferentially in liver, skeletal muscle and various regions of the brain, which differs from the pattern of tissue distribution of the other known PDE4B long forms, PDE4B1 and PDE4B3. Expression of PDE4B4 cDNA in COS7 cells produced a protein of 85 kDa under denaturing conditions. Subcellular fractionation of recombinant, COS7-cell expressed PDE4B4 showed that the protein was localized within the cytosol, which was confirmed by confocal microscopic analysis of living COS7 cells transfected with a green fluorescent protein-PDE4B4 chimaera. PDE4B4 exhibited a K(m) for cAMP of 5.4 microM and a V(max), relative to that of the long PDE4B1 isoform, of 2.1. PDE4B4 was inhibited by the prototypical PDE4 inhibitor rolipram [4-[3-(cyclopentoxyl)-4-methoxyphenyl]-2-pyrrolidinone] with an IC(50) of 83 nM. Treatment of COS7 cells with forskolin, to elevate cAMP levels, produced activation of PDE4B4, which was associated with the phosphorylation of PDE4B4 on Ser-56 within UCR1. The unique tissue distribution and intracellular targeting of PDE4B4 suggests that this isoform may have a distinct functional role in regulating cAMP levels in specific cell types. PMID:12441002

  16. The substrate-associated protein p45 of porcine endothelial cells: multiple isoforms, cytoskeletal-like properties and induction by hyperoxic stress.

    PubMed

    White, J E; Tsan, M F; Phillips, P G; Higgins, P J

    1990-01-01

    1. Cultured mesenchymal cells respond to hyperoxic (hyper-O2) stress with increased cell flattening/substrate adhesion and overall 47-69% reductions in total matrix-associated (i.e. saponin-resistant [SAP fraction]) protein. 2. Electrophoretic analysis revealed a selective hyper-O2-related 2.7- to 4-fold increase in SAP and cytoskeletal fraction deposition of the protein p45 beginning early (within 12 hr) after initial exposure of porcine endothelial cells to hyper-O2 and increasing over a 48 hr period. 3. p45 consisted of 8 distinct isoforms differing only in pI; hyper-O2-augmented matrix deposition of 3. p45 consisted of 8 distinct isoforms differing only in pI; hyper-02-augmented matrix deposition of p45 involved all 8 isoforms with the more basic subtypes exhibiting slightly greater net increases. 4. Both the specificity and time course of p45 induction, relative to the onset of hyper-O2 cytoarchitectural remodeling, indicate that p45 up-regulation constitutes an early aspect of the hyper-O2 adaptive response. PMID:2289622

  17. Identification, cloning and characterization of two thioredoxin h isoforms, HvTrxh1 and HvTrxh2, from the barley seed proteome.

    PubMed

    Maeda, Kenji; Finnie, Christine; ØStergaard, Ole; Svensson, Birte

    2003-06-01

    Two thioredoxin h isoforms, HvTrxh1 and HvTrxh2, were identified in two and one spots, respectively, in a proteome analysis of barley (Hordeum vulgare) seeds based on 2D gel electrophoresis and MS. HvTrxh1 was observed in 2D gel patterns of endosperm, aleurone layer and embryo of mature barley seeds, and HvTrxh2 was present mainly in the embryo. During germination, HvTrxh2 decreased in abundance and HvTrxh1 decreased in the aleurone layer and endosperm but remained at high levels in the embryo. On the basis of MS identification of the two isoforms, expressed sequence tag sequences were identified, and cDNAs encoding HvTrxh1 and HvTrxh2 were cloned by RT-PCR. The sequences were 51% identical, but showed higer similarity to thioredoxin h isoforms from other cereals, e.g. rice Trxh (74% identical with HvTrxh1) and wheat TrxTa (90% identical with HvTrxh2). Recombinant HvTrxh1, HvTrxh2 and TrxTa were produced in Escherichia coli and purified using a three-step procedure. The activity of the purified recombinant thioredoxin h isoforms was demonstrated using insulin and barley alpha-amylase/subtilisin inhibitor as substrates. HvTrxh1 and HvTrxh2 were also efficiently reduced by Arabidopsis thaliana NADP-dependent thioredoxin reductase (NTR). The biochemical properties of HvTrxh2 and TrxTa were similar, whereas HvTrxh1 had higher insulin-reducing activity and was a better substrate for Arabidopsis NTR than HvTrxh2, with a Km of 13 micro m compared with 44 micro m for HvTrxh2. Thus, barley seeds contain two distinct thioredoxin h isoforms which differ in temporal and spatial distribution and kinetic properties, suggesting that they may have different physiological roles. PMID:12787030

  18. Smooth muscle myosin expression, isoform composition, and functional activities in rat corpus cavernosum altered by the streptozotocin-induced type 1 diabetes

    PubMed Central

    Zhang, Xinhua; Kanika, Nirmala D.; Melman, Arnold

    2012-01-01

    Diabetes mellitus (DM) is a quite common chronic disease, and the prevalence of erectile dysfunction (ED) is three times higher in this large population. Although diabetes-related ED has been studied extensively, the actin-myosin contractile apparatus was not examined. The mRNAs encoding smooth muscle myosin (SMM) heavy chains (MHC) and essential light chains (LC17) exist as several different alternatively spliced isoforms with distinct contractile properties. Recently, we provided novel data that blebbistatin (BLEB), a specific myosin II inhibitor, potently relaxed corpus cavernosum smooth muscle (CCSM). In this study, we examine whether diabetes alters SMM expression, alternative splicing, and/or functional activities, including sensitivity to BLEB. By using streptozotocin (STZ)-induced 2-mo diabetic rats, functional activities were tested in vivo by intracavernous pressure (ICP) recording during cavernous nerve stimulation and in vitro via organ bath contractility studies. SMM isoform composition was analyzed by competitive RT-PCR and total SMM, myocardin, and embryonic SMM (SMemb) expression by real-time RT-PCR. Results revealed that the blood glucose level of STZ rats was 407.0 vs. 129.5 mg/dl (control). STZ rats exhibited ED confirmed by significantly increased CCSM contractile response to phenylephrine and decreased ICP response. For STZ rats, SM-B, LC17a and SM2 isoforms, total SMM, and myocardin expression increased, whereas SM-A, LC17b, and SM1 isoforms were decreased, with SMemb unchanged. BLEB was significantly more effective in relaxing STZ CCSM both in vitro and in vivo. Thus we demonstrated a novel diabetes-specific effect on alternative splicing of the SMM heavy chain and essential light chain genes to a SMM isoform composition favoring a heightened contractility and ED. A switch to a more contractile phenotype was supported further by total SMM expression increase. Moreover, the change in CCSM phenotype was associated with an increased sensitivity

  19. Distinct roles of PTCH2 splice variants in Hedgehog signalling.

    PubMed Central

    Rahnama, Fahimeh; Toftgård, Rune; Zaphiropoulos, Peter G

    2004-01-01

    The human PTCH2 gene is highly similar to PTCH1, a tumour suppressor gene frequently mutated in basal cell carcinoma and several other tumour types. PTCH1 is a transmembrane protein believed to inhibit another transmembrane protein SMO (Smoothened), which mediates HH (Hedgehog) signalling. In this study, we analysed the biological properties of several PTCH2 splice variants. An mRNA form that lacked the last exon was abundantly expressed in all tissues examined, in contrast with the one that included it. Moreover, a transcript lacking exon 9, which is a part of a conserved sterol-sensing domain, was identified in intestine, prostate and cerebellum. In ovary, spleen, testis, cerebellum and skin, an mRNA lacking both exons 9 and 10 could also be observed. The different PTCH2 isoforms localized in the cytoplasm were capable of internalizing the N-terminal fragment of Sonic HH (Shh-N). Additionally, the PTCH2 gene was found to be a target of HH signalling. PTCH2 promoter regulation assays demonstrated that only one of the PTCH2 variants could inhibit the activity of SHH-N, whereas none was capable of inhibiting the activated form of SMO (SMO-M2) and this contrasts with PTCH1. Despite the fact that the PTCH2 isoforms lacked the ability to inhibit SMO-M2 activity, all PTCH2 variants as well as PTCH1, on co-transfection with Smo, were able to change Smo localization from being largely dispersed in the cytoplasm to the juxtanuclear region. Furthermore, the PTCH2 isoforms and PTCH1 co-localized in doubly transfected cells and an interaction between them was confirmed using immunoprecipitation assays. Using Ptch1-/- mouse cells, it was shown that the PTCH2 variants and PTCH1 differentially act to reconstitute not only the SHH but also the Desert HH-dependent transcriptional response. We conclude that in spite of their structural similarities, the PTCH2 isoforms have distinct functional properties when compared with PTCH1. PMID:14613484

  20. A Novel Isoform of Sucrose Synthase Is Targeted to the Cell Wall during Secondary Cell Wall Synthesis in Cotton Fiber[C][W][OA

    PubMed Central

    Brill, Elizabeth; van Thournout, Michel; White, Rosemary G.; Llewellyn, Danny; Campbell, Peter M.; Engelen, Steven; Ruan, Yong-Ling; Arioli, Tony; Furbank, Robert T.

    2011-01-01

    Sucrose (Suc) synthase (Sus) is the major enzyme of Suc breakdown for cellulose biosynthesis in cotton (Gossypium hirsutum) fiber, an important source of fiber for the textile industry. This study examines the tissue-specific expression, relative abundance, and temporal expression of various Sus transcripts and proteins present in cotton. A novel isoform of Sus (SusC) is identified that is expressed at high levels during secondary cell wall synthesis in fiber and is present in the cell wall fraction. The phylogenetic relationships of the deduced amino acid sequences indicate two ancestral groups of Sus proteins predating the divergence of monocots and dicots and that SusC sequences form a distinct branch in the phylogeny within the dicot-specific clade. The subcellular location of the Sus isoforms is determined, and it is proposed that cell wall-localized SusC may provide UDP-glucose for cellulose and callose synthesis from extracellular sugars. PMID:21757635

  1. Evolutionary, environmental and tissue controls on the occurrence of multiple isoforms of acyl carrier protein

    SciTech Connect

    Battey, J.F.; Ohlrogge, J.B. )

    1989-04-01

    Previous research has revealed that several higher plant species have multiple isoforms of acyl carrier protein (ACP). We have examined the development of this trait in evolutionarily diverse species. Isoforms were resolved by Western blotting and native PAGE of {sup 3}H-palmitate labelled ACP's. Multiple isoforms of ACP were observed in primitive vascular plants including gymnosperms, ferns and Psilotum and the nonvascular liverworts and mosses. Therefore, the development of ACP isoforms occurred early in evolution. However, unicellular algae and bacteria such as Chlamydomonas, Dunaliella, Synechocystis and Agmnellum have only a single electrophoretic form of ACP. Thus, multiple forms of ACP do not occur in all photosynthetic organisms but may be associated with multicellular plants. We have also examined light and tissue control over the expression of ACP isoforms. The expression of multiple forms of ACP in leaf of Spinacia and Avena is altered very little by light. Rather, the different patterns of ACP isoforms are primarily dependant on tissue source.

  2. Myosin motor isoforms direct specification of actomyosin function by tropomyosins

    PubMed Central

    Clayton, Joseph E.; Pollard, Luther W.; Murray, George G.; Lord, Matthew

    2015-01-01

    Myosins and tropomyosins represent two cytoskeletal proteins that often work together with actin filaments in contractile and motile cellular processes. While the specialized role of tropomyosin in striated muscle myosin-II regulation is well characterized, its role in non-muscle myosin regulation is poorly understood. We previously showed that fission yeast tropomyosin (Cdc8p) positively regulates myosin-II (Myo2p) and myosin-V (Myo52p) motors. To understand the broader implications of this regulation we examined the role of two mammalian tropomyosins (Tpm3.1cy/Tm5NM1 and Tpm4.2cy/Tm4) recently implicated in cancer cell proliferation and metastasis. Like Cdc8p, the Tpm3.1cy and Tpm4.2cy isoforms significantly enhance Myo2p and Myo52p motor activity, converting non-processive Myo52p molecules into processive motors that can walk along actin tracks as single molecules. In contrast to the positive regulation of Myo2p and Myo52p, Cdc8p and the mammalian tropomyosins potently inhibited skeletal muscle myosin-II, while having negligible effects on the highly processive mammalian myosin-Va. In support of a conserved role for certain tropomyosins in regulating non-muscle actomyosin structures, Tpm3.1cy supported normal contractile ring function in fission yeast. Our work reveals that actomyosin regulation by tropomyosin is dependent on the myosin isoform, highlighting a general role for specific isoforms of tropomyosin in sorting myosin motor outputs. PMID:25712463

  3. PSA Isoforms' Velocities for Early Diagnosis of Prostate Cancer.

    PubMed

    Heidegger, Isabel; Klocker, Helmut; Pichler, Renate; Horninger, Wolfgang; Bektic, Jasmin

    2015-06-01

    Free prostate-specific antigen (fPSA) and its molecular isoforms are suggested for enhancement of PSA testing in prostate cancer (PCa). In the present study we evaluated whether PSA isoforms' velocities might serve as a tool to improve early PCa diagnosis. Our study population included 381 men who had undergone at least one ultrasound-guided prostate biopsy whose pathologic examination yielded PCa or showed no evidence of prostatic malignancy. Serial PSA, fPSA, and proPSA measurements were performed on serum samples covering 7 years prior to biopsy using Beckmann Coulter Access immunoassays. Afterwards, velocities of PSA (PSAV), fPSA% (fPSA%V), proPSA% (proPSA%V) and the ratio proPSA/PSA/V were calculated and their ability to discriminate cancer from benign disease was evaluated. Among 381 men included in the study, 202 (53%) were diagnosed with PCa and underwent radical prostatectomy at our Department. PSAV, fPSA%V, proPSA%V as well as proPSA/PSA/V were able to differentiate significantly between PCa and non-cancerous prostate. The highest discriminatory power between cancer and benign disease has been observed two and one year prior to diagnosis with all measured parameters. Among all measured parameters, fPSA%V showed the best cancer specificity of 45.3% with 90% of sensitivity. In summary, our results highlight the value of PSA isoforms' velocity for early detection of PCa. Especially fPSA%V should be used in the clinical setting to increase cancer detection specificity. PMID:26026127

  4. Different motifs regulate trafficking of SorCS1 isoforms.

    PubMed

    Nielsen, Morten S; Keat, Sady J; Hamati, Jida W; Madsen, Peder; Gutzmann, Jakob J; Engelsberg, Arne; Pedersen, Karen M; Gustafsen, Camilla; Nykjaer, Anders; Gliemann, Jørgen; Hermans-Borgmeyer, Irm; Kuhl, Dietmar; Petersen, Claus M; Hermey, Guido

    2008-06-01

    The type I transmembrane protein SorCS1 is a member of the Vps10p-domain receptor family comprised of Sortilin, SorLA and SorCS1, -2 and -3. Current information indicates that Sortilin and SorLA mediate intracellular protein trafficking and sorting, but little is known about the cellular functions of the SorCS subgroup. SorCS1 binds platelet-derived growth factor-BB (PDGF-BB) and is expressed in isoforms differing only in their cytoplasmic domains. Here, we identify two novel isoforms of mouse SorCS1 designated m-SorCS1c and -d. In situ hybridization revealed a combinatorial expression pattern of the variants in brain and embryonic tissues. We demonstrate that among the mouse variants, only SorCS1c mediates internalization and that the highly conserved SorCS1c is internalized through a canonical tyrosine-based motif. In contrast, human SorCS1a, whose cytoplasmic domain is completely different from mouse SorCS1a, is internalized through a DXXLL motif. We report that the human SorCS1a cytoplasmic domain interacts with the alphaC/sigma2 subunits of the adaptor protein (AP)-2 complex, and internalization of human SorCS1a and -c is mediated by AP-2. Our results suggest that the endocytic isoforms target internalized cargo to lysosomes but are not engaged in Golgi-endosomal transport to a significant degree. PMID:18315530

  5. Expression of Gls and Gls2 glutaminase isoforms in astrocytes.

    PubMed

    Cardona, Carolina; Sánchez-Mejías, Elisabeth; Dávila, José C; Martín-Rufián, Mercedes; Campos-Sandoval, José A; Vitorica, Javier; Alonso, Francisco J; Matés, José M; Segura, Juan A; Norenberg, Michael D; Rama Rao, Kakulavarapu V; Jayakumar, Arumugan R; Gutiérrez, Antonia; Márquez, Javier

    2015-03-01

    The expression of glutaminase in glial cells has been a controversial issue and matter of debate for many years. Actually, glutaminase is essentially considered as a neuronal marker in brain. Astrocytes are endowed with efficient and high capacity transport systems to recapture synaptic glutamate which seems to be consistent with the absence of glutaminase in these glial cells. In this work, a comprehensive study was devised to elucidate expression of glutaminase in neuroglia and, more concretely, in astrocytes. Immunocytochemistry in rat and human brain tissues employing isoform-specific antibodies revealed expression of both Gls and Gls2 glutaminase isozymes in glutamatergic and GABAergic neuronal populations as well as in astrocytes. Nevertheless, there was a different subcellular distribution: Gls isoform was always present in mitochondria while Gls2 appeared in two different locations, mitochondria and nucleus. Confocal microscopy and double immunofluorescence labeling in cultured astrocytes confirmed the same pattern previously seen in brain tissue samples. Astrocytic glutaminase expression was also assessed at the mRNA level, real-time quantitative RT-PCR detected transcripts of four glutaminase isozymes but with marked differences on their absolute copy number: the predominance of Gls isoforms over Gls2 transcripts was remarkable (ratio of 144:1). Finally, we proved that astrocytic glutaminase proteins possess enzymatic activity by in situ activity staining: concrete populations of astrocytes were labeled in the cortex, cerebellum and hippocampus of rat brain demonstrating functional catalytic activity. These results are relevant for the stoichiometry of the Glu/Gln cycle at the tripartite synapse and suggest novel functions for these classical metabolic enzymes. PMID:25297978

  6. Highly divergent strains of porcine reproductive and respiratory syndrome virus incorporate multiple isoforms of nonstructural protein 2 into virions.

    PubMed

    Kappes, Matthew A; Miller, Cathy L; Faaberg, Kay S

    2013-12-01

    Viral structural proteins form the critical intermediary between viral infection cycles within and between hosts, function to initiate entry, participate in immediate early viral replication steps, and are major targets for the host adaptive immune response. We report the identification of nonstructural protein 2 (nsp2) as a novel structural component of the porcine reproductive and respiratory syndrome virus (PRRSV) particle. A set of custom α-nsp2 antibodies targeting conserved epitopes within four distinct regions of nsp2 (the PLP2 protease domain [OTU], the hypervariable domain [HV], the putative transmembrane domain [TM], and the C-terminal region [C]) were obtained commercially and validated in PRRSV-infected cells. Highly purified cell-free virions of several PRRSV strains were isolated through multiple rounds of differential density gradient centrifugation and analyzed by immunoelectron microscopy (IEM) and Western blot assays using the α-nsp2 antibodies. Purified viral preparations were found to contain pleomorphic, predominantly spherical virions of uniform size (57.9 nm ± 8.1 nm diameter; n = 50), consistent with the expected size of PRRSV particles. Analysis by IEM indicated the presence of nsp2 associated with the viral particle of diverse strains of PRRSV. Western blot analysis confirmed the presence of nsp2 in purified viral samples and revealed that multiple nsp2 isoforms were associated with the virion. Finally, a recombinant PRRSV genome containing a myc-tagged nsp2 was used to generate purified virus, and these particles were also shown to harbor myc-tagged nsp2 isoforms. Together, these data identify nsp2 as a virion-associated structural PRRSV protein and reveal that nsp2 exists in or on viral particles as multiple isoforms. PMID:24089566

  7. Multiple phosphorylated forms of the Saccharomyces cerevisiae Mcm1 protein include an isoform induced in response to high salt concentrations.

    PubMed Central

    Kuo, M H; Nadeau, E T; Grayhack, E J

    1997-01-01

    The Saccharomyces cerevisiae Mcm1 protein is an essential multifunctional transcription factor which is highly homologous to human serum response factor. Mcm1 protein acts on a large number of distinctly regulated genes: haploid cell-type-specific genes, G2-cell-cycle-regulated genes, pheromone-induced genes, arginine metabolic genes, and genes important for cell wall and cell membrane function. We show here that Mcm1 protein is phosphorylated in vivo. Several (more than eight) isoforms of Mcm1 protein, resolved by isoelectric focusing, are present in vivo; two major phosphorylation sites lie in the N-terminal 17 amino acids immediately adjacent to the conserved MADS box DNA-binding domain. The implications of multiple species of Mcm1, particularly the notion that a unique Mcm1 isoform could be required for regulation of a specific set of Mcm1's target genes, are discussed. We also show here that Mcm1 plays an important role in the response to stress caused by NaCl. G. Yu, R. J. Deschenes, and J. S. Fassler (J. Biol. Chem. 270:8739-8743, 1995) showed that Mcm1 function is affected by mutations in the SLN1 gene, a signal transduction component implicated in the response to osmotic stress. We find that mcm1 mutations can confer either reduced or enhanced survival on high-salt medium; deletion of the N terminus or mutation in the primary phosphorylation site results in impaired growth on high-salt medium. Furthermore, Mcm1 protein is a target of a signal transduction system responsive to osmotic stress: a new isoform of Mcm1 is induced by NaCl or KCl; this result establishes that Mcm1 itself is regulated. PMID:9001236

  8. Expression of TP53 Isoforms p53β or p53γ Enhances Chemosensitivity in TP53null Cell Lines

    PubMed Central

    Silden, Elisabeth; Hjelle, Sigrun M.; Wergeland, Line; Sulen, André; Andresen, Vibeke; Bourdon, Jean-Christophe; Micklem, David R.; McCormack, Emmet; Gjertsen, Bjørn Tore

    2013-01-01

    The carboxy-terminal truncated p53 alternative spliced isoforms, p53β and p53γ, are expressed at disparate levels in cancer and are suggested to influence treatment response and therapy outcome. However, their functional role in cancer remains to be elucidated. We investigated their individual functionality in the p53null background of cell lines H1299 and SAOS-2 by stable retroviral transduction or transient transfection. Expression status of p53β and p53γ protein was found to correlate with increased response to camptothecin and doxorubicin chemotherapy. Decreased DNA synthesis and clonogenicity in p53β and p53γ congenic H1299 was accompanied by increased p21(CIP1/WAF1), Bax and Mdm2 proteins. Chemotherapy induced p53 isoform degradation, most prominent for p53γ. The proteasome inhibitor bortezomib substantially increased basal p53γ protein level, while the level of p53β protein was unaffected. Treatment with dicoumarol, a putative blocker of the proteasome-related NAD(P)H quinone oxidoreductase NQO1, effectively attenuated basal p53γ protein level in spite of bortezomib treatment. Although in vitro proliferation and clonogenicity assays indicated a weak suppressive effect by p53β and p53γ expression, studies of in vivo subcutaneous H1299 tumor growth demonstrated a significantly increased growth by expression of either p53 isoforms. This study suggests that p53β and p53γ share functionality in chemosensitizing and tumor growth enhancement but comprise distinct regulation at the protein level. PMID:23409163

  9. Expression of TP53 isoforms p53β or p53γ enhances chemosensitivity in TP53(null) cell lines.

    PubMed

    Silden, Elisabeth; Hjelle, Sigrun M; Wergeland, Line; Sulen, André; Andresen, Vibeke; Bourdon, Jean-Christophe; Micklem, David R; McCormack, Emmet; Gjertsen, Bjørn Tore

    2013-01-01

    The carboxy-terminal truncated p53 alternative spliced isoforms, p53β and p53γ, are expressed at disparate levels in cancer and are suggested to influence treatment response and therapy outcome. However, their functional role in cancer remains to be elucidated. We investigated their individual functionality in the p53(null) background of cell lines H1299 and SAOS-2 by stable retroviral transduction or transient transfection. Expression status of p53β and p53γ protein was found to correlate with increased response to camptothecin and doxorubicin chemotherapy. Decreased DNA synthesis and clonogenicity in p53β and p53γ congenic H1299 was accompanied by increased p21((CIP1/WAF1)), Bax and Mdm2 proteins. Chemotherapy induced p53 isoform degradation, most prominent for p53γ. The proteasome inhibitor bortezomib substantially increased basal p53γ protein level, while the level of p53β protein was unaffected. Treatment with dicoumarol, a putative blocker of the proteasome-related NAD(P)H quinone oxidoreductase NQO1, effectively attenuated basal p53γ protein level in spite of bortezomib treatment. Although in vitro proliferation and clonogenicity assays indicated a weak suppressive effect by p53β and p53γ expression, studies of in vivo subcutaneous H1299 tumor growth demonstrated a significantly increased growth by expression of either p53 isoforms. This study suggests that p53β and p53γ share functionality in chemosensitizing and tumor growth enhancement but comprise distinct regulation at the protein level. PMID:23409163

  10. Peroxisome Proliferator-activated Receptor γ Coactivator-1 α Isoforms Selectively Regulate Multiple Splicing Events on Target Genes.

    PubMed

    Martínez-Redondo, Vicente; Jannig, Paulo R; Correia, Jorge C; Ferreira, Duarte M S; Cervenka, Igor; Lindvall, Jessica M; Sinha, Indranil; Izadi, Manizheh; Pettersson-Klein, Amanda T; Agudelo, Leandro Z; Gimenez-Cassina, Alfredo; Brum, Patricia C; Dahlman-Wright, Karin; Ruas, Jorge L

    2016-07-15

    Endurance and resistance exercise training induces specific and profound changes in the skeletal muscle transcriptome. Peroxisome proliferator-activated receptor γ coactivator-1 α (PGC-1α) coactivators are not only among the genes differentially induced by distinct training methods, but they also participate in the ensuing signaling cascades that allow skeletal muscle to adapt to each type of exercise. Although endurance training preferentially induces PGC-1α1 expression, resistance exercise activates the expression of PGC-1α2, -α3, and -α4. These three alternative PGC-1α isoforms lack the arginine/serine-rich (RS) and RNA recognition motifs characteristic of PGC-1α1. Discrete functions for PGC-1α1 and -α4 have been described, but the biological role of PGC-1α2 and -α3 remains elusive. Here we show that different PGC-1α variants can affect target gene splicing through diverse mechanisms, including alternative promoter usage. By analyzing the exon structure of the target transcripts for each PGC-1α isoform, we were able to identify a large number of previously unknown PGC-1α2 and -α3 target genes and pathways in skeletal muscle. In particular, PGC-1α2 seems to mediate a decrease in the levels of cholesterol synthesis genes. Our results suggest that the conservation of the N-terminal activation and repression domains (and not the RS/RNA recognition motif) is what determines the gene programs and splicing options modulated by each PGC-1α isoform. By using skeletal muscle-specific transgenic mice for PGC-1α1 and -α4, we could validate, in vivo, splicing events observed in in vitro studies. These results show that alternative PGC-1α variants can affect target gene expression both quantitatively and qualitatively and identify novel biological pathways under the control of this system of coactivators. PMID:27231350

  11. Specialized compartments of cardiac nuclei exhibit distinct proteomic anatomy*

    PubMed Central

    Franklin, Sarah; Zhang, Michael J.; Chen, Haodong; Paulsson, Anna K.; Mitchell-Jordan, Scherise A.; Li, Yifeng; Ping, Peipei; Vondriska, Thomas M.

    2011-01-01

    As host to the genome, the nucleus plays a critical role as modulator of cellular phenotype. To understand the totality of proteins that regulate this organelle, we used proteomics to characterize the components of the cardiac nucleus. Following purification, cardiac nuclei were fractionated into biologically relevant fractions including acid-soluble proteins, chromatin-bound molecules and nucleoplasmic proteins. These distinct subproteomes were characterized by liquid chromatography-tandem MS. We report a cardiac nuclear proteome of 1048 proteins—only 146 of which are shared between the distinct subcompartments of this organelle. Analysis of genomic loci encoding these molecules gives insights into local hotspots for nuclear protein regulation. High mass accuracy and complementary analytical techniques allowed the discrimination of distinct protein isoforms, including 54 total histone variants, 17 of which were distinguished by unique peptide sequences and four of which have never been detected at the protein level. These studies are the first unbiased analysis of cardiac nuclear subcompartments and provide a foundation for exploration of this organelle's proteomes during disease. PMID:20807835

  12. Is Face Distinctiveness Gender Based?

    ERIC Educational Resources Information Center

    Baudouin, Jean-Yves; Gallay, Mathieu

    2006-01-01

    Two experiments were carried out to study the role of gender category in evaluations of face distinctiveness. In Experiment 1, participants had to evaluate the distinctiveness and the femininity-masculinity of real or artificial composite faces. The composite faces were created by blending either faces of the same gender (sexed composite faces,…

  13. The multigene families of actinoporins (part I): Isoforms and genetic structure.

    PubMed

    Valle, A; Alvarado-Mesén, J; Lanio, M E; Álvarez, C; Barbosa, J A R G; Pazos, I F

    2015-09-01

    Actinoporins are basic pore-forming proteins produced by sea anemones, with molecular weight around 20 kDa showing high affinity for sphingomyelin-containing membranes. Most sea anemones produce more than one actinoporin isoform differing in isoelectric point, molecular weigth and cytolytic activity. Examples of sea anemones with actinoporin isoforms are: Actinia equina with at least five isoform genes; Actinia tenebrosa, three isoforms; Actinia fragacea, five isoforms; Actineria villosa, Phyllodiscus semoni, Stichodactyla helianthus and Oulactis orientalis, with two isoforms each one, and Heteractis crispa with twenty-four isoforms. Additionally, thirty-four different amino acid sequences were deduced from fifty-two nucleotide sequences of Heteractis magnifica toxins suggesting the presence of a large number of isoforms or allelic variants. Many amino acidic changes in the isoforms are located in important regions for pore formation. The genetic structure of actinoporins comprises a pre-propeptide and a mature toxin region; therefore, actinoporins could be synthetized in the Golgi apparatus as precursor forms. The subsequent maturation of the toxins involves a proteolytic processing during secretion. Here we hypothesize that sea anemones could have suffered duplication, conversion and mutation of genes that produced multigene families as an efficient response to evolutionary pressure, leading to successful strategies of predatory and defensive function. PMID:26187849

  14. Male-Specific Fruitless Isoforms Target Neurodevelopmental Genes to Specify a Sexually Dimorphic Nervous System

    PubMed Central

    Neville, Megan C.; Nojima, Tetsuya; Ashley, Elizabeth; Parker, Darren J.; Walker, John; Southall, Tony; Van de Sande, Bram; Marques, Ana C.; Fischer, Bettina; Brand, Andrea H.; Russell, Steven; Ritchie, Michael G.; Aerts, Stein; Goodwin, Stephen F.

    2014-01-01

    Summary Background In Drosophila, male courtship behavior is regulated in large part by the gene fruitless (fru). fru encodes a set of putative transcription factors that promote male sexual behavior by controlling the development of sexually dimorphic neuronal circuitry. Little is known about how Fru proteins function at the level of transcriptional regulation or the role that isoform diversity plays in the formation of a male-specific nervous system. Results To characterize the roles of sex-specific Fru isoforms in specifying male behavior, we generated novel isoform-specific mutants and used a genomic approach to identify direct Fru isoform targets during development. We demonstrate that all Fru isoforms directly target genes involved in the development of the nervous system, with individual isoforms exhibiting unique binding specificities. We observe that fru behavioral phenotypes are specified by either a single isoform or a combination of isoforms. Finally, we illustrate the utility of these data for the identification of novel sexually dimorphic genomic enhancers and novel downstream regulators of male sexual behavior. Conclusions These findings suggest that Fru isoform diversity facilitates both redundancy and specificity in gene expression, and that the regulation of neuronal developmental genes may be the most ancient and conserved role of fru in the specification of a male-specific nervous system. PMID:24440396

  15. Optimal Distinctiveness Signals Membership Trust.

    PubMed

    Leonardelli, Geoffrey J; Loyd, Denise Lewin

    2016-07-01

    According to optimal distinctiveness theory, sufficiently small minority groups are associated with greater membership trust, even among members otherwise unknown, because the groups are seen as optimally distinctive. This article elaborates on the prediction's motivational and cognitive processes and tests whether sufficiently small minorities (defined by relative size; for example, 20%) are associated with greater membership trust relative to mere minorities (45%), and whether such trust is a function of optimal distinctiveness. Two experiments, examining observers' perceptions of minority and majority groups and using minimal groups and (in Experiment 2) a trust game, revealed greater membership trust in minorities than majorities. In Experiment 2, participants also preferred joining minorities over more powerful majorities. Both effects occurred only when minorities were 20% rather than 45%. In both studies, perceptions of optimal distinctiveness mediated effects. Discussion focuses on the value of relative size and optimal distinctiveness, and when membership trust manifests. PMID:27140657

  16. Transgenic complementation of MeCP2 deficiency: phenotypic rescue of Mecp2-null mice by isoform-specific transgenes

    PubMed Central

    Kerr, Bredford; Soto C, Jessica; Saez, Mauricio; Abrams, Alexander; Walz, Katherina; Young, Juan I

    2012-01-01

    Rett syndrome (RTT) is a disorder that affects patients' ability to communicate, move and behave. RTT patients are characterized by impaired language, stereotypic behaviors, frequent seizures, ataxia and sleep disturbances, with the onset of symptoms occurring after a period of seemingly normal development. RTT is caused by mutations in methyl-CpG binding protein 2 (MECP2), an X-chromosome gene encoding for MeCP2, a protein that regulates gene expression. MECP2 generates two alternative splice variants encoding two protein isoforms that differ only in the N-terminus. Although no functional differences have been identified for these splice variants, it has been suggested that the RTT phenotype may occur in the presence of a functional MeCP2-e2 protein. This suggests that the two isoforms might be functionally distinct. Supporting this notion, the two variants show regional and age-related differences in transcript abundance. Here, we show that transgenic expression of either the MeCP2-e1 or MeCP2-e2 splice variant results in prevention of development of RTT-like phenotypic manifestations in a mouse model lacking Mecp2. Our results indicate that the two MeCP2 splice variants can substitute for each other and fulfill the basic functions of MeCP2 in the mouse brain. PMID:21829232

  17. Discrete functions of GSK3α and GSK3β isoforms in prostate tumor growth and micrometastasis

    PubMed Central

    Gao, Fei; Al-Azayzih, Ahmad; Somanath, Payaningal R.

    2015-01-01

    Isoform specific function of glycogen synthase kinase-3 (GSK3) in cancer is not well defined. We report that silencing of GSK3α, but not GSK3β expression inhibited proliferation, survival and colony formation by the PC3, DU145 and LNCaP prostate cancer cells, and the growth of PC3 tumor xenografts in athymic nude mice. Silencing of GSK3α, but not GSK3β resulted in reduced proliferation and enhanced apoptosis in tumor xenografts. ShRNA-mediated knockdown of GSK3α and GSK3β equally inhibited the ability of prostate cancer cells to migrate and invade the endothelial-barrier in vitro, and PC3 cell micrometastasis to lungs in vivo. Mechanistically, whereas silencing GSK3α resulted in increased expression of pro-apoptotic markers cleaved caspase-3 and cleaved caspase-9 in LNCaP, PC3 and DU145 cells, silencing GSK3β resulted in the inhibition of cell scattering, establishment of cell-cell contacts, increased expression and membrane localization of β-catenin, and reduced expression of epithelial to mesenchymal transition (EMT) markers such as Snail and MMP-9. This indicated the specific role of GSK3β in EMT, acquisition of motility and invasive potential. Overall, our data demonstrated the isoform specific role of GSK3α and GSK3β in prostate cancer cells in vitro, and tumor growth and micrometastasis in vivo, via distinct molecular and cellular mechanisms. PMID:25714023

  18. The Drosophila Retinoblastoma Binding Protein 6 Family Member Has Two Isoforms and Is Potentially Involved in Embryonic Patterning

    PubMed Central

    Hull, Rodney; Oosthuysen, Brent; Cajee, Umar-Faruq; Mokgohloa, Lehlogonolo; Nweke, Ekene; Antunes, Ricardo Jorge; Coetzer, Theresa H. T.; Ntwasa, Monde

    2015-01-01

    The human retinoblastoma binding protein 6 (RBBP6) is implicated in esophageal, lung, hepatocellular and colon cancers. Furthermore, RBBP6 was identified as a strong marker for colon cancer prognosis and as a predisposing factor in familial myeloproliferative neoplasms. Functionally, the mammalian protein interacts with p53 and enhances the activity of Mdm2, the prototypical negative regulator of p53. However, since RBBP6 (known as PACT in mice) exists in multiple isoforms and pact−/− mice exhibit a more severe phenotype than mdm2−/− mutants, it must possess some Mdm2-independent functions. The function of the invertebrate homologue is poorly understood. This is complicated by the absence of the Mdm2 gene in both Drosophila and Caenorhabditis elegans. We have experimentally identified the promoter region of Snama, the Drosophila homologue, analyzed potential transcription factor binding sites and confirmed the existence of an additional isoform. Using band shift and co-immunoprecipitation assays combined with mass spectrometry, we found evidence that this gene may be regulated by, amongst others, DREF, which regulates hundreds of genes related to cell proliferation. The potential transcription factors for Snama fall into distinct functional groups, including anteroposterior embryonic patterning and nucleic acid metabolism. Significantly, previous work in mice shows that pact−/− induces an anteroposterior phenotype in embryos when rescued by simultaneous deletion of p53. Taken together, these observations indicate the significance of RBBP6 proteins in carcinogenesis and in developmental defects. PMID:25955646

  19. Transgenic complementation of MeCP2 deficiency: phenotypic rescue of Mecp2-null mice by isoform-specific transgenes.

    PubMed

    Kerr, Bredford; Soto C, Jessica; Saez, Mauricio; Abrams, Alexander; Walz, Katherina; Young, Juan I

    2012-01-01

    Rett syndrome (RTT) is a disorder that affects patients' ability to communicate, move and behave. RTT patients are characterized by impaired language, stereotypic behaviors, frequent seizures, ataxia and sleep disturbances, with the onset of symptoms occurring after a period of seemingly normal development. RTT is caused by mutations in methyl-CpG binding protein 2 (MECP2), an X-chromosome gene encoding for MeCP2, a protein that regulates gene expression. MECP2 generates two alternative splice variants encoding two protein isoforms that differ only in the N-terminus. Although no functional differences have been identified for these splice variants, it has been suggested that the RTT phenotype may occur in the presence of a functional MeCP2-e2 protein. This suggests that the two isoforms might be functionally distinct. Supporting this notion, the two variants show regional and age-related differences in transcript abundance. Here, we show that transgenic expression of either the MeCP2-e1 or MeCP2-e2 splice variant results in prevention of development of RTT-like phenotypic manifestations in a mouse model lacking Mecp2. Our results indicate that the two MeCP2 splice variants can substitute for each other and fulfill the basic functions of MeCP2 in the mouse brain. PMID:21829232

  20. Role of Rho kinase isoforms in murine allergic airway responses.

    PubMed

    Zhu, M; Liu, P-Y; Kasahara, D I; Williams, A S; Verbout, N G; Halayko, A J; Fedulov, A; Shoji, T; Williams, E S; Noma, K; Shore, S A; Liao, J K

    2011-10-01

    Inhibition of Rho-associated coiled-coil forming kinases (ROCKs) reduces allergic airway responses in mice. The purpose of this study was to determine the roles of the two ROCK isoforms, ROCK1 and ROCK2, in these responses. Wildtype (WT) mice and heterozygous ROCK1 and ROCK2 knockout mice (ROCK1(+/-) and ROCK2(+/-), respectively) were sensitised and challenged with ovalbumin. ROCK expression and activation were assessed by western blotting. Airway responsiveness was measured by forced oscillation. Bronchoalveolar lavage was performed and the lungs were fixed for histological assessment. Compared with WT mice, ROCK1 and ROCK2 expression were 50% lower in lungs of ROCK1(+/-) and ROCK2(+/-) mice, respectively, without changes in the other isoform. In WT lungs, ROCK activation increased after ovalbumin challenge and was sustained for several hours. This activation was reduced in ROCK1(+/-) and ROCK2(+/-) lungs. Airway responsiveness was comparable in WT, ROCK1(+/-), and ROCK2(+/-) mice challenged with PBS. Ovalbumin challenge caused airway hyperresponsiveness in WT, but not ROCK1(+/-) or ROCK2(+/-) mice. Lavage eosinophils and goblet cell hyperplasia were significantly reduced in ovalbumin-challenged ROCK1(+/-) and ROCK2(+/-) versus WT mice. Ovalbumin-induced changes in lavage interleukin-13, interleukin-5 and lymphocytes were also reduced in ROCK1(+/-) mice. In conclusion, both ROCK1 and ROCK2 are important in regulating allergic airway responses. PMID:21565918

  1. BDNF isoforms: a round trip ticket between neurogenesis and serotonin?

    PubMed

    Foltran, Rocío Beatriz; Diaz, Silvina Laura

    2016-07-01

    The brain-derived neurotrophic factor, BDNF, was discovered more than 30 years ago and, like other members of the neurotrophin family, this neuropeptide is synthetized as a proneurotrophin, the pro-BDNF, which is further cleaved to yield mature BDNF. The myriad of actions of these two BDNF isoforms in the central nervous system is constantly increasing and requires the development of sophisticated tools and animal models to refine our understanding. This review is focused on BDNF isoforms, their participation in the process of neurogenesis taking place in the hippocampus of adult mammals, and the modulation of their expression by serotonergic agents. Interestingly, around this triumvirate of BDNF, serotonin, and neurogenesis, a series of recent research has emerged with apparently counterintuitive results. This calls for an exhaustive analysis of the data published so far and encourages thorough work in the quest for new hypotheses in the field. BDNF is synthetized as a pre-proneurotrophin. After removal of the pre-region, proBDNF can be cleaved by intracellular or extracellular proteases. Mature BDNF can bind TrkB receptors, promoting their homodimerization and intracellular phosphorylation. Phosphorylated-TrkB can activate three different signaling pathways. Whereas G-protein-coupled receptors can transactivate TrkB receptors, truncated forms can inhibit mBDNF signaling. Pro-BDNF binds p75(NTR) by its mature domain, whereas the pro-region binds co-receptors. PMID:27167299

  2. GABAB(1) receptor subunit isoforms differentially regulate stress resilience.

    PubMed

    O'Leary, Olivia F; Felice, Daniela; Galimberti, Stefano; Savignac, Hélène M; Bravo, Javier A; Crowley, Tadhg; El Yacoubi, Malika; Vaugeois, Jean-Marie; Gassmann, Martin; Bettler, Bernhard; Dinan, Timothy G; Cryan, John F

    2014-10-21

    Stressful life events increase the susceptibility to developing psychiatric disorders such as depression; however, many individuals are resilient to such negative effects of stress. Determining the neurobiology underlying this resilience is instrumental to the development of novel and more effective treatments for stress-related psychiatric disorders. GABAB receptors are emerging therapeutic targets for the treatment of stress-related disorders such as depression. These receptors are predominantly expressed as heterodimers of a GABAB(2) subunit with either a GABAB(1a) or a GABAB(1b) subunit. Here we show that mice lacking the GABAB(1b) receptor isoform are more resilient to both early-life stress and chronic psychosocial stress in adulthood, whereas mice lacking GABAB(1a) receptors are more susceptible to stress-induced anhedonia and social avoidance compared with wild-type mice. In addition, increased hippocampal expression of the GABAB(1b) receptor subunit is associated with a depression-like phenotype in the helpless H/Rouen genetic mouse model of depression. Stress resilience in GABAB(1b)(-/-) mice is coupled with increased proliferation and survival of newly born cells in the adult ventral hippocampus and increased stress-induced c-Fos activation in the hippocampus following early-life stress. Taken together, the data suggest that GABAB(1) receptor subunit isoforms differentially regulate the deleterious effects of stress and, thus, may be important therapeutic targets for the treatment of depression. PMID:25288769

  3. Role of cysteines in mammalian VDAC isoforms' function.

    PubMed

    De Pinto, Vito; Reina, Simona; Gupta, Ankit; Messina, Angela; Mahalakshmi, Radhakrishnan

    2016-08-01

    In this mini-review, we analyze the influence of cysteines in the structure and activity of mitochondrial outer membrane mammalian VDAC isoforms. The three VDAC isoforms show conserved sequences, similar structures and the same gene organization. The meaning of three proteins encoded in different chromosomes must thus be searched for subtle differences at the amino acid level. Among others, cysteine content is noticeable. In humans, VDAC1 has 2, VDAC2 has 9 and VDAC3 has 6 cysteines. Recent works have shown that, at variance from VDAC1, VDAC2 and VDAC3 exhibit cysteines predicted to protrude towards the intermembrane space, making them a preferred target for oxidation by ROS. Mass spectrometry in VDAC3 revealed that a disulfide bridge can be formed and other cysteine oxidations are also detectable. Both VDAC2 and VDAC3 cysteines were mutagenized to highlight their role in vitro and in complementation assays in Δporin1 yeast. Chemico-physical techniques revealed an important function of cysteines in the structural stabilization of the pore. In conclusion, the works available on VDAC cysteines support the notion that the three proteins are paralogs with a similar pore-function and slightly different, but important, ancillary biological functions. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi. PMID:26947058

  4. Identification and characterization of a plastid-localized Arabidopsis glyoxylate reductase isoform: comparison with a cytosolic isoform and implications for cellular redox homeostasis and aldehyde detoxification

    PubMed Central

    Simpson, Jeffrey P.; Di Leo, Rosa; Dhanoa, Preetinder K.; Allan, Wendy L.; Makhmoudova, Amina; Clark, Shawn M.; Hoover, Gordon J.; Mullen, Robert T.; Shelp, Barry J.

    2008-01-01

    Enzymes that reduce the aldehyde chemical grouping (i.e. H-C=O) to its corresponding alcohol could be crucial in maintaining plant health. Recently, recombinant expression of a cytosolic enzyme from Arabidopsis thaliana (L.) Heynh (designated as glyoxylate reductase 1 or AtGR1) revealed that it effectively catalyses the in vitro reduction of both glyoxylate and succinic semialdehyde (SSA). In this paper, web-based bioinformatics tools revealed a second putative GR cDNA (GenBank Accession No. AAP42747; designated herein as AtGR2) that is 57% identical on an amino acid basis to GR1. Sequence encoding a putative targeting signal (N-terminal 43 amino acids) was deleted from the full-length GR2 cDNA and the resulting truncated gene was co-expressed with the molecular chaperones GroES/EL in Escherichia coli, enabling production and purification of soluble recombinant protein. Kinetic analysis revealed that recombinant GR2 catalysed the conversion of glyoxylate to glycolate (Km glyoxylate=34 μM), and SSA to γ-hydroxybutyrate (Km SSA=8.96 mM) via an essentially irreversible, NADPH-based mechanism. GR2 had a 350-fold higher preference for glyoxylate than SSA, based on the performance constants (kcat/Km). Fluorescence microscopic analysis of tobacco (Nicotiana tabacum L.) suspension cells transiently transformed with GR1 linked to the green fluorescent protein (GFP) revealed that GR1 was localized to the cytosol, whereas GR2-GFP was localized to plastids via targeting information contained within its N-terminal 45 amino acids. The identification and characterization of distinct plastidial and cytosolic glyoxylate reductase isoforms is discussed with respect to aldehyde detoxification and the plant stress response. PMID:18495639

  5. A novel sphingosine-dependent protein kinase (SDK1) specifically phosphorylates certain isoforms of 14-3-3 protein.

    PubMed

    Megidish, T; Cooper, J; Zhang, L; Fu, H; Hakomori, S

    1998-08-21

    Protein kinases activated by sphingosine or N,N'-dimethylsphingosine, but not by other lipids, have been detected and are termed sphingosine-dependent protein kinases (SDKs). These SDKs were previously shown to phosphorylate endogenous 14-3-3 proteins (Megidish, T., White, T., Takio, K., Titani, K., Igarashi, Y., and Hakomori, S. (1995) Biochem. Biophys. Res. Commun. 216, 739-747). We have now partially purified one SDK, termed SDK1, from cytosol of mouse Balb/c 3T3(A31) fibroblasts. SDK1 is a serine kinase with molecular mass 50-60 kDa that is strongly activated by N, N'-dimethylsphingosine and sphingosine, but not by ceramide, sphingosine 1-phosphate, or other sphingo-, phospho-, or glycerolipids tested. Its activity is inhibited by the protein kinase C activator phosphatidylserine. Activity of SDK1 is clearly distinct from other types of serine kinases tested, including casein kinase II, the alpha and zeta isoforms of protein kinase C, extracellular signal-regulated mitogene-activated protein kinase 1 (Erk-1), Erk-2, and Raf-1. SDK1 specifically phosphorylates certain isoforms of 14-3-3 (eta, beta, zeta) but not others (sigma, tau). The phosphorylation site was identified as Ser* in the sequence Arg-Arg-Ser-Ser*-Trp-Arg in 14-3-3 beta. The sigma and tau isoforms of 14-3-3 lack serine at this position, potentially explaining their lack of phosphorylation by SDK1. Interestingly, the phosphorylation site is located on the dimer interface of 14-3-3. Phosphorylation of this site by SDK1 was studied in 14-3-3 mutants. Mutation of a lysine residue, located 9 amino acids N-terminal to the phosphorylation site, abolished 14-3-3 phosphorylation. Furthermore, co-immunoprecipitation experiments demonstrate an association between an SDK and 14-3-3 in situ. Exogenous N, N'-dimethylsphingosine stimulates 14-3-3 phosphorylation in Balb/c 3T3 fibroblasts, suggesting that SDK1 may phosphorylate 14-3-3 in situ. These data support a biological role of SDK1 activation and consequent

  6. Functional Networks of Highest-Connected Splice Isoforms: From The Chromosome 17 Human Proteome Project.

    PubMed

    Li, Hong-Dong; Menon, Rajasree; Govindarajoo, Brandon; Panwar, Bharat; Zhang, Yang; Omenn, Gilbert S; Guan, Yuanfang

    2015-09-01

    Alternative splicing allows a single gene to produce multiple transcript-level splice isoforms from which the translated proteins may show differences in their expression and function. Identifying the major functional or canonical isoform is important for understanding gene and protein functions. Identification and characterization of splice isoforms is a stated goal of the HUPO Human Proteome Project and of neXtProt. Multiple efforts have catalogued splice isoforms as "dominant", "principal", or "major" isoforms based on expression or evolutionary traits. In contrast, we recently proposed highest connected isoforms (HCIs) as a new class of canonical isoforms that have the strongest interactions in a functional network and revealed their significantly higher (differential) transcript-level expression compared to nonhighest connected isoforms (NCIs) regardless of tissues/cell lines in the mouse. HCIs and their expression behavior in the human remain unexplored. Here we identified HCIs for 6157 multi-isoform genes using a human isoform network that we constructed by integrating a large compendium of heterogeneous genomic data. We present examples for pairs of transcript isoforms of ABCC3, RBM34, ERBB2, and ANXA7. We found that functional networks of isoforms of the same gene can show large differences. Interestingly, differential expression between HCIs and NCIs was also observed in the human on an independent set of 940 RNA-seq samples across multiple tissues, including heart, kidney, and liver. Using proteomic data from normal human retina and placenta, we showed that HCIs are a promising indicator of expressed protein isoforms exemplified by NUDFB6 and M6PR. Furthermore, we found that a significant percentage (20%, p = 0.0003) of human and mouse HCIs are homologues, suggesting their conservation between species. Our identified HCIs expand the repertoire of canonical isoforms and are expected to facilitate studying main protein products, understanding gene

  7. Differential sensitivity of rat voltage-sensitive sodium channel isoforms to pyrazoline-type insecticides

    SciTech Connect

    Silver, Kristopher S.; Soderlund, David M. . E-mail: dms6@cornell.edu

    2006-07-15

    Pyrazoline-type insecticides are potent inhibitors of insect and mammalian voltage-sensitive sodium channels. In mammals, there are nine sodium channel {alpha} subunit isoforms that have unique distributions and pharmacological properties, but no published data exist that compare the relative sensitivity of these different mammalian sodium channel isoforms to inhibition by pyrazoline-type insecticides. This study employed the Xenopus oocyte expression system to examine the relative sensitivity of rat Na{sub v}1.2a, Na{sub v}1.4, Na{sub v}1.5, and Na{sub v}1.8 sodium channel {alpha} subunit isoforms to the pyrazoline-type insecticides indoxacarb, DCJW, and RH 3421. Additionally, we assessed the effect of coexpression with the rat {beta}1 auxiliary subunit on the sensitivity of the Na{sub v}1.2a and Na{sub v}1.4 isoforms to these compounds. The relative sensitivity of the four sodium channel {alpha} subunits differed for each of the three compounds we examined. With DCJW, the order of sensitivity was Na{sub v}1.4 > Na{sub v}1.2a > Na{sub v}1.5 > Na{sub v}1.8. In contrast, the relative sensitivity of these isoforms to indoxacarb differed from that to DCJW: the Na{sub v}1.8 isoform was most sensitive, the Na{sub v}1.4 isoform was completely insensitive, and the sensitivities of the Na{sub v}1.5 and Na{sub v}1.2a isoforms were intermediate between these two extremes. Moreover, the pattern of sensitivity to RH 3421 among these four isoforms was different from that for either indoxacarb or DCJW: the Na{sub v}1.4 isoform was most sensitive to RH 3421, whereas the sensitivities of the remaining three isoforms were substantially less than that of the Na{sub v}1.4 isoform and were approximately equivalent. The only statistically significant effect of coexpression of either the Na{sub v}1.2a or Na{sub v}1.4 isoforms with the {beta}1 subunit was the modest reduction in the sensitivity of the Na{sub v}1.2a isoform to RH 3421. These results demonstrate that mammalian sodium

  8. Two temporally synthesized charge subunits interact to form the five isoforms of cottonseed (Gossypium hirsutum) catalase.

    PubMed Central

    Ni, W; Trelease, R N; Eising, R

    1990-01-01

    Five charge isoforms of tetrameric catalase were isolated from cotyledons of germinated cotton (Gossypium hirsutum L.) seedlings. Denaturing isoelectric focusing of the individual isoforms in polyacrylamide gels indicated that isoforms A (most anodic) and E (most cathodic) consisted of one subunit of different charge, whereas isoforms B, C and D each consisted of a mixture of these two subunits. Thus the five isoforms apparently were formed through combinations of two subunits in different ratios. Labelling cotyledons in vivo with [35S]methionine at three daily intervals in the dark, and translation in vivo of polyadenylated RNA isolated from cotyledons at the same ages, revealed synthesis of two different subunits. One of the subunits was synthesized in cotyledons at all ages studied (days 1-3), whereas the other subunit was detected only at days 2 and 3. This differential expression of two catalase subunits helped explain previous results from this laboratory showing that the two anodic forms (A and B) found in maturing seeds were supplemented with three cathodic forms (C-E) after the seeds germinated. These subunit data also helped clarify our new findings that proteins of isoforms A, B and C (most active isoforms) accumulated in cotyledons of plants kept in the dark for 3 days, then gradually disappeared during the next several days, whereas isoforms D and E (least active isoforms) remained in the cells. This shift in isoform pattern occurred whether seedlings were kept in the dark or exposed to continuous light after day 3, although exposure to light enhanced this process. These sequential molecular events were responsible for the characteristic developmental changes (rise and fall) in total catalase activity. We believe that the isoform changeover is physiologically related to the changeover in glyoxysome to leaf-type-peroxisome metabolism. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. PMID:1695843

  9. Amphibian ryanodine receptor isoforms are related to those of mammalian skeletal or cardiac muscle.

    PubMed

    Lai, F A; Liu, Q Y; Xu, L; el-Hashem, A; Kramarcy, N R; Sealock, R; Meissner, G

    1992-08-01

    The ryanodine receptor (RyR)-Ca2+ release channels of frog skeletal muscle have been purified as 30S protein complexes comprised of two high molecular weight polypeptides. The upper and lower bands of the frog doublet comigrated on sodium dodecyl sulfate polyacylamide gels with the mammalian skeletal and cardiac RyR polypeptides, respectively. Immunoblot analysis showed that a polyclonal antiserum to the rat skeletal RyR preferentially cross-reacted with the upper band, whereas monoclonal antibodies to the canine cardiac RyR preferentially cross-reacted with the lower band of the frog receptor doublet. Immunoprecipitation studies indicated the presence of two homooligomer 30S RyR complexes comprised of either the lower or upper polypeptide band of the frog doublet, and immunocytochemical staining revealed their colocalization in frog gastrocnemius muscle. After planar lipid bilayer reconstitution of the 30S frog RyR, single-channel currents were observed that exhibited a Na+ and Ca2+ conductance and pharmacological characteristics similar to those of the mammalian skeletal and cardiac Ca2+ release channels. These results suggest that amphibian skeletal muscle expresses two distinct RyR isoforms that share epitopes in common with the mammalian skeletal or cardiac RyR. PMID:1325114

  10. Different phosphoinositide 3-kinase isoforms mediate carrageenan nociception and inflammation

    PubMed Central

    Pritchard, Rory A.; Falk, Lovissa; Larsson, Mathilda; Leinders, Mathias; Sorkin, Linda S.

    2016-01-01

    Abstract Phosphoinositide 3-kinases (PI3Ks) participate in signal transduction cascades that can directly activate and sensitize nociceptors and enhance pain transmission. They also play essential roles in chemotaxis and immune cell infiltration leading to inflammation. We wished to determine which PI3K isoforms were involved in each of these processes. Lightly anesthetized rats (isoflurane) were injected subcutaneously with carrageenan in their hind paws. This was preceded by a local injection of 1% DMSO vehicle or an isoform-specific antagonist to PI3K-α (compound 15-e), -β (TGX221), -δ (Cal-101), or -γ (AS252424). We measured changes in the mechanical pain threshold and spinal c-Fos expression (4 hours after injection) as indices of nociception. Paw volume, plasma extravasation (Evans blue, 0.3 hours after injection), and neutrophil (myeloperoxidase; 1 hour after injection) and macrophage (CD11b+; 4 hour after injection) infiltration into paw tissue were the measured inflammation endpoints. Only PI3K-γ antagonist before treatment reduced the carrageenan-induced pain behavior and spinal expression of c-Fos (P ≤ 0.01). In contrast, pretreatment with PI3K-α, -δ, and-γ antagonists reduced early indices of inflammation. Plasma extravasation PI3K-α (P ≤ 0.05), -δ (P ≤ 0.05), and -γ (P ≤ 0.01), early (0-2 hour) edema -α (P ≤ 0.05), -δ (P ≤ 0.001), and -γ (P ≤ 0.05), and neutrophil infiltration (all P ≤ 0.001) were all reduced compared to vehicle pretreatment. Later (2-4 hour), edema and macrophage infiltration (P ≤ 0.05) were reduced by only the PI3K-δ and -γ isoform antagonists, with the PI3K-δ antagonist having a greater effect on edema. PI3K-β antagonism was ineffective in all paradigms. These data indicate that pain and clinical inflammation are pharmacologically separable and may help to explain clinical conditions in which inflammation naturally wanes or goes into remission, but pain continues unabated. PMID:26313408

  11. Different phosphoinositide 3-kinase isoforms mediate carrageenan nociception and inflammation.

    PubMed

    Pritchard, Rory A; Falk, Lovissa; Larsson, Mathilda; Leinders, Mathias; Sorkin, Linda S

    2016-01-01

    Phosphoinositide 3-kinases (PI3Ks) participate in signal transduction cascades that can directly activate and sensitize nociceptors and enhance pain transmission. They also play essential roles in chemotaxis and immune cell infiltration leading to inflammation. We wished to determine which PI3K isoforms were involved in each of these processes. Lightly anesthetized rats (isoflurane) were injected subcutaneously with carrageenan in their hind paws. This was preceded by a local injection of 1% DMSO vehicle or an isoform-specific antagonist to PI3K-α (compound 15-e), -β (TGX221), -δ (Cal-101), or -γ (AS252424). We measured changes in the mechanical pain threshold and spinal c-Fos expression (4 hours after injection) as indices of nociception. Paw volume, plasma extravasation (Evans blue, 0.3 hours after injection), and neutrophil (myeloperoxidase; 1 hour after injection) and macrophage (CD11b+; 4 hour after injection) infiltration into paw tissue were the measured inflammation endpoints. Only PI3K-γ antagonist before treatment reduced the carrageenan-induced pain behavior and spinal expression of c-Fos (P ≤ 0.01). In contrast, pretreatment with PI3K-α, -δ, and-γ antagonists reduced early indices of inflammation. Plasma extravasation PI3K-α (P ≤ 0.05), -δ (P ≤ 0.05), and -γ (P ≤ 0.01), early (0-2 hour) edema -α (P ≤ 0.05), -δ (P ≤ 0.001), and -γ (P ≤ 0.05), and neutrophil infiltration (all P ≤ 0.001) were all reduced compared to vehicle pretreatment. Later (2-4 hour), edema and macrophage infiltration (P ≤ 0.05) were reduced by only the PI3K-δ and -γ isoform antagonists, with the PI3K-δ antagonist having a greater effect on edema. PI3K-β antagonism was ineffective in all paradigms. These data indicate that pain and clinical inflammation are pharmacologically separable and may help to explain clinical conditions in which inflammation naturally wanes or goes into remission, but pain continues unabated. PMID:26313408

  12. Role of nuclear progesterone receptor isoforms in uterine pathophysiology

    PubMed Central

    Patel, Bansari; Elguero, Sonia; Thakore, Suruchi; Dahoud, Wissam; Bedaiwy, Mohamed; Mesiano, Sam

    2015-01-01

    BACKGROUND Progesterone is a key hormonal regulator of the female reproductive system. It plays a major role to prepare the uterus for implantation and in the establishment and maintenance of pregnancy. Actions of progesterone on the uterine tissues (endometrium, myometrium and cervix) are mediated by the combined effects of two progesterone receptor (PR) isoforms, designated PR-A and PR-B. Both receptors function primarily as ligand-activated transcription factors. Progesterone action on the uterine tissues is qualitatively and quantitatively determined by the relative levels and transcriptional activities of PR-A and PR-B. The transcriptional activity of the PR isoforms is affected by specific transcriptional coregulators and by PR post-translational modifications that affect gene promoter targeting. In this context, appropriate temporal and cell-specific expression and function of PR-A and PR-B are critical for normal uterine function. METHODS Relevant studies describing the role of PRs in uterine physiology and pathology (endometriosis, uterine leiomyoma, endometrial cancer, cervical cancer and recurrent pregnancy loss) were comprehensively searched using PubMed, Cochrane Library, Web of Science, and Google Scholar and critically reviewed. RESULTS Progesterone, acting through PR-A and PR-B, regulates the development and function of the endometrium and induces changes in cells essential for implantation and the establishment and maintenance of pregnancy. During pregnancy, progesterone via the PRs promotes myometrial relaxation and cervical closure. Withdrawal of PR-mediated progesterone signaling triggers menstruation and parturition. PR-mediated progesterone signaling is anti-mitogenic in endometrial epithelial cells, and as such, mitigates the tropic effects of estrogen on eutopic normal endometrium, and on ectopic implants in endometriosis. Similarly, ligand-activated PRs function as tumor suppressors in endometrial cancer cells through inhibition of key

  13. Explication of interactions between HMGCR isoform 2 and various statins through In silico modeling and docking.

    PubMed

    Karthik, M V K; Satya Deepak, M V K N; Shukla, Pratyoosh

    2012-02-01

    The enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) catalyzes the conversion of HMG-CoA to mevalonate, a four-electron oxidoreduction that is the rate-limiting step in the synthesis of cholesterol and other isoprenoids. This study was designed to understand the mode of interactions of HMGCR isoform 2 with other statins. Hence, ligands such as Atorvastatin (DB01076), Lovastatin (DB00227), Fluvastatin (DB01095), Simvastatin (DB00641), Pravastatin (DB00175), Rosuvastatin (DB01098) and Cerivastatin (DB00439) were docked with enzymes HMGCR isoform 1 (pdb: 1DQ8) and modeled HMGCR isoform 2 (gi|196049380). Our homology modeling results were further processed to model the structure of human HMGCR isoform 2 and its accuracy was confirmed through RMS Z-scores (1.249). These interactions revealed that binding residues such as Arg515, Asp516, Tyr517 and Asn518 are found to be conserved in HMGCR isoform 2 with various statins. Our studies further concluded that Atorvastatin is most efficient inhibitor against both the isoforms of HMGCR whereas HMGCR isoform 2 shows less effectiveness with statins when compared with HMGCR isoform 1. PMID:22177940

  14. Appearance of Novel Glucose-6-Phosphate Dehydrogenase Isoforms in Chlamydomonas reinhardtii during Growth on Nitrate.

    PubMed Central

    Huppe, H. C.; Turpin, D. H.

    1996-01-01

    Extractable glucose-6-phosphate dehydrogenase activity is higher from N-limited Chlamydomonas reinhardtii cells than from N-sufficient cells. Native gels reveal that the isoform complexity varies depending on the form of N supplied. The isoforms associated with NO3- growth appear within 2 h of switching cells from NH4+ to NO3-. PMID:12226271

  15. AN ENZYME LINKED IMMUNOSORBENT ASSAY FOR THE HO-1 ISOFORM OF HEME OXYGENASE

    EPA Science Inventory

    AN ENZYME LINKED IMMUNOSORBENT ASSAY FOR THE HO-1 ISOFORM OF HEME OXYGENASE

    Heme oxygenase (HO) occurs in biological tissues as two major isoforms HO-1 and HO-2. HO-1 is inducible by many treatments, particularly oxidative stress-related conditions such as depletion of gl...

  16. Activation of AMPK alpha and gamma-isoform complexes in the intact ischemic rat heart

    Technology Transfer Automated Retrieval System (TEKTRAN)

    AMP-activated protein kinase (AMPK) plays a key role in modulating cellular metabolic processes. AMPK, a serine-threonine kinase, is a heterotrimeric complex of catalytic alpha-subunits and regulatory beta- and gamma-subunits with multiple isoforms. Mutations in the cardiac gamma(2)-isoform have bee...

  17. Revisiting the Identification of Canonical Splice Isoforms through Integration of Functional Genomics and Proteomics Evidence

    PubMed Central

    Li, Hong-Dong; Menon, Rajasree; Omenn, Gilbert S.; Guan, Yuanfang

    2014-01-01

    Canonical isoforms in different databases have been defined as the most prevalent, most conserved, most expressed, longest, or the one with the clearest description of domains or post-translational modifications. In this article, we revisit these definitions of canonical isoforms based on functional genomics and proteomics evidence, focusing on mouse data. We report a novel functional relationship network-based approach for identifying the Highest Connected Isoforms (HCIs). We show that 46% of these HCIs are not the longest transcripts. In addition, this approach revealed many genes that have more than one highly connected isoforms. Averaged across 175 RNA-seq datasets covering diverse tissues and conditions, 65% of the HCIs show higher expression levels than non-highest connected isoforms (NCIs) at the transcript level. At the protein level, these HCIs highly overlap with the expressed splice variants, based on proteomic data from eight different normal tissues. These results suggest that a more confident definition of canonical isoforms can be made through integration of multiple lines of evidence, including highest connected isoforms defined by biological processes and pathways, expression prevalence at the transcript level, and relative or absolute abundance at the protein level. This integrative proteogenomics approach can successfully identify principal isoforms that are responsible for the canonical functions of genes. PMID:25265570

  18. Comprehensive analysis of tropomyosin isoforms in skeletal muscles by top-down proteomics.

    PubMed

    Jin, Yutong; Peng, Ying; Lin, Ziqing; Chen, Yi-Chen; Wei, Liming; Hacker, Timothy A; Larsson, Lars; Ge, Ying

    2016-04-01

    Mammalian skeletal muscles are heterogeneous in nature and are capable of performing various functions. Tropomyosin (Tpm) is a major component of the thin filament in skeletal muscles and plays an important role in controlling muscle contraction and relaxation. Tpm is known to consist of multiple isoforms resulting from different encoding genes and alternative splicing, along with post-translational modifications. However, a systematic characterization of Tpm isoforms in skeletal muscles is still lacking. Therefore, we employed top-down mass spectrometry (MS) to identify and characterize Tpm isoforms present in different skeletal muscles from multiple species, including swine, rat, and human. Our study revealed that Tpm1.1 and Tpm2.2 are the two major Tpm isoforms in swine and rat skeletal muscles, whereas Tpm1.1, Tpm2.2, and Tpm3.12 are present in human skeletal muscles. Tandem MS was utilized to identify the sequences of the major Tpm isoforms. Furthermore, quantitative analysis revealed muscle-type specific differences in the abundance of un-modified and modified Tpm isoforms in rat and human skeletal muscles. This study represents the first systematic investigation of Tpm isoforms in skeletal muscles, which not only demonstrates the capabilities of top-down MS for the comprehensive characterization of skeletal myofilament proteins but also provides the basis for further studies on these Tpm isoforms in muscle-related diseases. PMID:27090236

  19. A global comparison between nuclear and cytosolic transcriptomes reveals differential compartmentalization of alternative transcript isoforms

    PubMed Central

    Chen, Liang

    2010-01-01

    Transcriptome analyses have typically disregarded nucleocytoplasmic differences. This approach has ignored some post-transcriptional regulations and their effect on the ultimate protein expression levels. Despite a longstanding interest in the differences between the nuclear and cytosolic transcriptomes, it is only recently that data have become available to study such differences and their associated features on a genome-wide scale. Here, we compared the nuclear and cytosolic transcriptomes of HepG2 and HeLa cells. HepG2 and HeLa cells vary significantly in the differential compartmentalization of their transcript isoforms, indicating that nucleocytoplasmic compartmentalization is a cell-specific characteristic. The differential compartmentalization is manifested at the transcript isoform level instead of the gene level because alternative isoforms of one gene can display different nucleocytoplasmic distributions. The isoforms enriched in the cytosol tend to have more introns and longer introns in their pre-mRNAs. They have more functional RNA folds and unique exons in the 3′ regions. These isoforms are more conserved than the isoforms enriched in the nucleus. Surprisingly, the presence of microRNAs does not have a significant impact on the nucleocytoplasmic distribution of their target isoforms. In contrast, nonsense-mediated decay is significantly more associated with the isoforms enriched in the nucleus than those enriched in the cytosol. PMID:19969546

  20. Myosin X and its motorless isoform differentially modulate dendritic spine development by regulating trafficking and retention of vasodilator-stimulated phosphoprotein

    PubMed Central

    Lin, Wan-Hsin; Hurley, Joshua T.; Raines, Alexander N.; Cheney, Richard E.; Webb, Donna J.

    2013-01-01

    Summary Myosin X (Myo10) is an unconventional myosin with two known isoforms: full-length (FL)-Myo10 that has motor activity, and a recently identified brain-expressed isoform, headless (Hdl)-Myo10, which lacks most of the motor domain. FL-Myo10 is involved in the regulation of filopodia formation in non-neuronal cells; however, the biological function of Hdl-Myo10 remains largely unknown. Here, we show that FL- and Hdl-Myo10 have important, but distinct, roles in the development of dendritic spines and synapses in hippocampal neurons. FL-Myo10 induces formation of dendritic filopodia and modulates filopodia dynamics by trafficking the actin-binding protein vasodilator-stimulated phosphoprotein (VASP) to the tips of filopodia. By contrast, Hdl-Myo10 acts on dendritic spines to enhance spine and synaptic density as well as spine head expansion by increasing the retention of VASP in spines. Thus, this study demonstrates a novel biological function for Hdl-Myo10 and an important new role for both Myo10 isoforms in the development of dendritic spines and synapses. PMID:23943878

  1. Beyond the one-locus-one-miRNA paradigm: microRNA isoforms enable deeper insights into breast cancer heterogeneity

    PubMed Central

    Telonis, Aristeidis G.; Loher, Phillipe; Jing, Yi; Londin, Eric; Rigoutsos, Isidore

    2015-01-01

    Here we describe our study of miRNA isoforms (isomiRs) in breast cancer (BRCA) and normal breast data sets from the Cancer Genome Atlas (TCGA) repository. We report that the full isomiR profiles, from both known and novel human-specific miRNA loci, are particularly rich in information and can distinguish tumor from normal tissue much better than the archetype miRNAs. IsomiR expression is also dependent on the patient's race, exemplified by miR-183-5p, several isomiRs of which are upregulated in triple negative BRCA in white but not black women. Additionally, we find that an isomiR's 5′ endpoint and length, but not the genomic origin, are key determinants of the regulation of its expression. Overexpression of distinct miR-183-5p isomiRs in MDA-MB-231 cells followed by microarray analysis revealed that each isomiR has a distinct impact on the cellular transcriptome. Parallel integrative analysis of mRNA expression from BRCA data sets of the TCGA repository demonstrated that isomiRs can distinguish between the luminal A and luminal B subtypes and explain in more depth the molecular differences between them than the archetype molecules. In conclusion, our findings provide evidence that post-transcriptional studies of BRCA will benefit from transcending the one-locus-one-miRNA paradigm and taking into account all isoforms from each miRNA locus as well as the patient's race. PMID:26400174

  2. Alternative splice isoforms of small conductance calcium-activated SK2 channels differ in molecular interactions and surface levels

    PubMed Central

    Scholl, Elizabeth Storer; Pirone, Antonella; Cox, Daniel H; Duncan, R Keith; Jacob, Michele H

    2014-01-01

    Small conductance Ca2+-sensitive potassium (SK2) channels are voltage-independent, Ca2+-activated ion channels that conduct potassium cations and thereby modulate the intrinsic excitability and synaptic transmission of neurons and sensory hair cells. In the cochlea, SK2 channels are functionally coupled to the highly Ca2+ permeant α9/10-nicotinic acetylcholine receptors (nAChRs) at olivocochlear postsynaptic sites. SK2 activation leads to outer hair cell hyperpolarization and frequency-selective suppression of afferent sound transmission. These inhibitory responses are essential for normal regulation of sound sensitivity, frequency selectivity, and suppression of background noise. However, little is known about the molecular interactions of these key functional channels. Here we show that SK2 channels co-precipitate with α9/10-nAChRs and with the actin-binding protein α-actinin-1. SK2 alternative splicing, resulting in a 3 amino acid insertion in the intracellular 3′ terminus, modulates these interactions. Further, relative abundance of the SK2 splice variants changes during developmental stages of synapse maturation in both the avian cochlea and the mammalian forebrain. Using heterologous cell expression to separately study the 2 distinct isoforms, we show that the variants differ in protein interactions and surface expression levels, and that Ca2+ and Ca2+-bound calmodulin differentially regulate their protein interactions. Our findings suggest that the SK2 isoforms may be distinctly modulated by activity-induced Ca2+ influx. Alternative splicing of SK2 may serve as a novel mechanism to differentially regulate the maturation and function of olivocochlear and neuronal synapses. PMID:24394769

  3. Exo70 Isoform Switching upon Epithelial-Mesenchymal Transition Mediates Cancer Cell Invasion

    PubMed Central

    Lu, Hezhe; Liu, Jianglan; Liu, Shujing; Zeng, Jingwen; Ding, Deqiang; Carstens, Russ P.; Cong, Yusheng; Xu, Xiaowei; Guo, Wei

    2014-01-01

    Summary Epithelial-mesenchymal transition (EMT) is an important developmental process hijacked by cancer cells for their dissemination. Here we show that Exo70, a component of the exocyst complex, undergoes isoform switching mediated by ESRP1, a pre-mRNA splicing factor that regulates EMT. Expression of the epithelial isoform of Exo70 affects the levels of key EMT transcriptional regulators such as Snail and ZEB2, and is sufficient to drive the transition to epithelial phenotypes. Differential Exo70 isoforms expression in human tumors correlates with cancer progression, and increased expression of the epithelial isoform of Exo70 inhibits tumor metastasis in mice. At the molecular level, the mesenchymal but not the epithelial isoform of Exo70 interacts with the Arp2/3 complex and stimulates actin polymerization for tumor invasion. Our findings provide a mechanism by which the exocyst function and actin dynamics are modulated for EMT and tumor invasion. PMID:24331928

  4. The mouse dead-end gene isoform alpha is necessary for germ cell and embryonic viability.

    PubMed

    Bhattacharya, Chitralekha; Aggarwal, Sita; Zhu, Rui; Kumar, Madhu; Zhao, Ming; Meistrich, Marvin L; Matin, Angabin

    2007-03-30

    Inactivation of the dead-end (Dnd1) gene in the Ter mouse strain results in depletion of primordial germ cells (PGCs) so that mice become sterile. However, on the 129 mouse strain background, loss of Dnd1 also increases testicular germ cell tumor incidence in parallel to PGC depletion. We report that inactivation of Dnd1 also affects embryonic viability in the 129 strain. Mouse Dnd1 encodes two protein isoforms, DND1-isoform alpha (DND1-alpha) and DND1-isoform beta (DND1-beta). Using isoform-specific antibodies, we determined DND1-alpha is expressed in embryos and embryonic gonads whereas DND1-beta expression is restricted to germ cells of the adult testis. Our data implicate DND1-alpha isoform to be necessary for germ cell viability and therefore its loss in Ter mice results in PGC depletion, germ cell tumor development and partial embryonic lethality in the 129 strain. PMID:17291453

  5. Differential recruitment of PKC isoforms in HeLa cells during redox stress

    PubMed Central

    Rimessi, Alessandro; Rizzuto, Rosario; Pinton, Paolo

    2007-01-01

    The protein kinase C (PKC) family is a major transducer of several intracellular pathways. In confirmation of this important role, PKCs exhibit high molecular heterogeneity, because they occur in at least 10 different isoforms differing in biochemical properties and sensitivity to activators. In this report we focused on the ability of different redox agents to induce modification of intracellular distribution of specific PKC isoforms in HeLa cells. To this end we utilized a panel of green fluorescent protein (GFP) chimeras and a high-speed digital imaging system. We observed a remarkable complexity of PKC signalling patterns occurring during redox stress with marked differences among PKC isoforms also belonging to the same subgroup. Moreover our results suggest that modifications of the intracellular redox state can modulate the responsiveness of specific PKC isoforms and, in turn, change the sensitivity of the different isoforms to cell stimulation. PMID:18229448

  6. Pharmacological targeting of PI3K isoforms as a therapeutic strategy in chronic lymphocytic leukaemia

    PubMed Central

    Blunt, Matthew D.; Steele, Andrew J.

    2015-01-01

    PI3Kδ inhibitors such as idelalisib are providing improved therapeutic options for the treatment of chronic lymphocytic leukaemia (CLL). However under certain conditions, inhibition of a single PI3K isoform can be compensated by the other PI3K isoforms, therefore PI3K inhibitors which target multiple PI3K isoforms may provide greater efficacy. The development of compounds targeting multiple PI3K isoforms (α, β, δ, and γ) in CLL cells, in vitro, resulted in sustained inhibition of BCR signalling but with enhanced cytotoxicity and the potential for improve clinical responses. This review summarises the progress of PI3K inhibitor development and describes the rationale and potential for targeting multiple PI3K isoforms. PMID:26500849

  7. Isoforms, structures, and functions of versatile spectraplakin MACF1

    PubMed Central

    Hu, Lifang; Su, Peihong; Li, Runzhi; Yin, Chong; Zhang, Yan; Shang, Peng; Yang, Tuanmin; Qian, Airong

    2016-01-01

    Spectraplakins are crucially important communicators, linking cytoskeletal components to each other and cellular junctions. Microtubule actin crosslinking factor 1 (MACF1), also known as actin crosslinking family 7 (ACF7), is a member of the spectraplakin family. It is expressed in numerous tissues and cells as one extensively studied spectraplakin. MACF1 has several isoforms with unique structures and well-known function to be able to crosslink F-actin and microtubules. MACF1 is one versatile spectraplakin with various functions in cell processes, embryo development, tissue-specific functions, and human diseases. The importance of MACF1 has become more apparent in recent years. Here, we summarize the current knowledge on the presence and function of MACF1 and provide perspectives on future research of MACF1 based on our studies and others. [BMB Reports 2016; 49(1): 37-44] PMID:26521939

  8. Quantitative isoform-profiling of highly diversified recognition molecules

    PubMed Central

    Schreiner, Dietmar; Simicevic, Jovan; Ahrné, Erik; Schmidt, Alexander; Scheiffele, Peter

    2015-01-01

    Complex biological systems rely on cell surface cues that govern cellular self-recognition and selective interactions with appropriate partners. Molecular diversification of cell surface recognition molecules through DNA recombination and complex alternative splicing has emerged as an important principle for encoding such interactions. However, the lack of tools to specifically detect and quantify receptor protein isoforms is a major impediment to functional studies. We here developed a workflow for targeted mass spectrometry by selected reaction monitoring that permits quantitative assessment of highly diversified protein families. We apply this workflow to dissecting the molecular diversity of the neuronal neurexin receptors and uncover an alternative splicing-dependent recognition code for synaptic ligands. DOI: http://dx.doi.org/10.7554/eLife.07794.001 PMID:25985086

  9. Extracellular rigidity sensing by talin isoform-specific mechanical linkages.

    PubMed

    Austen, Katharina; Ringer, Pia; Mehlich, Alexander; Chrostek-Grashoff, Anna; Kluger, Carleen; Klingner, Christoph; Sabass, Benedikt; Zent, Roy; Rief, Matthias; Grashoff, Carsten

    2015-12-01

    The ability of cells to adhere and sense differences in tissue stiffness is crucial for organ development and function. The central mechanisms by which adherent cells detect extracellular matrix compliance, however, are still unknown. Using two single-molecule-calibrated biosensors that allow the analysis of a previously inaccessible but physiologically highly relevant force regime in cells, we demonstrate that the integrin activator talin establishes mechanical linkages following cell adhesion, which are indispensable for cells to probe tissue stiffness. Talin linkages are exposed to a range of piconewton forces and bear, on average, 7-10 pN during cell adhesion depending on their association with F-actin and vinculin. Disruption of talin's mechanical engagement does not impair integrin activation and initial cell adhesion but prevents focal adhesion reinforcement and thus extracellular rigidity sensing. Intriguingly, talin mechanics are isoform specific so that expression of either talin-1 or talin-2 modulates extracellular rigidity sensing. PMID:26523364

  10. Differential expression of two scribble isoforms during Drosophila embryogenesis.

    PubMed

    Li, M; Marhold, J; Gatos, A; Török, I; Mechler, B M

    2001-10-01

    The tumour suppressor gene scribble (scrib) is required for epithelial polarity and growth control in Drosophila. Here, we report the identification and embryonic expression pattern of two Scrib protein isoforms resulting from alternative splicing during scrib transcription. Both proteins are first ubiquitously expressed during early embryogenesis. Then, during morphogenesis each Scrib protein displays a specific pattern of expression in the central and peripheral nervous systems, CNS and PNS, respectively. During germ band extension, the expression of the longer form Scrib1 occurs predominantly in the neuroblasts derived from the neuro-ectoderm and becomes later restricted to CNS neurones as well as to the pole cells in the gonads. By contrast, the shorter form Scrib2 is strongly expressed in the PNS and a subset of CNS neurones. PMID:11578873

  11. Spontaneous Hepatocellular Carcinoma after the Combined Deletion of Akt Isoforms.

    PubMed

    Wang, Qi; Yu, Wan-Ni; Chen, Xinyu; Peng, Xiao-Ding; Jeon, Sang-Min; Birnbaum, Morris J; Guzman, Grace; Hay, Nissim

    2016-04-11

    Akt is frequently hyperactivated in human cancers and is targeted for cancer therapy. However, the physiological consequences of systemic Akt isoform inhibition were not fully explored. We showed that while combined Akt1 and Akt3 deletion in adult mice is tolerated, combined Akt1 and Akt2 deletion induced rapid mortality. Akt2(-/-) mice survived hepatic Akt1 deletion but all developed spontaneous hepatocellular carcinoma (HCC), which is associated with FoxO-dependent liver injury and inflammation. The gene expression signature of HCC-bearing livers is similar to aggressive human HCC. Consistently, neither Akt1(-/-) nor Akt2(-/-) mice are resistant to diethylnitrosamine-induced hepatocarcinogenesis, and Akt2(-/-) mice display a high incidence of lung metastasis. Thus, in contrast to other cancers, hepatic Akt inhibition induces liver injury that could promote HCC. PMID:26996309

  12. Locomotion in Lymphocytes is Altered by Differential PKC Isoform Expression

    NASA Technical Reports Server (NTRS)

    Sundaresan, A.; Risin, D.; Pellis, N. R.

    1999-01-01

    Lymphocyte locomotion is critical for proper elicitation of the immune response. Locomotion of immune cells via the interstitium is essential for optimal immune function during wound healing, inflammation and infection. There are conditions which alter lymphocyte locomotion and one of them is spaceflight. Lymphocyte locomotion is severely inhibited in true spaceflight (true microgravity) and in rotating wall vessel culture (modeled microgravity). When lymphocytes are activated prior to culture in modeled microgravity, locomotion is not inhibited and the levels are comparable to those of static cultured lymphocytes. When a phorbol ester (PMA) is used in modeled microgravity, lymphocyte locomotion is restored by 87%. This occurs regardless if PMA is added after culture in the rotating wall vessel or during culture. Inhibition of DNA synthesis also does not alter restoration of lymphocyte locomotion by PMA. PMA is a direct activator of (protein kinase C) PKC . When a calcium ionophore, ionomycin is used it does not possess any restorative properties towards locomotion either alone or collectively with PMA. Since PMA brings about restoration without help from calcium ionophores (ionomycin), it is infer-red that calcium independent PKC isoforms are involved. Changes were perceived in the protein levels of PKC 6 where levels of the protein were downregulated at 24,72 and 96 hours in untreated rotated cultures (modeled microgravity) compared to untreated static (1g) cultures. At 48 hours there is an increase in the levels of PKC & in the same experimental set up. Studies on transcriptional and translational patterns of calcium independent isoforms of PKC such as 8 and E are presented in this study.

  13. Isoform-selective Inhibition of Facilitative Glucose Transporters

    PubMed Central

    Hresko, Richard C.; Kraft, Thomas E.; Tzekov, Anatoly; Wildman, Scott A.; Hruz, Paul W.

    2014-01-01

    Pharmacologic HIV protease inhibitors (PIs) and structurally related oligopeptides are known to reversibly bind and inactivate the insulin-responsive facilitative glucose transporter 4 (GLUT4). Several PIs exhibit isoform selectivity with little effect on GLUT1. The ability to target individual GLUT isoforms in an acute and reversible manner provides novel means both to investigate the contribution of individual GLUTs to health and disease and to develop targeted treatment of glucose-dependent diseases. To determine the molecular basis of transport inhibition, a series of chimeric proteins containing transmembrane and cytosolic domains from GLUT1 and GLUT4 and/or point mutations were generated and expressed in HEK293 cells. Structural integrity was confirmed via measurement of N-[2-[2-[2-[(N-biotinylcaproylamino)ethoxy)ethoxyl]-4-[2-(trifluoromethyl)-3H-diazirin-3-yl]benzoyl]-1,3-bis(mannopyranosyl-4-yloxy)-2-propylamine (ATB-BMPA) labeling of the chimeric proteins in low density microsome fractions isolated from stably transfected 293 cells. Functional integrity was assessed via measurement of zero-trans 2-deoxyglucose (2-DOG) uptake. ATB-BMPA labeling studies and 2-DOG uptake revealed that transmembrane helices 1 and 5 contain amino acid residues that influence inhibitor access to the transporter binding domain. Substitution of Thr-30 and His-160 in GLUT1 to the corresponding positions in GLUT4 is sufficient to completely transform GLUT1 into GLUT4 with respect to indinavir inhibition of 2-DOG uptake and ATB-BMPA binding. These data provide a structural basis for the selectivity of PIs toward GLUT4 over GLUT1 that can be used in ongoing novel drug design. PMID:24706759

  14. p53 isoforms regulate astrocyte-mediated neuroprotection and neurodegeneration.

    PubMed

    Turnquist, C; Horikawa, I; Foran, E; Major, E O; Vojtesek, B; Lane, D P; Lu, X; Harris, B T; Harris, C C

    2016-09-01

    Bidirectional interactions between astrocytes and neurons have physiological roles in the central nervous system and an altered state or dysfunction of such interactions may be associated with neurodegenerative diseases, such as Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS). Astrocytes exert structural, metabolic and functional effects on neurons, which can be either neurotoxic or neuroprotective. Their neurotoxic effect is mediated via the senescence-associated secretory phenotype (SASP) involving pro-inflammatory cytokines (e.g., IL-6), while their neuroprotective effect is attributed to neurotrophic growth factors (e.g., NGF). We here demonstrate that the p53 isoforms Δ133p53 and p53β are expressed in astrocytes and regulate their toxic and protective effects on neurons. Primary human astrocytes undergoing cellular senescence upon serial passaging in vitro showed diminished expression of Δ133p53 and increased p53β, which were attributed to the autophagic degradation and the SRSF3-mediated alternative RNA splicing, respectively. Early-passage astrocytes with Δ133p53 knockdown or p53β overexpression were induced to show SASP and to exert neurotoxicity in co-culture with neurons. Restored expression of Δ133p53 in near-senescent, otherwise neurotoxic astrocytes conferred them with neuroprotective activity through repression of SASP and induction of neurotrophic growth factors. Brain tissues from AD and ALS patients possessed increased numbers of senescent astrocytes and, like senescent astrocytes in vitro, showed decreased Δ133p53 and increased p53β expression, supporting that our in vitro findings recapitulate in vivo pathology of these neurodegenerative diseases. Our finding that Δ133p53 enhances the neuroprotective function of aged and senescent astrocytes suggests that the p53 isoforms and their regulatory mechanisms are potential targets for therapeutic intervention in neurodegenerative diseases. PMID:27104929

  15. Subunit isoform selectivity in assembly of Na,K-ATPase α-β heterodimers.

    PubMed

    Tokhtaeva, Elmira; Clifford, Rebecca J; Kaplan, Jack H; Sachs, George; Vagin, Olga

    2012-07-27

    To catalyze ion transport, the Na,K-ATPase must contain one α and one β subunit. When expressed by transfection in various expression systems, each of the four α subunit isoforms can assemble with each of the three β subunit isoforms and form an active enzyme, suggesting the absence of selective α-β isoform assembly. However, it is unknown whether in vivo conditions the α-β assembly is random or isoform-specific. The α(2)-β(2) complex was selectively immunoprecipitated by both anti-α(2) and anti-β(2) antibodies from extracts of mouse brain, which contains cells co-expressing multiple Na,K-ATPase isoforms. Neither α(1)-β(2) nor α(2)-β(1) complexes were detected in the immunoprecipitates. Furthermore, in MDCK cells co-expressing α(1), β(1), and β(2) isoforms, a greater fraction of the β(2) subunits was unassembled with α(1) as compared with that of the β(1) subunits, indicating preferential association of the α(1) isoform with the β(1) isoform. In addition, the α(1)-β(2) complex was less resistant to various detergents than the α(1)-β(1) complex isolated from MDCK cells or the α(2)-β(2) complex isolated from mouse brain. Therefore, the diversity of the α-β Na,K-ATPase heterodimers in vivo is determined not only by cell-specific co-expression of particular isoforms, but also by selective association of the α and β subunit isoforms. PMID:22696220

  16. Dissecting signalling by individual Akt/PKB isoforms, three steps at once.

    PubMed

    Osorio-Fuentealba, Cesar; Klip, Amira

    2015-09-01

    The serine/threonine kinase Akt/PKB (protein kinase B) is key for mammalian cell growth, survival, metabolism and oncogenic transformation. The diverse level and tissue expression of its three isoforms, Akt1/PKBα, Akt2/PKBβ and Akt3/PKBγ, make it daunting to identify isoform-specific actions in vivo and even in isolated tissues/cells. To date, isoform-specific knockout and knockdown have been the best strategies to dissect their individual overall functions. In a recent article in the Biochemical Journal, Kajno et al. reported a new strategy to study isoform selectivity in cell lines. Individual Akt/PKB isoforms in 3T3-L1 pre-adipocytes are first silenced via shRNA and stable cellular clones lacking one or the other isoform are selected. The stably silenced isoform is then replaced by a mutant engineered to be refractory to inhibition by MK-2206 (Akt1(W80A) or Akt2(W80A)). Akt1(W80A) or Akt2(W80A) are functional and effectively recruited to the plasma membrane in response to insulin. The system affords the opportunity to acutely control the activity of the endogenous non-silenced isoform through timely addition of MK-2206. Using this approach, it is confirmed that Akt1/PKBα is the preferred isoform sustaining adipocyte differentiation, but both Akt1/PKBα and Akt2/PKBβ can indistinctly support insulin-dependent FoxO1 (forkhead box O1) nuclear exclusion. Surprisingly, either isoform can also support insulin-dependent glucose transporter (GLUT) 4 translocation to the membrane, in contrast with the preferential role of Akt2/PKBβ assessed by knockdown studies. The new strategy should allow analysis of the plurality of Akt/PKB functions in other cells and in response to other stimuli. It should also be amenable to high-throughput studies to speed up advances in signal transmission by this pivotal kinase. PMID:26348913

  17. Distinct purinergic signaling pathways in prepubescent mouse spermatogonia.

    PubMed

    Fleck, David; Mundt, Nadine; Bruentgens, Felicitas; Geilenkirchen, Petra; Machado, Patricia A; Veitinger, Thomas; Veitinger, Sophie; Lipartowski, Susanne M; Engelhardt, Corinna H; Oldiges, Marco; Spehr, Jennifer; Spehr, Marc

    2016-09-01

    Spermatogenesis ranks among the most complex, yet least understood, developmental processes. The physiological principles that control male germ cell development in mammals are notoriously difficult to unravel, given the intricate anatomy and complex endo- and paracrinology of the testis. Accordingly, we lack a conceptual understanding of the basic signaling mechanisms within the testis, which control the seminiferous epithelial cycle and thus govern spermatogenesis. Here, we address paracrine signal transduction in undifferentiated male germ cells from an electrophysiological perspective. We identify distinct purinergic signaling pathways in prepubescent mouse spermatogonia, both in vitro and in situ. ATP-a dynamic, widespread, and evolutionary conserved mediator of cell to cell communication in various developmental contexts-activates at least two different spermatogonial purinoceptor isoforms. Both receptors operate within nonoverlapping stimulus concentration ranges, display distinct response kinetics and, in the juvenile seminiferous cord, are uniquely expressed in spermatogonia. We further find that spermatogonia express Ca(2+)-activated large-conductance K(+) channels that appear to function as a safeguard against prolonged ATP-dependent depolarization. Quantitative purine measurements additionally suggest testicular ATP-induced ATP release, a mechanism that could increase the paracrine radius of initially localized signaling events. Moreover, we establish a novel seminiferous tubule slice preparation that allows targeted electrophysiological recordings from identified testicular cell types in an intact epithelial environment. This unique approach not only confirms our in vitro findings, but also supports the notion of purinergic signaling during the early stages of spermatogenesis. PMID:27574293

  18. Targeting Human Central Nervous System Protein Kinases: An Isoform Selective p38αMAPK Inhibitor That Attenuates Disease Progression in Alzheimer’s Disease Mouse Models

    PubMed Central

    2015-01-01

    The first kinase inhibitor drug approval in 2001 initiated a remarkable decade of tyrosine kinase inhibitor drugs for oncology indications, but a void exists for serine/threonine protein kinase inhibitor drugs and central nervous system indications. Stress kinases are of special interest in neurological and neuropsychiatric disorders due to their involvement in synaptic dysfunction and complex disease susceptibility. Clinical and preclinical evidence implicates the stress related kinase p38αMAPK as a potential neurotherapeutic target, but isoform selective p38αMAPK inhibitor candidates are lacking and the mixed kinase inhibitor drugs that are promising in peripheral tissue disease indications have limitations for neurologic indications. Therefore, pursuit of the neurotherapeutic hypothesis requires kinase isoform selective inhibitors with appropriate neuropharmacology features. Synaptic dysfunction disorders offer a potential for enhanced pharmacological efficacy due to stress-induced activation of p38αMAPK in both neurons and glia, the interacting cellular components of the synaptic pathophysiological axis, to be modulated. We report a novel isoform selective p38αMAPK inhibitor, MW01-18-150SRM (=MW150), that is efficacious in suppression of hippocampal-dependent associative and spatial memory deficits in two distinct synaptic dysfunction mouse models. A synthetic scheme for biocompatible product and positive outcomes from pharmacological screens are presented. The high-resolution crystallographic structure of the p38αMAPK/MW150 complex documents active site binding, reveals a potential low energy conformation of the bound inhibitor, and suggests a structural explanation for MW150’s exquisite target selectivity. As far as we are aware, MW150 is without precedent as an isoform selective p38MAPK inhibitor or as a kinase inhibitor capable of modulating in vivo stress related behavior. PMID:25676389

  19. Murine laminin alpha3A and alpha3B isoform chains are generated by usage of two promoters and alternative splicing.

    PubMed

    Ferrigno, O; Virolle, T; Galliano, M F; Chauvin, N; Ortonne, J P; Meneguzzi, G; Aberdam, D

    1997-08-15

    We already identified two distinct laminin alpha3A and alpha3B chain isoforms which differ in their amino-terminal ends and display different tissue-specific expression patterns. In this study we have investigated whether these two different isoforms are products of the same laminin alpha3 (lama3) gene and transcribed from one or two separate promoters. Genomic clones were isolated that encompass the sequences upstream to the 5' ends of both the alpha3A and the alpha3B cDNAs. Sequence analysis of the region upstream to the alpha3A open reading frame revealed the presence of a TATA box and potential binding sites for responsive elements. By primer extension analysis, the transcription start site of the alpha3B mRNA isoform was defined. The sequences upstream to the alpha3B mRNA transcription start site do not contain a TATA box near the transcription initiation sites, but AP-1, AP-2, and Sp1 consensus binding site sequences were identified. The genomic regions located immediately upstream of the alpha3A and alpha3B transcription start sites were shown to possess promoter activities in transfection experiments. In the promoter regions, response elements for the acute phase reactant signal and NF-interleukin 6 were found, and their possible relevance in the context of inflammation and wound healing is discussed. Our results demonstrate that the lama3 gene produces the two polypeptides by alternative splicing and contains two promoters, which regulate the production of the two isoforms alpha3A and alpha3B. PMID:9252362

  20. Expression and identification of 10 sarcomeric MyHC isoforms in human skeletal muscles of different embryological origin. Diversity and similarity in mammalian species.

    PubMed

    Mascarello, Francesco; Toniolo, Luana; Cancellara, Pasqua; Reggiani, Carlo; Maccatrozzo, Lisa

    2016-09-01

    In the mammalian genome, among myosin heavy chain (MyHC) isoforms a family can be identified as sarcomeric based on their molecular structure which allows thick filament formation. In this study we aimed to assess the expression of the 10 sarcomeric isoforms in human skeletal muscles, adopting this species as a reference for comparison with all other mammalian species. To this aim, we set up the condition for quantitative Real Time PCR assay to detect and quantify MyHC mRNA expression in a wide variety of human muscles from somitic, presomitic and preotic origin. Specific patterns of expression of the following genes MYH1, MYH2, MYH3, MYH4, MYH6, MYH7, MYH8, MYH13, MYH14/7b and MYH15 were demonstrated in various muscle samples. On the same muscle samples which were analysed for mRNA expression, the corresponding MyHC proteins were studied with SDS PAGE and Western blot. The mRNA-protein comparison allowed the identification of 10 distinct proteins based on the electrophoretic migration rate. Three groups were formed based on the migration rate: fast migrating comprising beta/slow/1, alpha cardiac and fast 2B, slow migrating comprising fast 2X, fast 2A and two developmental isoforms (NEO and EMB), intermediate migrating comprising EO MyHC, slow B (product of MYH15), slow tonic (product of MYH14/7b). Of special interest was the demonstration of a protein band corresponding to 2B-MyHC in laryngeal muscles and the finding that all 10 isoforms are expressed in extraocular muscles. These latter muscles are the unique localization for extraocular, slow B (product of MYH15) and slow tonic (product of MYH14/7b). PMID:26970499

  1. Distinct regulation of dopamine D2S and D2L autoreceptor signaling by calcium.

    PubMed

    Gantz, Stephanie C; Robinson, Brooks G; Buck, David C; Bunzow, James R; Neve, Rachael L; Williams, John T; Neve, Kim A

    2015-01-01

    D2 autoreceptors regulate dopamine release throughout the brain. Two isoforms of the D2 receptor, D2S and D2L, are expressed in midbrain dopamine neurons. Differential roles of these isoforms as autoreceptors are poorly understood. By virally expressing the isoforms in dopamine neurons of D2 receptor knockout mice, this study assessed the calcium-dependence and drug-induced plasticity of D2S and D2L receptor-dependent G protein-coupled inwardly rectifying potassium (GIRK) currents. The results reveal that D2S, but not D2L receptors, exhibited calcium-dependent desensitization similar to that exhibited by endogenous autoreceptors. Two pathways of calcium signaling that regulated D2 autoreceptor-dependent GIRK signaling were identified, which distinctly affected desensitization and the magnitude of D2S and D2L receptor-dependent GIRK currents. Previous in vivo cocaine exposure removed calcium-dependent D2 autoreceptor desensitization in wild type, but not D2S-only mice. Thus, expression of D2S as the exclusive autoreceptor was insufficient for cocaine-induced plasticity, implying a functional role for the co-expression of D2S and D2L autoreceptors. PMID:26308580

  2. Merlin Isoforms 1 and 2 Both Act as Tumour Suppressors and Are Required for Optimal Sperm Maturation

    PubMed Central

    Zoch, Ansgar; Mayerl, Steffen; Schulz, Alexander; Greither, Thomas; Frappart, Lucien; Rübsam, Juliane; Heuer, Heike; Giovannini, Marco; Morrison, Helen

    2015-01-01

    The tumour suppressor Merlin, encoded by the gene NF2, is frequently mutated in the autosomal dominant disorder neurofibromatosis type II, characterised primarily by the development of schwannoma and other glial cell tumours. However, NF2 is expressed in virtually all analysed human and rodent organs, and its deletion in mice causes early embryonic lethality. Additionally, NF2 encodes for two major isoforms of Merlin of unknown functionality. Specifically, the tumour suppressor potential of isoform 2 remains controversial. In this study, we used Nf2 isoform-specific knockout mouse models to analyse the function of each isoform during development and organ homeostasis. We found that both isoforms carry full tumour suppressor functionality and can completely compensate the loss of the other isoform during development and in most adult organs. Surprisingly, we discovered that spermatogenesis is strictly dependent on the presence of both isoforms. While the testis primarily expresses isoform 1, we noticed an enrichment of isoform 2 in spermatogonial stem cells. Deletion of either isoform was found to cause decreased sperm quality as observed by maturation defects and head/midpiece abnormalities. These defects led to impaired sperm functionality as assessed by decreased sperm capacitation. Thus, we describe spermatogenesis as a new Nf2-dependent process. Additionally, we provide for the first time in vivo evidence for equal tumour suppressor potentials of Merlin isoform 1 and isoform 2. PMID:26258444

  3. Splice variants of the SWR1-type nucleosome remodeling factor Domino have distinct functions during Drosophila melanogaster oogenesis.

    PubMed

    Börner, Kenneth; Becker, Peter B

    2016-09-01

    SWR1-type nucleosome remodeling factors replace histone H2A by variants to endow chromatin locally with specialized functionality. In Drosophila melanogaster a single H2A variant, H2A.V, combines functions of mammalian H2A.Z and H2A.X in transcription regulation and the DNA damage response. A major role in H2A.V incorporation for the only SWR1-like enzyme in flies, Domino, is assumed but not well documented in vivo. It is also unclear whether the two alternatively spliced isoforms, DOM-A and DOM-B, have redundant or specialized functions. Loss of both DOM isoforms compromises oogenesis, causing female sterility. We systematically explored roles of the two DOM isoforms during oogenesis using a cell type-specific knockdown approach. Despite their ubiquitous expression, DOM-A and DOM-B have non-redundant functions in germline and soma for egg formation. We show that chromatin incorporation of H2A.V in germline and somatic cells depends on DOM-B, whereas global incorporation in endoreplicating germline nurse cells appears to be independent of DOM. By contrast, DOM-A promotes the removal of H2A.V from stage 5 nurse cells. Remarkably, therefore, the two DOM isoforms have distinct functions in cell type-specific development and H2A.V exchange. PMID:27578180

  4. Dysregulation of miRNA isoform level at 5' end in Alzheimer's disease.

    PubMed

    Wang, Shengqin; Xu, Yuming; Li, Musheng; Tu, Jing; Lu, Zuhong

    2016-06-15

    Alzheimer's disease (AD) is the most common form of dementia, whose mechanism is still not yet fully understood. A miRNA-based signature method, commonly according to the changes of expression levels, is widely used for AD analysis in previous studies. Recently, miRNA isoforms called as isomiR variants, which is considered to play important biological roles, have been demonstrated as the applications of high throughput sequencing platforms. Here, we presented an entropy-based model to detect the miRNA isoform level at the 5' end, and found many miRNAs with significant changes of isoform levels between the early stage and the late stage of AD by the application of this model to the public data. The statistical significance of the overlap between isoform-level changed miRNAs and AD related miRNAs extracted from HMDD2 supports that these miRNA isoforms are not degradation products. Based on the most common isomiR seed analysis of isoform-level changed AD related miRNAs, the predicted targets are also found to be enriched for genes involved in transcriptional regulation and the nervous system. After comparing with the expression level based method, we detected that changes of 5' isoform levels are more stable than those of expression levels for AD related miRNA detecting. PMID:26899870

  5. The isolation of parvalbumin isoforms from the tail muscle of the American alligator (Alligator mississipiensis).

    PubMed

    Laney, E L; Shabanowitz, J; King, G; Hunt, D F; Nelson, D J

    1997-04-01

    Multiple parvalbumin isoforms have been detected in the tail (skeletal) muscle of the American alligator (Alligator mississipiensis). One of these isoforms (APV-1) has been highly purified and partially characterized. Protein purification involved mainly gel filtration and anion exchange chromatography, and characterization included gel electrophoresis, amino acid composition analysis, metal ion analysis, MALDI-TOF and ESI mass spectrometry, ultraviolet and fluorescence spectroscopy, and one- and two-dimensional 500 MHz proton NMR spectroscopy. The alligator isoforms are rich in phenylalanine and deficient in the other aromatic residues as is typical for parvalbumins. In fact, the one highly purified isoform that forms the basis of this study has only phenyl-alanine as an aromatic residue. Ion exchange chromatography further indicates that this isoform has a relatively high isoelectric point (pl approximately 5.0), indicating that it is an alpha-lineage parvalbumin. This alligator parvalbumin isoform is unusual in that it has an atypically high Ca2+ content (almost 3.0 mole of Ca2+ per mole of protein) following purification, a fact supported by terbium fluorescence titration experiments. Preliminary comparative analysis of the highly purified alligator parvalbumin isoform (in the Ca2-loaded state) by two-dimensional 1H-NMR (2D 1H TOCSY and 2D 1H NOESY) indicates that there is considerable similarity in structure between the alligator protein and a homologous protein obtained from the silver hake (a saltwater fish species). PMID:9076974

  6. Cloning and Characterisation of Multiple Ferritin Isoforms in the Atlantic Salmon (Salmo salar)

    PubMed Central

    Lee, Jun-Hoe; Pooley, Nicholas J.; Mohd-Adnan, Adura; Martin, Samuel A. M.

    2014-01-01

    Ferritin is a highly-conserved iron-storage protein that has also been identified as an acute phase protein within the innate immune system. The iron-storage function is mediated through complementary roles played by heavy (H)-chain subunit as well as the light (L) in mammals or middle (M)-chain in teleosts, respectively. In this study, we report the identification of five ferritin subunits (H1, H2, M1, M2, M3) in the Atlantic salmon that were supported by the presence of iron-regulatory regions, gene structure, conserved domains and phylogenetic analysis. Tissue distribution analysis across eight different tissues showed that each of these isoforms is differentially expressed. We also examined the expression of the ferritin isoforms in the liver and kidney of juvenile Atlantic salmon that was challenged with Aeromonas salmonicida as well as in muscle cell culture stimulated with interleukin-1β. We found that each isoform displayed unique expression profiles, and in certain conditions the expressions between the isoforms were completely diametrical to each other. Our study is the first report of multiple ferritin isoforms from both the H- and M-chains in a vertebrate species, as well as ferritin isoforms that showed decreased expression in response to infection. Taken together, the results of our study suggest the possibility of functional differences between the H- and M-chain isoforms in terms of tissue localisation, transcriptional response to bacterial exposure and stimulation by specific immune factors. PMID:25078784

  7. Identification of Cardiac Myofilament Protein Isoforms Using Multiple Mass Spectrometry Based Approaches

    PubMed Central

    Kirk, Jonathan A.; Ubaida-Mohien, Ceereena; Graham, David R.; Faber, Matthijs J.; Van Eyk, Jennifer E.

    2014-01-01

    Purpose The identification of protein isoforms in complex biological samples is challenging. We, therefore, used a mass spectrometry (MS) approach to unambiguously identify cardiac myofilament protein isoforms based on the observation of a tryptic peptide consisting of a sequence unique to a particular isoform. Experimental design Three different workflows were used to isolate and fractionate rat cardiac myofilament subproteomes. All fractions were analyzed on an LTQ-Orbitrap MS, proteins were identified using various search engines (Mascot, X!Tandem, X!Tandem Kscore and OMSSA) with results combined via PepArML Meta-Search Engine, and a post-search analysis was performed by MASPECTRAS. Results The combination of multiple workflows and search engines resulted in a larger number of non-redundant proteins identified than with individual methods. A total of 102 myofilament annotated proteins were observed overlapping in two or three of the workflows. Literature search for myofilament presence with manual validation of the MS spectra was carried out for unambiguous identification: 10 cardiac myofilament and 17 cardiac myofilament-associated proteins were identified with 39 isoforms and sub-isoforms. Conclusion and clinical relevance We have identified multiple isoforms of myofilament proteins that are present in cardiac tissue using unique tryptic peptides. Changes in distribution of these protein isoforms under pathological conditions could ultimately allow for clinical diagnostics or as therapeutic targets. PMID:24974818

  8. CK-MB isoforms for early risk stratification of emergency department patients.

    PubMed

    Green, G B; Dehlinger, E; McGrievey, T S; Li, D J; Jones, K A; Kelen, G D; Chan, D W

    2000-10-01

    The potential clinical utility of single sample CK-MB isoforms measurement for early risk stratification of Emergency Department (ED) patients with possible myocardial ischemia was evaluated among 405 patients presenting to two urban EDs. Clinical and serologic data were prospectively collected and the occurrence of adverse events (AEs) and myocardial infarction (MI) during the 14-day outcome period was recorded and utilized to calculate and compare relative risks (RR) and predictive values of isoforms and CK-MB alone. Among the 405 patients, 67 accrued 105 AEs. Both isoforms and CK-MB alone were predictive of AEs with RR of 3.32 (2.09, 5.27) and 6.28 (4.64, 8.52), respectively. Isoforms had higher sensitivity for AEs compared to CK-MB (65.7% [54.3, 77.0] vs. 14.9% [6.4, 23.5]; p<0. 01) but lower specificity (69.2% [64.3, 74.2] vs. 99.7% [99.1,100. 0]; p<0.01). Isoforms' superior sensitivity allowed identification of many high risk patients missed by CK-MB alone. Further, for the prediction of MI, isoforms had superior diagnostic sensitivity and equivalent specificity. This investigation supports the emergency department use of early, single sample CK-MB isoform testing. PMID:10958863

  9. TMPRSS2 Isoform 1 Activates Respiratory Viruses and Is Expressed in Viral Target Cells

    PubMed Central

    Zmora, Pawel; Moldenhauer, Anna-Sophie; Hofmann-Winkler, Heike; Pöhlmann, Stefan

    2015-01-01

    The cellular protease TMPRSS2 cleaves and activates the influenza virus hemagglutinin (HA) and TMPRSS2 expression is essential for viral spread and pathogenesis in mice. Moreover, severe acute respiratory syndrome coronavirus (SARS-CoV) and other respiratory viruses are activated by TMPRSS2. However, previous studies on viral activation by TMPRSS2 focused on a 492 amino acids comprising form of the protein (isoform 2) while other TMPRSS2 isoforms, generated upon alternative splicing of the tmprss2 mRNA, have not been characterized. Here, we show that the mRNA encoding a TMPRSS2 isoform with an extended N-terminal cytoplasmic domain (isoform 1) is expressed in lung-derived cell lines and tissues. Moreover, we demonstrate that TMPRSS2 isoform 1 colocalizes with HA and cleaves and activates HA. Finally, we show that isoform 1 activates the SARS-CoV spike protein for cathepsin L-independent entry into target cells. Our results indicate that TMPRSS2 isoform 1 is expressed in viral target cells and might contribute to viral activation in the host. PMID:26379044

  10. Diversification of importin-α isoforms in cellular trafficking and disease states

    PubMed Central

    Pumroy, Ruth A.; Cingolani, Gino

    2015-01-01

    The human genome encodes seven isoforms of importin α which are grouped into three subfamilies known as α1, α2 and α3. All isoforms share a fundamentally conserved architecture that consists of an N-terminal, autoinhibitory, importin-β-binding (IBB) domain and a C-terminal Arm (Armadillo)-core that associates with nuclear localization signal (NLS) cargoes. Despite striking similarity in amino acid sequence and 3D structure, importin-α isoforms display remarkable substrate specificity in vivo. In the present review, we look at key differences among importin-α isoforms and provide a comprehensive inventory of known viral and cellular cargoes that have been shown to associate preferentially with specific isoforms. We illustrate how the diversification of the adaptor importin α into seven isoforms expands the dynamic range and regulatory control of nucleocytoplasmic transport, offering unexpected opportunities for pharmacological intervention. The emerging view of importin α is that of a key signalling molecule, with isoforms that confer preferential nuclear entry and spatiotemporal specificity on viral and cellular cargoes directly linked to human diseases. PMID:25656054

  11. Distinctive Characteristics of Educational Donors

    ERIC Educational Resources Information Center

    James, Russell N., III.

    2008-01-01

    Examining the charitable behavior of 56,663 US households, this paper evaluates the distinctive characteristics of educational donors as compared with donors to noneducational charitable organizations and with nondonors. In general, educational donors had significantly greater income, wealth, and education than other donors. Educational donors…

  12. China English: Its Distinctive Features

    ERIC Educational Resources Information Center

    Yang, Wei-dong; Dai, Wei-ping

    2011-01-01

    This paper attempts to expound that China English boasting its own distinctive features on the levels of phonology, words, sentences and discourse has been playing an irreplaceable role in intercultural activities, though still in its infancy and in the process of developing and perfecting itself, and it now makes every effort to move towards…

  13. Distinctiveness Maps for Image Matching

    NASA Technical Reports Server (NTRS)

    Manduchi, Roberto; Tomasi, Carlo

    2000-01-01

    Stereo correspondence is hard because different image features can look alike. We propose a measure for the ambiguity of image points that allows matching distinctive points first and breaks down the matching task into smaller and separate subproblems. Experiments with an algorithm based on this measure demonstrate the ensuing efficiency and low likelihood of incorrect matches.

  14. Educational Psychology: The Distinctive Contribution

    ERIC Educational Resources Information Center

    Cameron, R. J.

    2006-01-01

    This paper, written in the twenty-first anniversary year of the journal "Educational Psychology in Practice", attempts to uncover those distinctive aspects of the discipline and the practice of applied psychology in general and educational psychology in particular. After considering some of the reasons for attempting this task at this point in…

  15. Integrated transducer for color distinction

    NASA Astrophysics Data System (ADS)

    Kato, Hisao; Kojima, Masahiko; Yoshida, Akira

    1983-06-01

    A method for fabricating the improved version of an integrated transducer for color distinction is proposed. It consists of three SnO2(n)-Si(n) photodiodes on a Si wafer and a trichromatic filter prepared by arranging filters of the three primary colors (Eastman Kodak gelatin filters; Nos. 47B, 58, and 25) on an infrared glass filter (Hoya Glass; B-460). Its photoelectric characteristics and dependence of error of color distinction on the dimension of the transducer are reported. The photodiodes employed in this conversion assembly are produced by the simple spray method and are most suitable for detecting the low illumination. Since this transducer adapts the trichromatic color resolution method, highly accurate color distinction is possible. With this type of transducer, three photodiodes sensitive to the primary colors, red, green, and blue, respectively, are arranged on a plane. An error in color distinction can occur due to the differences in the strength of the light incident upon the respective photodiodes. This problem is reduced by making the transducer more compact. Finally, 11 kinds of colors are discerned in this experiment as an application of the transducer.

  16. Distinctive Qualities of Expert Teachers

    ERIC Educational Resources Information Center

    Tsui, Amy B. M.

    2009-01-01

    This paper attempts to identify the distinctive qualities of successful veteran teachers, referred to as "expert teachers", which separates them not only from novice teachers but more importantly from experienced non-expert teachers. Based on earlier case studies, this paper maintains that the critical differences between expert and non-expert…

  17. Myosin isoform fiber type and fiber size in the tail of the Virginia opossum (Didelphis virginiana).

    PubMed

    Hazimihalis, P J; Gorvet, M A; Butcher, M T

    2013-01-01

    Muscle fiber type is a well studied property in limb muscles, however, much less is understood about myosin heavy chain (MHC) isoform expression in caudal muscles of mammalian tails. Didelphid marsupials are an interesting lineage in this context as all species have prehensile tails, but show a range of tail-function depending on either their arboreal or terrestrial locomotor habits. Differences in prehensility suggest that MHC isoform fiber types may also be different, in that terrestrial opossums may have a large distribution of oxidative fibers for object carrying tasks instead of faster, glycolytic fiber types expected in mammals with long tails. To test this hypothesis, MHC isoform fiber type and their regional distribution (proximal/transitional/distal) were determined in the tail of the Virginia opossum (Didelphis virginiana). Fiber types were determined by a combination of myosin-ATPase histochemistry, immunohistochemistry, and SDS-PAGE. Results indicate a predominance of the fast MHC-2A and -2X isoforms in each region of the tail. The presence of two fast isoforms, in addition to the slow MHC-1 isoform, was confirmed by SDS-PAGE analysis. The overall MHC isoform fiber type distribution for the tail was: 25% MHC-1, 71% MHC-2A/X hybrid, and 4% MHC-1/2A hybrid. Oxidative MHC-2A/X isoform fibers were found to be relatively large in cross-section compared to slow, oxidative MHC-1 and MHC-1/2A hybrid fibers. A large percentage of fast MHC-2A/X hybrids fibers may be suggestive of an evolutionary transition in MHC isoform distribution (fast-to-slow fiber type) in the tail musculature of an opossum with primarily a terrestrial locomotor habit and adaptive tail-function. PMID:23152195

  18. Profilin Isoforms Modulate Astrocytic Morphology and the Motility of Astrocytic Processes

    PubMed Central

    Schweinhuber, Stefanie K.; Meßerschmidt, Tania; Hänsch, Robert; Korte, Martin; Rothkegel, Martin

    2015-01-01

    The morphology of astrocytic processes determines their close structural association with synapses referred to as the ‘tripartite synapse’. Concerted morphological plasticity processes at tripartite synapses are supposed to shape neuronal communication. Morphological changes in astrocytes as well as the motility of astrocytic processes require remodeling of the actin cytoskeleton. Among the regulators of fast timescale actin-based motility, the actin binding protein profilin 1 has recently been shown to control the activity-dependent outgrowth of astrocytic processes. Here, we demonstrate that cultured murine astrocytes in addition to the ubiquitous profilin 1 also express the neuronal isoform profilin 2a. To analyze the cellular function of both profilins in astrocytes, we took advantage of a shRNA mediated isoform-specific downregulation. Interestingly, consistent with earlier results in neurons, we found redundant as well as isoform-specific functions of both profilins in modulating cellular physiology. The knockdown of either profilin 1 or profilin 2a led to a significant decrease in cell spreading of astrocytes. In contrast, solely the knockdown of profilin 2a resulted in a significantly reduced morphological complexity of astrocytes in both dissociated and slice culture astrocytes. Moreover, both isoforms proved to be crucial for forskolin-induced astrocytic stellation. Furthermore, forskolin treatment resulted in isoform-specific changes in the phosphorylation level of profilin 1 and profilin 2a, leading to a PKA-dependent phosphorylation of profilin 2a. In addition, transwell assays revealed an involvement of both isoforms in the motility of astrocytic processes, while FRAP analysis displayed an isoform-specific role of profilin 1 in the regulation of actin dynamics in peripheral astrocytic processes. Taken together, we suggest profilin isoforms to be important modulators of astrocytic morphology and motility with overlapping as well as isoform

  19. NSMAP: A method for spliced isoforms identification and quantification from RNA-Seq

    PubMed Central

    2011-01-01

    Background The development of techniques for sequencing the messenger RNA (RNA-Seq) enables it to study the biological mechanisms such as alternative splicing and gene expression regulation more deeply and accurately. Most existing methods employ RNA-Seq to quantify the expression levels of already annotated isoforms from the reference genome. However, the current reference genome is very incomplete due to the complexity of the transcriptome which hiders the comprehensive investigation of transcriptome using RNA-Seq. Novel study on isoform inference and estimation purely from RNA-Seq without annotation information is desirable. Results A Nonnegativity and Sparsity constrained Maximum APosteriori (NSMAP) model has been proposed to estimate the expression levels of isoforms from RNA-Seq data without the annotation information. In contrast to previous methods, NSMAP performs identification of the structures of expressed isoforms and estimation of the expression levels of those expressed isoforms simultaneously, which enables better identification of isoforms. In the simulations parameterized by two real RNA-Seq data sets, more than 77% expressed isoforms are correctly identified and quantified. Then, we apply NSMAP on two RNA-Seq data sets of myelodysplastic syndromes (MDS) samples and one normal sample in order to identify differentially expressed known and novel isoforms in MDS disease. Conclusions NSMAP provides a good strategy to identify and quantify novel isoforms without the knowledge of annotated reference genome which can further realize the potential of RNA-Seq technique in transcriptome analysis. NSMAP package is freely available at https://sites.google.com/site/nsmapforrnaseq. PMID:21575225

  20. Complex Interplay of the UL136 Isoforms Balances Cytomegalovirus Replication and Latency

    PubMed Central

    Caviness, Katie; Bughio, Farah; Crawford, Lindsey B.; Streblow, Daniel N.; Nelson, Jay A.; Caposio, Patrizia

    2016-01-01

    ABSTRACT Human cytomegalovirus (HCMV), a betaherpesvirus, persists indefinitely in the human host through poorly understood mechanisms. The UL136 gene is carried within a genetic locus important to HCMV latency termed the UL133/8 locus, which also carries UL133, UL135, and UL138. Previously, we demonstrated that UL136 is expressed as five protein isoforms ranging from 33-kDa to 19-kDa, arising from alternative transcription and, likely, translation initiation mechanisms. We previously showed that the UL136 isoforms are largely dispensable for virus infection in fibroblasts, a model for productive virus replication. In our current work, UL136 has emerged as a complex regulator of HCMV infection in multiple contexts of infection relevant to HCMV persistence: in an endothelial cell (EC) model of chronic infection, in a CD34+ hematopoietic progenitor cell (HPC) model of latency, and in an in vivo NOD-scid IL2Rγcnull humanized (huNSG) mouse model for latency. The 33- and 26-kDa isoforms promote replication, while the 23- and 19-kDa isoforms suppress replication in ECs, in CD34+ HPCs, and in huNSG mice. The role of the 25-kDa isoform is context dependent and influences the activity of the other isoforms. These isoforms localize throughout the secretory pathway, and loss of the 33- and 26-kDa UL136 isoforms results in virus maturation defects in ECs. This work reveals an intriguing functional interplay between protein isoforms that impacts virus replication, latency, and dissemination, contributing to the overall role of the UL133/8 locus in HCMV infection. PMID:26933055

  1. PML isoforms in response to arsenic: high-resolution analysis of PML body structure and degradation.

    PubMed

    Hands, Katherine J; Cuchet-Lourenco, Delphine; Everett, Roger D; Hay, Ronald T

    2014-01-15

    Arsenic is a clinically effective treatment for acute promyelocytic leukaemia (APL) in which the promyelocytic leukaemia (PML) protein is fused to retinoic receptor alpha (RARα). PML-RARα is degraded by the proteasome by a SUMO-dependent, ubiquitin-mediated pathway in response to arsenic treatment, curing the disease. Six major PML isoforms are expressed as a result of alternative splicing, each of which encodes a unique C-terminal region. Using a system in which only a single EYFP-linked PML isoform is expressed, we demonstrate that PMLI, PMLII and PMLVI accumulate in the cytoplasm following arsenic treatment, whereas PMLIII, PMLIV and PMLV do not. 3D structured illumination was used to obtain super-resolution images of PML bodies, revealing spherical shells of PML along with associated SUMO. Arsenic treatment results in dramatic isoform-specific changes to PML body ultrastructure. After extended arsenic treatment most PML isoforms are degraded, leaving SUMO at the core of the nuclear bodies. A high-content imaging assay identifies PMLV as the isoform most readily degraded following arsenic treatment, and PMLIV as relatively resistant to degradation. Immunoprecipitation analysis demonstrates that all PML isoforms are modified by SUMO and ubiquitin after arsenic treatment, and by using siRNA, we demonstrate that arsenic-induced degradation of all PML isoforms is dependent on the ubiquitin E3 ligase RNF4. Intriguingly, depletion of RNF4 results in marked accumulation of PMLV, suggesting that this isoform is an optimal substrate for RNF4. Thus the variable C-terminal domain influences the rate and location of degradation of PML isoforms following arsenic treatment. PMID:24190887

  2. Isoform analysis of LC-MS/MS data from multidimensional fractionation of the serum proteome.

    PubMed

    Krasnoselsky, Alexei L; Faca, Vitor M; Pitteri, Sharon J; Zhang, Qing; Hanash, Samir M

    2008-06-01

    We developed a visualization approach for the identification of protein isoforms, precursor/mature protein combinations, and fragments from LC-MS/MS analysis of multidimensional fractionation of serum and plasma proteins. We also describe a pattern recognition algorithm to automatically detect and flag potentially heterogeneous species of proteins in proteomic experiments that involve extensive fractionation and result in a large number of identified serum or plasma proteins in an experiment. Examples are given of proteins with known isoforms that validate our approach and present a subset of precursor/mature protein pairs that were detected with this approach. Potential applications include identification of differentially expressed isoforms in disease states. PMID:18419151

  3. A novel alternative splicing isoform of NF2 identified in human Schwann cells

    PubMed Central

    Su, Fang; Zhou, Zhengguang; Su, Wen; Wang, Zishu; Wu, Qiong

    2016-01-01

    Vestibular schwannoma (VS) is a benign, slow-growing cranial tumor that originates from the hypertrophy of Schwann cells. The majority of sporadic VS are unilateral, and the mechanisms underlying VS tumorigenesis are not fully understood. The human neurofibromin 2 (NF2) gene encodes the tumor suppressor protein merlin and the NF2 transcript can be alternatively spliced to form numerous isoforms. The present study investigated human Schwann cells (HSCs) at the mRNA and protein level to understand the function of the alternative splicing (AS) isoform of NF2. The total RNA of HSCs was isolated and the full-length coding sequence of NF2 was amplified. The amplified products were excised from agarose gels, purified and sequenced. NF2 at a protein level was assayed by immunoprecipitation and western blot analysis. The full-length and spliced NF2 forms were amplified by polymerase chain reaction (PCR) from the HSC complementary DNA and ligated into eukaryotic expression vector pcDNA3.1(+). The plasmids were transfected into the HSC HEI-193 cell line and cell proliferation assays were performed using Cell Counting Kit-8. PCR analysis using HSC total RNA as a template revealed the presence of a shortened NF2 transcript, which was due to splicing at the 3′-end of the NF2 mRNA. Sequence analysis confirmed that this AS isoform omitted exons 11, 12, 13, 14, 15 and 16. Immunoprecipitation and western blot analysis demonstrated that the AS isoform was highly expressed in the HSCs at 38 kDa, while the wild-type (WT) isoform, which was expected at 66 kDa, was undetectable. Transfection and cell proliferation assays revealed that the WT isoform exhibited significant growth inhibition, while the AS isoform did not suppress cell growth. In conclusion, the present study detected AS NF2 isoforms in HSC for the first time, and investigated the function of the principle AS isoform. The present study suggests that although HSCs have an undetectable level of WT isoform of the NF2 protein

  4. Nitric oxide synthases activation and inhibition by metallacarborane cluster-based isoform-specific affectors

    PubMed Central

    Kaplánek, Robert; Martásek, Pavel; Grüner, Bohumír; Panda, Satya; Rak, Jakub; Masters, Bettie Sue Siler; Král, Vladimír; Roman, Linda J.

    2012-01-01

    A small library of boron cluster and metallacarborane cluster-based ligands was designed, prepared and tested for isoform-selective activation or inhibition of the three nitric oxide synthase isoforms. Based on the concept of creating a hydrophobic analog of a natural substrate, a stable and non-toxic basic boron cluster system, previously used for boron neutron capture therapy, was modified by the addition of positively charged moieties to its periphery, providing hydrophobic and non-classical hydrogen bonding interactions with the protein. Several of these compounds show efficacy for inhibition of NO synthesis with differential effects on the various nitric oxide synthase isoforms. PMID:23075390

  5. Application of denaturing gradient gel electrophoresis to detect DNA sequence differences encoding apolipoprotein E isoforms

    SciTech Connect

    Parker, S.; Angelico, M.C.; Laffel, L.; Krolewski, A.S. Harvard Medical School, Boston, MA )

    1993-04-01

    Apolipoprotein E (apoE) plays an important role in plasma lipid metabolism. Three common isoforms of this protein have been identified by the isoelectric focusing method. In this report the authors describe a new method for distinguishing these isoforms. Their method employs PCR amplification of the DNA sequence of exon 4 in the apoE gene followed by denaturing gradient gel electrophoresis (DGGE) to distinguish its different melting characteristics. Identification of the ApoE isoforms through DNA melting behavior rather than protein charge differences eliminates the problems associated with isoelectric focusing and facilitates screening for additional mutations at the apoE locus. 12 refs., 2 figs.

  6. An alternatively spliced isoform of PECAM-1 is expressed at high levels in human and murine tissues, and suggests a novel role for the C-terminus of PECAM-1 in cytoprotective signaling.

    PubMed

    Bergom, Carmen; Paddock, Cathy; Gao, Cunji; Holyst, Trudy; Newman, Debra K; Newman, Peter J

    2008-04-15

    The Ig-ITIM family member PECAM-1 is expressed in vascular and endothelial cells, and its functions include suppression of mitochondria-dependent apoptosis. Previous studies have identified distinct PECAM-1 cytoplasmic domain splice variants at the mRNA, but not protein, level. Several relatively abundant mRNA isoforms lack exon 15 (Delta15) and would theoretically encode a protein with a truncated cytoplasmic domain and a unique C-terminal sequence. Using a novel rabbit polyclonal antibody that specifically recognizes Delta15 PECAM-1, we found that the Delta15 PECAM-1 isoform was expressed in human tissues, including brain, testes and ovary. This isoform was also expressed on the cell surface of human platelets, human umbilical vein endothelial cells (HUVECs) and the Jurkat T-cell leukemia, human erythroleukemia (HEL) and U937 histiocytic lymphoma cell lines. Furthermore, murine platelets and lung lysates demonstrated abundant amounts of exon-15-deficient PECAM-1. Functional studies revealed that Delta15 PECAM-1 retains both its homophilic binding capacity and its ability to signal by means of its immunoreceptor tyrosine-based inhibitory motif (ITIM) domains. Delta15 PECAM-1 was unable, however, to protect against apoptosis induced by overexpression of Bax or treatment with the chemotherapy agent etoposide. These studies suggest a novel role for the PECAM-1 C-terminus in cytoprotective signaling and highlight a need for further characterization of expression of PECAM-1 isoforms in normal and malignant tissues. PMID:18388311

  7. Cytoplasmic Dynein Antagonists with Improved Potency and Isoform Selectivity

    PubMed Central

    2015-01-01

    Cytoplasmic dyneins 1 and 2 are related members of the AAA+ superfamily (ATPases associated with diverse cellular activities) that function as the predominant minus-end-directed microtubule motors in eukaryotic cells. Dynein 1 controls mitotic spindle assembly, organelle movement, axonal transport, and other cytosolic, microtubule-guided processes, whereas dynein 2 mediates retrograde trafficking within motile and primary cilia. Small-molecule inhibitors are important tools for investigating motor protein-dependent mechanisms, and ciliobrevins were recently discovered as the first dynein-specific chemical antagonists. Here, we demonstrate that ciliobrevins directly target the heavy chains of both dynein isoforms and explore the structure–activity landscape of these inhibitors in vitro and in cells. In addition to identifying chemical motifs that are essential for dynein blockade, we have discovered analogs with increased potency and dynein 2 selectivity. These antagonists effectively disrupt Hedgehog signaling, intraflagellar transport, and ciliogenesis, making them useful probes of these and other cytoplasmic dynein 2-dependent cellular processes. PMID:26555042

  8. Autophagosome-associated variant isoforms of cytosolic enzymes.

    PubMed Central

    Fengsrud, M; Raiborg, C; Berg, T O; Strømhaug, P E; Ueno, T; Erichsen, E S; Seglen, P O

    2000-01-01

    In a search for autophagosome-associated proteins, two-dimensional gel separations of proteins from purified autophagosomes, postnuclear supernatant, cytosol, lysosomes, mitochondria, endosomes and a cytomembrane fraction (mostly endoplasmic reticulum) were compared. Three proteins, with monomeric molecular masses of 43, 35 and 31 kDa, were enriched in total or sedimentable fractions of autophagosomes relative to the corresponding fractions of postnuclear supernatant, suggesting an association with the autophagosomal delimiting membrane. These proteins were also present on lysosomal membranes, but they were absent from mitochondria, and detected only in small amounts in the cytomembrane fraction and in endosomes, indicating that they were not associated with organelles sequestered by autophagy. However, all three proteins were present in the cytosol, suggesting that they were cytosolic proteins binding peripherally to the delimiting membrane of autophagosomes, probably to its innermost surface as indicated by their resistance to treatment of intact autophagosomes with proteinase or protein-stripping agents. Amino acid sequencing identified these proteins as an isoform of argininosuccinate synthase, an N-truncated variant of glyceraldehyde-3-phosphate dehydrogenase, and a sequence variant of short-chain 2-enoyl-CoA hydratase. PMID:11104685

  9. RAS isoforms and mutations in cancer at a glance.

    PubMed

    Hobbs, G Aaron; Der, Channing J; Rossman, Kent L

    2016-04-01

    RAS proteins (KRAS4A, KRAS4B, NRAS and HRAS) function as GDP-GTP-regulated binary on-off switches, which regulate cytoplasmic signaling networks that control diverse normal cellular processes. Gain-of-function missense mutations in RAS genes are found in ∼25% of human cancers, prompting interest in identifying anti-RAS therapeutic strategies for cancer treatment. However, despite more than three decades of intense effort, no anti-RAS therapies have reached clinical application. Contributing to this failure has been an underestimation of the complexities of RAS. First, there is now appreciation that the four human RAS proteins are not functionally identical. Second, with >130 different missense mutations found in cancer, there is an emerging view that there are mutation-specific consequences on RAS structure, biochemistry and biology, and mutation-selective therapeutic strategies are needed. In this Cell Science at a Glance article and accompanying poster, we provide a snapshot of the differences between RAS isoforms and mutations, as well as the current status of anti-RAS drug-discovery efforts. PMID:26985062

  10. Splice isoform estrogen receptors as integral transmembrane proteins

    PubMed Central

    Kim, Kyung Hee; Toomre, Derek; Bender, Jeffrey R.

    2011-01-01

    In addition to enhancing or repressing transcription, steroid hormone receptors rapidly transduce kinase activation signals. On ligand engagement, an N-terminus–truncated splice isoform of estrogen receptor (ER) α, ER46, triggers membrane-initiated signals, resulting in endothelial nitric oxide synthase (eNOS) activation and endothelial NO production. The orientation of ER46 at the plasma membrane is incompletely defined. With the use of ecliptic pHluorin-fused ER46, total internal reflection fluorescence microscopy in live human endothelial cells illustrates that ER46 can topologically conform to a type I transmembrane protein structure. Mutation of isoleucine-386 at the center of ER46's transmembrane hydrophobic core prevents membrane spanning, obscures the N-terminal ectodomain, and effects a marked reduction in membrane-impermeant estrogen binding with diminished rapid eNOS activation and NO production, despite maintained genomic induction of an estrogen response element–luciferase reporter. Thus there exist pools of transmembrane steroid hormone receptors that are efficient signaling molecules and potential novel therapeutic targets. PMID:21937726

  11. Identification and Characterization of Lipoxygenase Isoforms in Senescing Carnation Petals

    PubMed Central

    Rouet-Mayer, Marie-Aude; Bureau, Jean-Marc; Laurière, Christiane

    1992-01-01

    A membrane-associated lipoxygenase and a soluble lipoxygenase have been identified in carnation (Dianthus caryophyllus L. cv Rêve) petals. Treatments of microsomal membranes by nonionic or zwitterionic detergents indicated that lipoxygenase is tightly bound to the membranes. By phase separation in Triton X-114, microsomal lipoxygenase can be identified in part as an integral membrane protein. Soluble lipoxygenase had an optimum pH range of 4.9 to 5.8, whereas microsomal lipoxygenase exhibited maximum activity at pH 6.1. Both soluble and membrane-associated lipoxygenases produced carbonyl compounds and hydroperoxides simultaneously, in the presence of oxygen. The membranous enzyme was fully inhibited by 0.1 millimolar n-propyl gallate, nordihydroguaiaretic acid, or salicylhydroxamic acid, but the effect of the three inhibitors on the soluble enzyme was much lower. The soluble lipoxygenase is polymorphic and three isoforms greatly differing by their isoelectric points were identified. Lipoxygenase activity in flowers was maximal at the beginning of withering, both in the microsomal and the soluble fractions. Substantial variations in the ratio of the two forms of lipoxygenase were noted at different sampling dates. Our results allowed us to formulate the hypothesis of a strong association of one soluble form with defined membrane constituents. ImagesFigure 1 PMID:16668773

  12. Assessment of orthologous splicing isoforms in human and mouse orthologous genes

    PubMed Central

    2010-01-01

    Background Recent discoveries have highlighted the fact that alternative splicing and alternative transcripts are the rule, rather than the exception, in metazoan genes. Since multiple transcript and protein variants expressed by the same gene are, by definition, structurally distinct and need not to be functionally equivalent, the concept of gene orthology should be extended to the transcript level in order to describe evolutionary relationships between structurally similar transcript variants. In other words, the identification of true orthology relationships between gene products now should progress beyond primary sequence and "splicing orthology", consisting in ancestrally shared exon-intron structures, is required to define orthologous isoforms at transcript level. Results As a starting step in this direction, in this work we performed a large scale human- mouse gene comparison with a twofold goal: first, to assess if and to which extent traditional gene annotations such as RefSeq capture genuine splicing orthology; second, to provide a more detailed annotation and quantification of true human-mouse orthologous transcripts defined as transcripts of orthologous genes exhibiting the same splicing patterns. Conclusions We observed an identical exon/intron structure for 32% of human and mouse orthologous genes. This figure increases to 87% using less stringent criteria for gene structure similarity, thus implying that for about 13% of the human RefSeq annotated genes (and about 25% of the corresponding transcripts) we could not identify any mouse transcript showing sufficient similarity to be confidently assigned as a splicing ortholog. Our data suggest that current gene and transcript data may still be rather incomplete - with several splicing variants still unknown. The observation that alternative splicing produces large numbers of alternative transcripts and proteins, some of them conserved across species and others truly species-specific, suggests that, still

  13. Endothelin-2, the forgotten isoform: emerging role in the cardiovascular system, ovarian development, immunology and cancer.

    PubMed

    Ling, Lowell; Maguire, Janet J; Davenport, Anthony P

    2013-01-01

    Endothelin-2 [ET-2; also known as vasoactive intestinal contractor (VIC), in rodents] differs from endothelin-1 (ET-1) by only two amino acids, and unlike the third isoform, endothelin-3 (ET-3), it has the same affinity as ET-1 for both ET(A) and ET(B) receptors. It is often assumed that ET-2 would mimic the actions of the more abundant ET-1 and current pharmacological interventions used to inhibit the ET system would also block the actions of ET-2. These assumptions have focused research on ET-1 with ET-2 studied in much less detail. Recent research suggests that our understanding of the ET family requires re-evaluation. Although ET-2 is very similar in structure as well as pharmacology to ET-1, and may co-exist in the same tissue compartments, there is converging evidence for an important and distinct ET-2 pathway. Specifically is has been demonstrated that ET-2 has a key role in ovarian physiology, with ET-2-mediated contraction proposed as a final signal facilitating ovulation. Furthermore, ET-2 may also have a pathophysiological role in heart failure, immunology and cancer. Comparison of ET-2 versus ET-1 mRNA expression suggests this may be accomplished at the level of gene expression but differences may also exist in peptide synthesis by enzymes such as endothelin converting enzymes (ECEs) and chymase, which may allow the two pathways to be distinguished pharmacologically and become separate drug targets. LINKED ARTICLES This article is part of a themed section on Endothelin. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2013.168.issue-1. PMID:22118774

  14. Isoform 5 of PIPKIγ regulates the endosomal trafficking and degradation of E-cadherin

    PubMed Central

    Schill, Nicholas J.; Hedman, Andrew C.; Choi, Suyong; Anderson, Richard A.

    2014-01-01

    ABSTRACT Phosphatidylinositol phosphate kinases (PIPKs) have distinct cellular targeting, allowing for site-specific synthesis of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] to activate specific signaling cascades required for cellular processes. Several C-terminal splice variants of PIPKIγ (also known as PIP5K1C) exist, and have been implicated in a multitude of cellular roles. PI(4,5)P2 serves as a fundamental regulator of E-cadherin transport, and PI(4,5)P2-generating enzymes are important signaling relays in these pathways. We present evidence that the isoform 5 splice variant of PIPKIγ (PIPKIγi5) associates with E-cadherin and promotes its lysosomal degradation. Additionally, we show that the endosomal trafficking proteins SNX5 and SNX6 associate with PIPKIγi5 and inhibit PIPKIγi5-mediated E-cadherin degradation. Following HGF stimulation, activated Src directly phosphorylates PIPKIγi5. Phosphorylation of the PIPKIγi5 C-terminus regulates its association with SNX5 and, consequently, E-cadherin degradation. Additionally, this PIPKIγi5-mediated pathway requires Rab7 to promote degradation of internalized E-cadherin. Taken together, the data indicate that PIPKIγi5 and SNX5 are crucial regulators of E-cadherin sorting and degradation. PIPKIγi5, SNX and phosphoinositide regulation of lysosomal sorting represent a novel area of PI(4,5)P2 signaling and research. PIPKIγi5 regulation of E-cadherin sorting for degradation might have broad implications in development and tissue maintenance, and enhanced PIPKIγi5 function might have pathogenic consequences due to downregulation of E-cadherin. PMID:24610942

  15. Expression of the p40 isoform of p63 has high specificity for cutaneous sarcomatoid squamous cell carcinoma.

    PubMed

    Ha Lan, Thanh T; Chen, Stephanie J T; Arps, David P; Fullen, Douglas R; Patel, Rajiv M; Siddiqui, Javed; Carskadon, Shannon; Palanisamy, Nallasivam; Harms, Paul W

    2014-11-01

    Cutaneous spindle cell malignancies such as sarcomatoid squamous cell carcinoma (SCC), leiomyosarcoma, desmoplastic melanoma (DM) and atypical fibroxanthoma (AFX) may be morphologically indistinguishable, yet accurate diagnosis is important for appropriate clinical management. The distinction among these entities relies on immunohistochemical evaluation for epidermal, muscle or melanocytic differentiation. Epidermal differentiation markers include cytokeratins and p63. p63 is expressed as two distinct isoforms, ΔNp63 (p40) and TAp63. p40 positivity is highly specific for pulmonary SCC and head and neck sarcomatoid SCC. We examined the utility of p40 vs. p63 immunostaining in the differentiation of a variety of cutaneous spindle cell malignancies, including sarcomatoid SCC (n = 27), AFX (n = 34) and DM (n = 10). p40 was less sensitive than p63 for detecting sarcomatoid SCC (56% and 81%, respectively). p63 and p40 were comparably specific for sarcomatoid SCC relative to AFX, with only rare weak staining of tumor cells for p63 and/or p40 in a minority of AFX cases, including one case with approximately 10% of cells staining weakly for p40. All cases of DM were negative for p40 and p63. Our results support continued use of p63 for diagnosis of cutaneous sarcomatoid SCC because of greater sensitivity relative to p40. PMID:25263756

  16. Divergent N-Terminal Sequences Target an Inducible Testis Deubiquitinating Enzyme to Distinct Subcellular Structures

    PubMed Central

    Lin, Haijiang; Keriel, Anne; Morales, Carlos R.; Bedard, Nathalie; Zhao, Qing; Hingamp, Pascal; Lefrançois, Stephane; Combaret, Lydie; Wing, Simon S.

    2000-01-01

    Ubiquitin-specific processing proteases (UBPs) presently form the largest enzyme family in the ubiquitin system, characterized by a core region containing conserved motifs surrounded by divergent sequences, most commonly at the N-terminal end. The functions of these divergent sequences remain unclear. We identified two isoforms of a novel testis-specific UBP, UBP-t1 and UBP-t2, which contain identical core regions but distinct N termini, thereby permitting dissection of the functions of these two regions. Both isoforms were germ cell specific and developmentally regulated. Immunocytochemistry revealed that UBP-t1 was induced in step 16 to 19 spermatids while UBP-t2 was expressed in step 18 to 19 spermatids. Immunoelectron microscopy showed that UBP-t1 was found in the nucleus while UBP-t2 was extranuclear and was found in residual bodies. For the first time, we show that the differential subcellular localization was due to the distinct N-terminal sequences. When transfected into COS-7 cells, the core region was expressed throughout the cell but the UBP-t1 and UBP-t2 isoforms were concentrated in the nucleus and the perinuclear region, respectively. Fusions of each N-terminal end with green fluorescent protein yielded the same subcellular localization as the native proteins, indicating that the N-terminal ends were sufficient for determining differential localization. Interestingly, UBP-t2 colocalized with anti-γ-tubulin immunoreactivity, indicating that like several other components of the ubiquitin system, a deubiquitinating enzyme is associated with the centrosome. Regulated expression and alternative N termini can confer specificity of UBP function by restricting its temporal and spatial loci of action. PMID:10938131

  17. A Synaptotagmin Isoform Switch during the Development of an Identified CNS Synapse.

    PubMed

    Kochubey, Olexiy; Babai, Norbert; Schneggenburger, Ralf

    2016-06-01

    Various Synaptotagmin (Syt) isoform genes are found in mammals, but it is unknown whether Syts can function redundantly in a given nerve terminal, or whether isoforms can be switched during the development of a nerve terminal. Here, we investigated the possibility of a developmental Syt isoform switch using the calyx of Held as a model synapse. At mature calyx synapses, fast Ca(2+)-driven transmitter release depended entirely on Syt2, but the release phenotype of Syt2 knockout (KO) mice was weaker at immature calyces, and absent at pre-calyceal synapses early postnatally. Instead, conditional genetic inactivation shows that Syt1 mediates fast release at pre-calyceal synapses, as well as a fast release component resistant to Syt2 deletion in immature calyces. This demonstrates a developmental Syt1-Syt2 isoform switch at an identified synapse, a mechanism that could fine-tune the speed, reliability, and plasticity of transmitter release at fast releasing CNS synapses. PMID:27210552

  18. Rational Quantitative Structure-Activity Relationship (RQSAR) Screen for PXR and CAR Isoform-Specific Nuclear Receptor Ligands

    PubMed Central

    Dring, Ann M.; Anderson, Linnea E.; Qamar, Saima; Stoner, Matthew A.

    2010-01-01

    Constitutive androstane receptor (CAR) and pregnane X receptor (PXR) are closely related orphan nuclear receptor proteins that share several ligands and target overlapping sets of genes involved in homeostasis and all phases of drug metabolism. CAR and PXR are involved in the development of certain diseases, including diabetes, metabolic syndrome and obesity. Ligand screens for these receptors so far have typically focused on steroid hormone analogs with pharmacophore-based approaches, only to find relatively few new hits. Multiple CAR isoforms have been detected in human liver, with the most abundant being the constitutively active reference, CAR1, and the ligand-dependent isoform CAR3. It has been assumed that any compound that binds CAR1 should also activate CAR3, and so CAR3 can be used as a ligand-activated surrogate for CAR1 studies. The possibility of CAR3-specific ligands has not, so far, been addressed. To investigate the differences between CAR1, CAR3 and PXR, and to look for more CAR ligands that may be of use in quantitative structure-activity relationship (QSAR) studies, we performed a luciferase transactivation assay screen of 60 mostly non-steroid compounds. Known active compounds with different core chemistries were chosen as starting points and structural variants were rationally selected for screening. Distinct differences in agonist versus inverse agonist/antagonist effects were seen in 49 compounds that had some ligand effect on at least one receptor and 18 that had effects on all three receptors; eight were CAR1 ligands only, three were CAR3 only ligands and four affected PXR only. This work provides evidence for new CAR ligands, some of which have CAR3-specific effects, and provides observational data on CAR and PXR ligands with which to inform in silico strategies. Compounds that demonstrated unique activity on any one receptor are potentially valuable diagnostic tools for the investigation of in vivo molecular targets. PMID:20869355

  19. Antibodies to T- and L-isoforms of the cytoskeletal protein, fimbrin, in patients with systemic lupus erythematosus.

    PubMed Central

    De Mendonca Neto, E C; Kumar, A; Shadick, N A; Michon, A M; Matsudaira, P; Eaton, R B; Kumar, P; Schur, P H

    1992-01-01

    The cytoskeleton is a complex network of proteins that maintain cell shape, mobility, and organelle function. Its components can be divided into three distinct classes: microfilaments, microtubules, and intermediate filaments. Fimbrins are microfilament proteins, a family of cytoplasmic phosphoproteins. Expression of the L-fimbrin isoform is restricted to replicating blood cells and expression of the T-fimbrin isoform to replicating cells of solid tissues. Sera from normals and from patients with systemic lupus erythematosus (SLE), juvenile arthritis, rheumatoid arthritis, Sjögren's syndrome, osteoarthritis, vasculitis, scleroderma, and mixed connective tissue disease were tested for the presence of antibodies to T- and L-fimbrin by ELISA, using purified recombinant fimbrin. The mean OD of sera from SLE patients was significantly higher than in normals (T-fimbrin, P less than 0.0001; L-fimbrin, P less than 0.001). 48 of 98 SLE sera had antibodies to T-fimbrin; 32 had antibodies to L-fimbrin; 20 had antibodies to both; 28 had only anti-T, and 12 had only anti-L-fimbrin. The mean OD for sera of the other rheumatic diseases was not significantly different from normals. The presence of either L- or T-fimbrin antibody was associated with pleuropericarditis (P = 0.015), photosensitivity (P = 0.011), and anti-Sm antibody (P = 0.010). Central nervous system SLE was associated with the presence of the L-fimbrin antibody alone (P = 0.016). There was a strong association between DR7 (but not other MHC alleles) and anti-L-fimbrin antibodies in SLE patients (chi square = 18; P less than 0.00002). No MHC association was observed with anti-T-fimbrin antibodies. Images PMID:1522211

  20. Structural and functional differences between KRIT1A and KRIT1B isoforms: A framework for understanding CCM pathogenesis

    SciTech Connect

    Francalanci, Floriana; Avolio, Maria; De Luca, Elisa; Longo, Dario; Menchise, Valeria; Guazzi, Paolo; Sgro, Francesco; Marino, Marco; Goitre, Luca; Balzac, Fiorella; Trabalzini, Lorenza; Retta, Saverio Francesco

    2009-01-15

    KRIT1 is a disease gene responsible for Cerebral Cavernous Malformations (CCM). It encodes for a protein containing distinct protein-protein interaction domains, including three NPXY/F motifs and a FERM domain. Previously, we isolated KRIT1B, an isoform characterized by the alternative splicing of the 15th coding exon and suspected to cause CCM when abnormally expressed. Combining homology modeling and docking methods of protein-structure and ligand binding prediction with the yeast two-hybrid assay of in vivo protein-protein interaction and cellular biology analyses we identified both structural and functional differences between KRIT1A and KRIT1B isoforms. We found that the 15th exon encodes for the distal {beta}-sheet of the F3/PTB-like subdomain of KRIT1A FERM domain, demonstrating that KRIT1B is devoid of a functional PTB binding pocket. As major functional consequence, KRIT1B is unable to bind Rap1A, while the FERM domain of KRIT1A is even sufficient for this function. Furthermore, we found that a functional PTB subdomain enables the nucleocytoplasmic shuttling of KRIT1A, while its alteration confers a restricted cytoplasmic localization and a dominant negative role to KRIT1B. Importantly, we also demonstrated that KRIT1A, but not KRIT1B, may adopt a closed conformation through an intramolecular interaction involving the third NPXY/F motif at the N-terminus and the PTB subdomain of the FERM domain, and proposed a mechanism whereby an open/closed conformation switch regulates KRIT1A nuclear translocation and interaction with Rap1A in a mutually exclusive manner. As most mutations found in CCM patients affect the KRIT1 FERM domain, the new insights into the structure-function relationship of this domain may constitute a useful framework for understanding molecular mechanisms underlying CCM pathogenesis.

  1. Purification and Characterization of Cinnamyl Alcohol Dehydrogenase Isoforms from the Periderm of Eucalyptus gunnii Hook.

    PubMed Central

    Hawkins, S. W.; Boudet, A. M.

    1994-01-01

    Cinnamyl alcohol dehydrogenase (CAD, EC 1.1.1.195) isoforms were purified from the periderm (containing both suberized and lignified cell layers) of Eucalyptus gunnii Hook stems. Two isoforms (CAD 1P and CAD 2P) were initially characterized, and the major form, CAD 2P, was resolved into three further isoforms by ion-exchange chromatography. Crude extracts contained two aliphatic alcohol dehydrogenases (ADH) and one aromatic ADH, which was later resolved into two further isoforms. Aliphatic ADHs did not use hydroxycinnamyl alcohols as substrates, whereas both aromatic ADH isoforms used coniferyl and sinapyl alcohol as substrates but with a much lower specific activity when compared with benzyl alcohol. The minor form, CAD 1P, was a monomer with a molecular weight of 34,000 that did not co-elute with either aromatic or aliphatic ADH activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis demonstrated that this protein was very similar to another CAD isoform purified from Eucalyptus xylem tissue. CAD 2P had a native molecular weight of approximately 84,000 and was a dimer consisting of two heterogenous subunits (with molecular weights of 42,000 and 44,000). These subunits were differentially combined to give the heterodimer and two homodimers. SDS-PAGE, western blots, and nondenaturing PAGE indicated that the CAD 2P heterodimer was very similar to the main CAD isoform previously purified in our laboratory from differentiating xylem tissue of E. gunnii (D. Goffner, I. Joffroy, J. Grima-Pettenati, C. Halpin, M.E. Knight, W. Schuch, A.M. Boudet [1992] Planta 188: 48-53). Kinetic data indicated that the different CAD 2P isoforms may be implicated in the preferential production of different monolignols used in the synthesis of lignin and/or suberin. PMID:12232063

  2. Structural basis for the superior activity of the large isoform of snow flea antifreeze protein.

    PubMed

    Mok, Yee-Foong; Lin, Feng-Hsu; Graham, Laurie A; Celik, Yeliz; Braslavsky, Ido; Davies, Peter L

    2010-03-23

    The snow flea (Hypogastrum harveyi) is protected from freezing at sub-zero temperatures by a glycine-rich antifreeze protein (AFP) that binds to seed ice crystals and prevents them from growing larger. This AFP is hyperactive and comprises two isoforms [Graham, L. A., and Davies, P. L. (2005) Science 310, 461]. The larger isoform (15.7 kDa) exhibits several-fold higher activity than the smaller isoform (6.5 kDa), although it is considerably less abundant. To establish the molecular basis for this difference in activity, we determined the sequence of the large isoform. The primary sequences of these two isoforms are surprisingly divergent. However, both contain tripeptide repeats and turn motifs that enabled us to build a three-dimensional model of the large isoform based upon the six-polyproline helix structure of the small isoform. Our model contains 13 polyproline type II helices connected by proline-containing loops stacked into two flat sheets oriented antiparallel to one another. The structure is strictly amphipathic, with a hydrophilic surface on one side and a hydrophobic, putative ice-binding surface on the other. The putative ice-binding site is approximately twice as large in area as that of the small isoform, providing an explanation for the difference in activity that is consistent with other examples noted. By tagging the recombinant AFP with green fluorescent protein, we observed its binding to multiple planes of ice, especially the basal plane. This finding supports the correlation between AFP hyperactivity and basal plane binding first observed with spruce budworm AFP. PMID:20158269

  3. Recombinant Erythropoietin in Humans Has a Prolonged Effect on Circulating Erythropoietin Isoform Distribution

    PubMed Central

    Aachmann-Andersen, Niels Jacob; Just Christensen, Søren; Lisbjerg, Kristian; Oturai, Peter; Meinild-Lundby, Anne-Kristine; Holstein-Rathlou, Niels-Henrik; Lundby, Carsten; Vidiendal Olsen, Niels

    2014-01-01

    The membrane-assisted isoform immunoassay (MAIIA) quantitates erythropoietin (EPO) isoforms as percentages of migrated isoforms (PMI). We evaluated the effect of recombinant human EPO (rhEPO) on the distribution of EPO isoforms in plasma in a randomized, placebo-controlled, double-blinded, cross-over study. 16 healthy subjects received either low-dose Epoetin beta (5000 IU on days 1, 3, 5, 7, 9, 11 and 13); high-dose Epoetin beta (30.000 IU on days 1, 2 and 3 and placebo on days 5, 7, 9, 11 and 13); or placebo on all days. PMI on days 4, 11 and 25 was determined by interaction of N-acetyl glucosamine with the glycosylation dependent desorption of EPO isoforms. At day 25, plasma-EPO in both rhEPO groups had returned to values not different from the placebo group. PMI with placebo, reflecting the endogenous EPO isoforms, averaged 82.5 (10.3) % (mean (SD)). High-dose Epoetin beta decreased PMI on days 4 and 11 to 31.0 (4.2)% (p<0.00001) and 45.2 (7.3)% (p<0.00001). Low-dose Epoetin beta decreased PMI on days 4 and 11 to 46.0 (12.8)% (p<0.00001) and 46.1 (10.4)% (p<0.00001). In both rhEPO groups, PMI on day 25 was still decreased (high-dose Epoetin beta: 72.9 (19.4)% (p = 0.029); low-dose Epoetin beta: 73.1 (17.8)% (p = 0.039)). In conclusion, Epoetin beta leaves a footprint in the plasma-EPO isoform pattern. MAIIA can detect changes in EPO isoform distribution up til at least three weeks after administration of Epoetin beta even though the total EPO concentration has returned to normal. PMID:25335123

  4. Differential distribution of aggrecan isoforms in perineuronal nets of the human cerebral cortex

    PubMed Central

    Virgintino, Daniela; Perissinotto, Daniela; Girolamo, Francesco; Mucignat, Maria T; Montanini, Luisa; Errede, Mariella; Kaneiwa, Tomoyuki; Yamada, Shushei; Sugahara, Kazuyuki; Roncali, Luisa; Perris, Roberto

    2009-01-01

    Aggrecan is a component of the CNS extracellular matrix (ECM) and we show here that the three primary alternative spliced transcripts of the aggrecan gene found in cartilage are also present in the adult CNS. Using a unique panel of core protein-directed antibodies against human aggrecan we further show that different aggrecan isoforms are deposited in perineuronal nets (PNNs) and neuropil ECM of Brodmann’s area 6 of the human adult cerebral cortex. According to their distribution pattern, the identified cortical aggrecan isoforms were subdivided into five clusters spanning from cluster 1, comprised isoforms that appeared widespread throughout the cortex, to cluster 5, which was an aggrecan-free subset. Comparison of brain and cartilage tissues showed a different relative abundance of aggrecan isoforms, with cartilage-specific isoforms characterizing cluster 5, and PNN-associated isoforms lacking keratan sulphate chains. In the brain, isoforms of cluster 1 were disclosed in PNNs surrounding small-medium interneurons of layers II–V, small-medium pyramidal neurons of layers III and V and large interneurons of layer VI. Aggrecan PNNs enveloped both neuron bodies and neuronal processes, encompassing pre-terminal nerve fibres, synaptic boutons and terminal processes of glial cells and aggrecan was also observed in continuous ‘coats’ associated with satellite, neuron-associated cells of a putative glial nature. Immunolabelling for calcium-binding proteins and glutamate demonstrated that aggrecan PNNs were linked to defined subsets of cortical interneurons and pyramidal cells. We suggest that in the human cerebral cortex, discrete, layer-specific PNNs are assembled through the participation of selected aggrecan isoforms that characterize defined subsets of cortical neurons. PMID:19220578

  5. Expression analysis of ATAD3 isoforms in rodent and human cell lines and tissues.

    PubMed

    Li, Shuijie; Lamarche, Fredéric; Charton, Romain; Delphin, Christian; Gires, Olivier; Hubstenberger, Arnaud; Schlattner, Uwe; Rousseau, Denis

    2014-02-01

    ATAD3 (ATPase family AAA-Domain containing protein 3) is a mitochondrial inner membrane ATPase with unknown but vital functions. Initial researches have focused essentially on the major p66-ATAD3 isoform, but other proteins and mRNAs are described in the data banks. Using a set of anti-peptide antibodies and by the use of rodent and human cell lines and organs, we tried to detail ATAD3 gene expression profiles and to verify the existence of the various ATAD3 isoforms. In rodent, the single ATAD3 gene is expressed as a major isoform of 67 kDa, (ATAD3l; long), in all cells and organs studied. A second isoform, p57-ATAD3s (small), is expressed specifically throughout brain development and in adult, and overexpressed around the peri-natal period. p57-ATAD3s is also expressed in neuronal and glial rodent cell lines, and during in vitro differentiation of primary cultured rat oligodendrocytes. Other smaller isoforms were also detected in a tissue-specific manner. In human and primates, ATAD3 paralogues are encoded by three genes (ATAD3A, 3B and 3C), each of them presenting several putative variants. Analyzing the expression of ATAD3A and ATAD3B with four specific anti-peptide antibodies, and comparing their expressions with in vitro expressed ATAD3 cDNAs, we were able to observe and define five isoforms. In particular, the previously described p72-ATAD3B is confirmed to be in certain cases a phosphorylated form of ATAD3As. Moreover, we observed that the ATAD3As phosphorylation level is regulated by insulin and serum. Finally, exploring ATAD3 mRNA expression, we confirmed the existence of an alternative splicing in rodent and of several mRNA isoforms in human. Considering these observations, we propose the development of a uniform denomination for ATAD3 isoforms in rodent and human. PMID:24239551

  6. Sodium pump isoform specificity for the digitalis-like factor isolated from human peritoneal dialysate.

    PubMed

    Tao, Q F; Hollenberg, N K; Price, D A; Graves, S W

    1997-03-01

    We have isolated a labile, specific sodium pump inhibitor or digitalis-like factor from the peritoneal dialysate of volume-expanded renal failure patients whose levels correlated closely with volume status and blood pressure. This study characterizes the inhibitory profile of this agent compared with that of ouabain against the three alpha-isoforms of the sodium pump. We prepared microsomal Na,K-ATPase from rat tissues representing the highest proportion of one of the alpha-isoforms. Both Northern and Western blot analyses confirmed that kidney had predominantly the alpha1-isoform, skeletal muscle the alpha2-isoform, and fetal brain the alpha3-isoform. Ouabain (5 x 10(-6) mol/L) produced little inhibition of kidney Na,K-ATPase (3.4+/-2.0%) but significant inhibition of skeletal muscle (37.2+/-3.7%, P<.001) and fetal brain (38.8+/-3.5%, P<.001) activity. In contrast, the labile digitalis-like factor, causing comparable inhibition of fetal brain Na,K-ATPase activity (33.3+/-4.7%), produced markedly greater inhibition of kidney (42.5+/-5.6%, P<.001) and moderately greater inhibition of skeletal muscle pump activity (57.7+/-6.3%, P<.05). In addition, the labile digitalis-like factor produced a marked concentration-dependent inhibition of the alpha2- and alpha3-isoforms (r=.79, P=.00005). Experiments combining the labile digitalis-like factor and ouabain confirmed that digitalis-like factor, unlike ouabain, was an effective inhibitor of all three isoforms in rat, in particular alpha2. The different pattern of isoform sensitivity displayed by the labile digitalis-like factor and ouabain further differentiates the two agents and raises some interesting possibilities about the functional implications of the endogenous factor. PMID:9052901

  7. Network-Based Isoform Quantification with RNA-Seq Data for Cancer Transcriptome Analysis

    PubMed Central

    Zhang, Wei; Chang, Jae-Woong; Lin, Lilong; Minn, Kay; Wu, Baolin; Chien, Jeremy; Yong, Jeongsik; Zheng, Hui; Kuang, Rui

    2015-01-01

    High-throughput mRNA sequencing (RNA-Seq) is widely used for transcript quantification of gene isoforms. Since RNA-Seq data alone is often not sufficient to accurately identify the read origins from the isoforms for quantification, we propose to explore protein domain-domain interactions as prior knowledge for integrative analysis with RNA-Seq data. We introduce a Network-based method for RNA-Seq-based Transcript Quantification (Net-RSTQ) to integrate protein domain-domain interaction network with short read alignments for transcript abundance estimation. Based on our observation that the abundances of the neighboring isoforms by domain-domain interactions in the network are positively correlated, Net-RSTQ models the expression of the neighboring transcripts as Dirichlet priors on the likelihood of the observed read alignments against the transcripts in one gene. The transcript abundances of all the genes are then jointly estimated with alternating optimization of multiple EM problems. In simulation Net-RSTQ effectively improved isoform transcript quantifications when isoform co-expressions correlate with their interactions. qRT-PCR results on 25 multi-isoform genes in a stem cell line, an ovarian cancer cell line, and a breast cancer cell line also showed that Net-RSTQ estimated more consistent isoform proportions with RNA-Seq data. In the experiments on the RNA-Seq data in The Cancer Genome Atlas (TCGA), the transcript abundances estimated by Net-RSTQ are more informative for patient sample classification of ovarian cancer, breast cancer and lung cancer. All experimental results collectively support that Net-RSTQ is a promising approach for isoform quantification. Net-RSTQ toolbox is available at http://compbio.cs.umn.edu/Net-RSTQ/. PMID:26699225

  8. Network-Based Isoform Quantification with RNA-Seq Data for Cancer Transcriptome Analysis.

    PubMed

    Zhang, Wei; Chang, Jae-Woong; Lin, Lilong; Minn, Kay; Wu, Baolin; Chien, Jeremy; Yong, Jeongsik; Zheng, Hui; Kuang, Rui

    2015-12-01

    High-throughput mRNA sequencing (RNA-Seq) is widely used for transcript quantification of gene isoforms. Since RNA-Seq data alone is often not sufficient to accurately identify the read origins from the isoforms for quantification, we propose to explore protein domain-domain interactions as prior knowledge for integrative analysis with RNA-Seq data. We introduce a Network-based method for RNA-Seq-based Transcript Quantification (Net-RSTQ) to integrate protein domain-domain interaction network with short read alignments for transcript abundance estimation. Based on our observation that the abundances of the neighboring isoforms by domain-domain interactions in the network are positively correlated, Net-RSTQ models the expression of the neighboring transcripts as Dirichlet priors on the likelihood of the observed read alignments against the transcripts in one gene. The transcript abundances of all the genes are then jointly estimated with alternating optimization of multiple EM problems. In simulation Net-RSTQ effectively improved isoform transcript quantifications when isoform co-expressions correlate with their interactions. qRT-PCR results on 25 multi-isoform genes in a stem cell line, an ovarian cancer cell line, and a breast cancer cell line also showed that Net-RSTQ estimated more consistent isoform proportions with RNA-Seq data. In the experiments on the RNA-Seq data in The Cancer Genome Atlas (TCGA), the transcript abundances estimated by Net-RSTQ are more informative for patient sample classification of ovarian cancer, breast cancer and lung cancer. All experimental results collectively support that Net-RSTQ is a promising approach for isoform quantification. Net-RSTQ toolbox is available at http://compbio.cs.umn.edu/Net-RSTQ/. PMID:26699225

  9. Myosin heavy-chain isoforms in the flight and leg muscles of hummingbirds and zebra finches.

    PubMed

    Velten, Brandy P; Welch, Kenneth C

    2014-06-01

    Myosin heavy chain (MHC) isoform complement is intimately related to a muscle's contractile properties, yet relatively little is known about avian MHC isoforms or how they may vary with fiber type and/or the contractile properties of a muscle. The rapid shortening of muscles necessary to power flight at the high wingbeat frequencies of ruby-throated hummingbirds and zebra finches (25-60 Hz), along with the varied morphology and use of the hummingbird hindlimb, provides a unique opportunity to understand how contractile and morphological properties of avian muscle may be reflected in MHC expression. Isoforms of the hummingbird and zebra finch flight and hindlimb muscles were electrophoretically separated and compared with those of other avian species representing different contractile properties and fiber types. The flight muscles of the study species operate at drastically different contraction rates and are composed of different histochemically defined fiber types, yet each exhibited the same, single MHC isoform corresponding to the chicken adult fast isoform. Thus, despite quantitative differences in the contractile demands of flight muscles across species, this isoform appears necessary for meeting the performance demands of avian powered flight. Variation in flight muscle contractile performance across species may be due to differences in the structural composition of this conserved isoform and/or variation within other mechanically linked proteins. The leg muscles were more varied in their MHC isoform composition across both muscles and species. The disparity in hindlimb MHC expression between hummingbirds and the other species highlights previously observed differences in fiber type composition and thrust production during take-off. PMID:24671242

  10. Tau isoform regulation is region- and cell-specific in mouse brain.

    PubMed

    McMillan, Pamela; Korvatska, Elena; Poorkaj, Parvoneh; Evstafjeva, Zana; Robinson, Linda; Greenup, Lynne; Leverenz, James; Schellenberg, Gerard D; D'Souza, Ian

    2008-12-20

    Tau is a microtubule-associated protein implicated in neurodegenerative tauopathies. Alternative splicing of the tau gene (MAPT) generates six tau isoforms, distinguishable by the exclusion or inclusion of a repeat region of exon 10, which are referred to as 3-repeat (3R) and 4-repeat (4R) tau, respectively. We developed transgenic mouse models that express the entire human MAPT gene in the presence and absence of the mouse Mapt gene and compared the expression and regulation of mouse and human tau isoforms during development and in the young adult. We found differences between mouse and human tau in the regulation of exon 10 inclusion. Despite these differences, the isoform splicing pattern seen in normal human brain is replicated in our mouse models. In addition, we found that all tau, both in the neonate and young adult, is phosphorylated. We also examined the normal anatomic distribution of mouse and human tau isoforms in mouse brain. We observed developmental and species-specific variations in the expression of 3R- and 4R-tau within the frontal cortex and hippocampus. In addition, there were differences in the cellular distribution of the isoforms. Mice transgenic for the human MAPT gene exhibited higher levels of neuronal cell body expression of tau compared to wildtype mice. This neuronal cell body expression of tau was limited to the 3R isoform, whereas expression of 4R-tau was more "synaptic like," with granular staining of neuropil rather than in neuronal cell bodies. These developmental and species-specific differences in the regulation and distribution of tau isoforms may be important to the understanding of normal and pathologic tau isoform expression. PMID:18925637

  11. Tau isoform regulation is region and cell-specific in mouse brain

    PubMed Central

    McMillan, Pamela; Korvatska, Elena; Poorkaj, Parvoneh; Evstafjeva, Zana; Robinson, Linda; Greenup, Lynne; Leverenz, James; Schellenberg, Gerard D.; D’Souza, Ian

    2008-01-01

    Tau is a microtubule-associated protein implicated in neurodegenerative tauopathies. Alternative splicing of the tau gene (MAPT) generates six tau isoforms, distinguishable by the exclusion or inclusion of a repeat region of exon 10, that are referred to as 3-repeat (3R) and 4-repeat (4R) tau, respectively. We developed transgenic mouse models that express the entire human MAPT gene in the presence and absence of the mouse Mapt gene and compared the expression and regulation of mouse and human tau isoforms during development and in the young adult. We found differences between mouse and human tau in the regulation of exon 10 inclusion. Despite these differences, the isoform splicing pattern seen in normal human brain is replicated in our mouse models. In addition, we found that all tau, both in the neonate and young adult, is phosphorylated. We also examined the normal anatomic distribution of mouse and human tau isoforms in mouse brain. We observed developmental and species-specific variations in the expression of 3R and 4R-tau within the frontal cortex and hippocampus. In addition, there were differences in the cellular distribution of the isoforms. Mice transgenic for the human MAPT gene exhibited higher levels of neuronal cell body expression of tau compared to wild-type mice. This neuronal cell body expression of tau was limited to the 3R isoform, whereas expression of 4R tau was more “synaptic like”, with granular staining of neuropil rather than in neuronal cell bodies. These developmental and species-specific differences in the regulation and distribution of tau isoforms may be important to the understanding of normal and pathologic tau isoform expression. PMID:18925637

  12. A novel tropomyosin isoform functions at the mitotic spindle and Golgi in Drosophila

    PubMed Central

    Goins, Lauren M.; Mullins, R. Dyche

    2015-01-01

    Most eukaryotic cells express multiple isoforms of the actin-binding protein tropomyosin that help construct a variety of cytoskeletal networks. Only one nonmuscle tropomyosin (Tm1A) has previously been described in Drosophila, but developmental defects caused by insertion of P-elements near tropomyosin genes imply the existence of additional, nonmuscle isoforms. Using biochemical and molecular genetic approaches, we identified three tropomyosins expressed in Drosophila S2 cells: Tm1A, Tm1J, and Tm2A. The Tm1A isoform localizes to the cell cortex, lamellar actin networks, and the cleavage furrow of dividing cells—always together with myosin-II. Isoforms Tm1J and Tm2A colocalize around the Golgi apparatus with the formin-family protein Diaphanous, and loss of either isoform perturbs cell cycle progression. During mitosis, Tm1J localizes to the mitotic spindle, where it promotes chromosome segregation. Using chimeras, we identified the determinants of tropomyosin localization near the C-terminus. This work 1) identifies and characterizes previously unknown nonmuscle tropomyosins in Drosophila, 2) reveals a function for tropomyosin in the mitotic spindle, and 3) uncovers sequence elements that specify isoform-specific localizations and functions of tropomyosin. PMID:25971803

  13. Role of acyl carrier protein isoforms in plant lipid metabolism: Progress report

    SciTech Connect

    Ohlrogge, J.B.

    1989-01-01

    Previous research from my lab has revealed that several higher plant species have multiple isoforms of acyl carrier protein (ACP) and therefore this trait appears highly conserved among higher plants. This level of conservation suggests that the existence of ACP isoforms is not merely the results of neutral gene duplications. We have developed techniques to examine a wider range of species. Acyl carrier proteins can be labelled very specifically and to high specific activity using H-palmitate and the E. coli enzyme acyl-ACP synthetase. Isoforms were then resolved by western blotting and native PAGE of H-palmitate labelled ACP's. Multiple isoforms of ACP were observed the leaf tissue of the monocots Avena sativa and Hordeum vulgare and dicots including Arabidopsis thallina, Cuphea wrightii, and Brassica napus. Lower vascular plants including the cycad, Dioon edule, Ginkgo biloba, the gymnosperm Pinus, the fern Anernia phyllitidis and Psilotum nudum, the most primitive known extant vascular plant, were also found to have multiple ACP isoforms as were the nonvascular liverwort, Marchantia and moss, Polytrichum. Therefore, the development of ACP isoforms occurred early in evolution. However, the uniellular alge Chlamydomonas and Dunaliella and the photosynthetic cyanobacteria Synechocystis and Agmnellum have only a single elecrophotetic form of ACP. Thus, multiple forms of ACP do not occur in all photosynthetic organisms but may be associated with multicellular plants.

  14. FTICR-MS analysis of 14-3-3 isoform substrate selection.

    PubMed

    Cardasis, Helene L; Sehnke, Paul C; Laughner, Beth; Eyler, John R; Powell, David H; Ferl, Robert J

    2007-07-01

    The 14-3-3s are a ubiquitous class of eukaryotic proteins that participate in a second regulatory step in many phosphorylation-based signal transduction systems. The Arabidopsis family of 14-3-3 proteins represents a rather large 14-3-3 gene family. The biological motive for such diversity within a single protein family is not yet completely understood. The work presented here utilizes 14-3-3 micro-affinity chromatography in conjunction with Fourier transform ion cyclotron resonance mass spectrometry to survey the substrate sequence selectivity of two Arabidopsis 14-3-3 isoforms that represent the two major subclasses of this protein family. A method was developed to compare the relative binding of eight synthetic phosphopeptide sequences. The degree to which each phosphopeptide bound to either isoform was assigned a relative value, defined here as the binding ratio. The method provided a simple means for visualizing differences in substrate sequence selection among different 14-3-3 isoforms. A reproducible preference for specific phosphopeptide sequences was measured for both isoforms. This binding preference was consistent among the two classes of isoforms, suggesting that any pressure for isoform selectivity must reside outside the central core that interacts with the phosphopeptide sequence of the client. PMID:17569603

  15. Regulated Expression of a Calmodulin Isoform Alters Growth and Development in Potato

    NASA Technical Reports Server (NTRS)

    Poovaiah, B. W.; Takezawa, D.; An, G.; Han, T.-J.

    1996-01-01

    A transgene approach was taken to study the consequences of altered expression of a calmodutin iso-form on plant growth and development. Eight genomic clones of potato calmodulin (PCM 1 to 8) have been isolated and characterized. Among the potato calmodulin isoforms studied, PCM 1 differs from the other isoforms because of its unique amino acid substitutions. Transgenic potato plants were produced carrying sense construct of PCM 1 fused to the CAMV 35S promoter. Transgenic plants showing a moderate increase in PCM 1 MRNA exhibited strong apical dominance, produced elongated tubers, and were taller than the controls. Interestingly, the plants expressing the highest level of PCM 1 MRNA did not form underground tubers. Instead, these transgenic plants produced aerial tubers when allowed to grow for longer periods. The expression of different calmodulin isoforms (PCM 1, 5, 6, and 8) was studied in transgenic plants. Among the four potato calmodulin isoforms, only the expression of PCM 1 MRNA was altered in transgenic plants, while the expression of other isoforms was not significantly altered. Western analysis revealed increased PCM 1 protein in transgenic plants, indicating that the expression of both MRNA and protein are altered in transgenic plants. These results suggest that increasing the expression of PCM 1 alters growth and development in potato plants.

  16. PECAM-1 isoform-specific functions in PECAM-1-deficient brain microvascular endothelial cells.

    PubMed

    DiMaio, Terri A; Sheibani, Nader

    2008-03-01

    Platelet endothelial cell adhesion molecule-1 (PECAM-1) is alternatively spliced generating eight isoforms that only differ in the length of their cytoplasmic domain. Multiple isoforms of PECAM-1 are present in the endothelium and their expression levels are regulated during vascular development and angiogenesis. However, the functional significance of PECAM-1 isoforms during these processes remains largely unknown. We recently showed that mouse brain endothelial (bEND) cells prepared from PECAM-1-deficient (PECAM-1-/-) mice differ in their cell adhesive and migratory properties compared to PECAM-1+/+ bEND cells. Here we demonstrate that the restoration of PECAM-1 expression in these cells affects their adhesive and migratory properties in an isoform-specific manner. Expression of Delta14&15 PECAM-1, the predominant isoform present in the mouse endothelium, in PECAM-1-/- bEND cells activated MAPK/ERKs, disrupted adherens junctions, and enhanced cell migration and capillary morphogenesis in Matrigel. In contrast, expression of Delta15 PECAM-1 in PECAM-1-/- bEND cells had minimal effects on their activation of MAPK/ERKs, migration, and capillary morphogenesis. The effects of PECAM-1 on cell adhesive and migratory properties were mediated in an isoform-specific manner, at least in part, through its interactions with intracellular signaling proteins, including SHP-2 and Src. These results suggest that the impact of PECAM-1 on EC adhesion, migration, and capillary morphogenesis is modulated by alternative splicing of its cytoplasmic domain. PMID:18029285

  17. Characterization of 14-3-3 isoforms expressed in the Echinococcus granulosus pathogenic larval stage.

    PubMed

    Teichmann, Aline; Vargas, Daiani M; Monteiro, Karina M; Meneghetti, Bruna V; Dutra, Cristine S; Paredes, Rodolfo; Galanti, Norbel; Zaha, Arnaldo; Ferreira, Henrique B

    2015-04-01

    The 14-3-3 protein family of eukaryotic regulators was studied in Echinococcus granulosus, the causative agent of cystic hydatid disease. These proteins mediate important cellular processes in eukaryotes and are expected to play important roles in parasite biology. Six isoforms of E. granulosus 14-3-3 genes and proteins (Eg14-3-3.1-6) were analyzed, and their phylogenetic relationships were established with bona fide 14-3-3 orthologous proteins from eukaryotic species. Eg14-3-3 isoforms with previous evidence of expression (Eg14-3-3.1-4) in E. granulosus pathogenic larval stage (metacestode) were cloned, and recombinant proteins were used for functional studies. These protein isoforms were detected in different components of E. granulosus metacestode, including interface components with the host. The roles that are played by Eg14-3-3 proteins in parasite biology were inferred from the repertoires of interacting proteins with each isoform, as assessed by gel overlay, cross-linking, and affinity chromatography assays. A total of 95 Eg14-3-3 protein ligands were identified by mass spectrometry. Eg14-3-3 isoforms have shared partners (44 proteins), indicating some overlapping functions; however, they also bind exclusive partners (51 proteins), suggesting Eg14-3-3 functional specialization. These ligand repertoires indicate the involvement of Eg14-3-3 proteins in multiple biochemical pathways in the E. granulosus metacestode and note some degree of isoform specialization. PMID:25748451

  18. Inhibition of various isoforms of rat liver glutathione S-transferases by tannic acid and butein.

    PubMed

    Zhang, K; Mack, P; Wong, K P

    1997-07-01

    Glutathione S-transferases (EC.2.5.1.18, GSTs) were purified from rat liver by S-hexylglutathione affinity chromatography and six isoforms, namely C-1, C-2, C-3, C-4, A-2 and A-1, were isolated by CM-cellulose and DEAE-cellulose ion-exchange columns. Tannic acid and butein showed varying degrees of inhibition on the six individual GST isoforms. When 1-chloro-2,4-dinitrobenzene (CDNB) was used as a substrate, butein exerted significantly more potent inhibition on the cationic isoforms C-2, C-3 and C-4 with IC50 values of 6.8, 8.5 and 8.0 muM respectively. All the isoforms showed lower activity towards p-nitrobenzyt chloride when compared to CDNB and inhibition of the p-nitrobenzyl chloride-activity by tannic acid and butein was also weaker. The inhibitory effects of tannic acid and butein on each isoform decreased generally with increasing pH in the range of 6.0 to 8.0. The optimum pHs for inhibitions by tannic acid and butein on the six individual isoforms lie in the pH range of 6.0 to 6.5. PMID:19856286

  19. Profiling alternatively spliced mRNA isoforms for prostate cancer classification

    PubMed Central

    Zhang, Chaolin; Li, Hai-Ri; Fan, Jian-Bing; Wang-Rodriguez, Jessica; Downs, Tracy; Fu, Xiang-Dong; Zhang, Michael Q

    2006-01-01

    Background Prostate cancer is one of the leading causes of cancer illness and death among men in the United States and world wide. There is an urgent need to discover good biomarkers for early clinical diagnosis and treatment. Previously, we developed an exon-junction microarray-based assay and profiled 1532 mRNA splice isoforms from 364 potential prostate cancer related genes in 38 prostate tissues. Here, we investigate the advantage of using splice isoforms, which couple transcriptional and splicing regulation, for cancer classification. Results As many as 464 splice isoforms from more than 200 genes are differentially regulated in tumors at a false discovery rate (FDR) of 0.05. Remarkably, about 30% of genes have isoforms that are called significant but do not exhibit differential expression at the overall mRNA level. A support vector machine (SVM) classifier trained on 128 signature isoforms can correctly predict 92% of the cases, which outperforms the classifier using overall mRNA abundance by about 5%. It is also observed that the classification performance can be improved using multivariate variable selection methods, which take correlation among variables into account. Conclusion These results demonstrate that profiling of splice isoforms is able to provide unique and important information which cannot be detected by conventional microarrays. PMID:16608523

  20. Single-cell polyadenylation site mapping reveals 3′ isoform choice variability

    PubMed Central

    Velten, Lars; Anders, Simon; Pekowska, Aleksandra; Järvelin, Aino I; Huber, Wolfgang; Pelechano, Vicent; Steinmetz, Lars M

    2015-01-01

    Cell-to-cell variability in gene expression is important for many processes in biology, including embryonic development and stem cell homeostasis. While heterogeneity of gene expression levels has been extensively studied, less attention has been paid to mRNA polyadenylation isoform choice. 3′ untranslated regions regulate mRNA fate, and their choice is tightly controlled during development, but how 3′ isoform usage varies within genetically and developmentally homogeneous cell populations has not been explored. Here, we perform genome-wide quantification of polyadenylation site usage in single mouse embryonic and neural stem cells using a novel single-cell transcriptomic method, BATSeq. By applying BATBayes, a statistical framework for analyzing single-cell isoform data, we find that while the developmental state of the cell globally determines isoform usage, single cells from the same state differ in the choice of isoforms. Notably this variation exceeds random selection with equal preference in all cells, a finding that was confirmed by RNA FISH data. Variability in 3′ isoform choice has potential implications on functional cell-to-cell heterogeneity as well as utility in resolving cell populations. PMID:26040288

  1. C/EBPβ Isoforms Expression in the Rat Brain during the Estrous Cycle

    PubMed Central

    Hansberg-Pastor, Valeria; Piña-Medina, Ana Gabriela; González-Arenas, Aliesha; Camacho-Arroyo, Ignacio

    2015-01-01

    The CCAAT/enhancer-binding protein beta (C/EBPβ) is a transcription factor expressed in different areas of the brain that regulates the expression of several genes involved in cell differentiation and proliferation. This protein has three isoforms (LAP1, LAP2, and LIP) with different transcription activation potential. The role of female sex hormones in the expression pattern of C/EBPβ isoforms in the rat brain has not yet been described. In this study we demonstrate by western blot that the expression of the three C/EBPβ isoforms changes in different brain areas during the estrous cycle. In the cerebellum, LAP2 content diminished on diestrus and proestrus and LIP content diminished on proestrus and estrus days. In the prefrontal cortex, LIP content was higher on proestrus and estrus days. In the hippocampus, LAP isoforms presented a switch on diestrus day, since LAP1 content was the highest while that of LAP2 was the lowest. The LAP2 isoform was the most abundant one in all the three brain areas. The LAP/LIP ratio changed throughout the cycle and was tissue specific. These results suggest that C/EBPβ isoforms expression changes in a tissue-specific manner in the rat brain due to the changes in sex steroid hormone levels presented during the estrous cycle. PMID:26064112

  2. Myosin II isoform co-assembly and differential regulation in mammalian systems.

    PubMed

    Beach, Jordan R; Hammer, John A

    2015-05-15

    Non-muscle myosin 2 (NM2) is a major force-producing, actin-based motor in mammalian non-muscle cells, where it plays important roles in a broad range of fundamental biological processes, including cytokinesis, cell migration, and epithelial barrier function. This breadth of function at the tissue and cellular levels suggests extensive diversity and differential regulation of NM2 bipolar filaments, the major, if not sole, functional form of NM2s in vivo. Previous in vitro, cellular and animal studies indicate that some of this diversity is supported by the existence of multiple NM2 isoforms. Moreover, two recent studies have shown that these isoforms can co-assemble to form heterotypic filaments, further expanding functional diversity. In addition to isoform co-assembly, cells may differentially regulate NM2 function via isoform-specific expression, RLC phosphorylation, MHC phosphorylation or regulation via binding partners. Here, we provide a brief summary of NM2 filament assembly, summarize the recent findings regarding NM2 isoform co-assembly, consider the mechanisms cells might utilize to differentially regulate NM2 isoforms, and review the data available to support these mechanisms. PMID:25655283

  3. Palmitoylation of the three isoforms of human endothelin-converting enzyme-1.

    PubMed Central

    Schweizer, A; Löffler, B M; Rohrer, J

    1999-01-01

    Endothelin-converting enzyme-1 (ECE-1) is a membrane-bound metalloprotease that catalyses the conversion of inactive big endothelins into active endothelins. Here we have examined whether the three isoforms of human ECE-1 (ECE-1a, ECE-1b and ECE-1c) are modified by the covalent attachment of the fatty acid palmitate and have evaluated a potential functional role of this modification. To do this, wild-type and mutant enzymes were expressed and analysed by metabolic labelling with [3H]palmitate, immunoprecipitation and SDS/PAGE. All three ECE-1 isoforms were found to be palmitoylated via hydroxylamine-sensitive thioester bonds. In addition, the isoforms showed similar levels of acylation. Cys46 in ECE-1a, Cys58 in ECE-1b and Cys42 in ECE-1c were identified as sites of palmitoylation and each of these cysteines accounted for all the palmitoylation that occured in the corresponding isoform. Immunofluorescence analysis demonstrated further that palmitoylated and non-palmitoylated ECE-1 isoforms had the same subcellular localizations. Moreover, complete solubility of the three isoforms in Triton X-100 revealed that palmitoylation does not target ECE-1 to cholesterol and sphingolipid-rich membrane domains or caveolae. The enzymic activities of ECE-1a, ECE-1b and ECE-1c were also not significantly affected by the absence of palmitoylation. PMID:10359648

  4. Distribution of tropomyosin isoforms in different types of single fibers isolated from bovine skeletal muscles.

    PubMed

    Oe, M; Ojima, K; Nakajima, I; Chikuni, K; Shibata, M; Muroya, S

    2016-08-01

    To clarify the relationship between myosin heavy chain (MyHC) isoforms and tropomyosin (TPM) isoforms in single fibers, 64 single fibers were isolated from each of bovine three muscles (masseter, semispinalis and semitendinosus). mRNA expressions of MyHC and TPM isoforms were analyzed by real-time PCR. All single fibers from the masseter expressed MyHC-slow. The fibers from the semispinalis expressed both MyHC-slow and 2a. The fibers from the semitendinosus expressed MyHC-slow, 2a and 2x. TPM-1 and TPM-2 were co-expressed in 2a and 2x type fibers, and TPM-2 and TPM-3 were co-expressed in slow type fibers. The expression pattern of TPM isoforms in each fiber type was similar between fibers isolated from different muscles. These results suggest that TPM-1 and TPM-3 isoforms correspond to the function of 2a or 2x type fibers and slow type fibers, respectively, with TPM-2 in common. Furthermore, the patterns of MyHC and TPM isoform combinations did not vary among single fibers isolated from the individual muscles examined. PMID:27105153

  5. N-Domain Isoform of Angiotensin I Converting Enzyme as a Marker of Hypertension: Populational Study

    PubMed Central

    Maluf-Meiken, Leila C. V.; Fernandes, Fernanda B.; Aragão, Danielle S.; Ronchi, Fernanda A.; Andrade, Maria C. C.; Franco, Maria C.; Febba, Andreia C. S.; Plavnik, Frida L.; Krieger, José E.; Mill, Jose G.; Sesso, Ricardo C. C.; Casarini, Dulce E.

    2012-01-01

    The aim of this paper was to investigate the presence of the urinary 90 kDa N-domain ACE in a cohort of the population from Vitoria, Brazil, to verify its association with essential hypertension since this isoform could be a possible genetic marker of hypertension. Anthropometric, clinical, and laboratory parameters of the individuals were evaluated (n = 1150) and the blood pressure (BP) was measured. The study population was divided according to ACE isoforms in urine as follows: ACE 65/90/190, presence of three ACE isoforms (n = 795), ACE 90+ (65/90) (n = 186), and ACE 90− (65/190) (n = 169) based on the presence (+) or absence (−) of the 90 kDa ACE isoform. The anthropometric parameters, lipid profile, serum levels of uric acid, glucose, and the systolic and diastolic BP were significantly greater in the ACE 90+ compared with the ACE 90− and ACE 65/90/190 individuals. We found that 98% of individuals from the ACE 90+ group and 38% from the ACE 65/90/190 group had hypertension, compared to only 1% hypertensive individuals in the ACE 90− group. There is a high presence of the 90 kDa N-domain ACE isoform (85%) in the studied population. The percentile of normotensive subjects with three isoforms was 62%. Our findings could contribute to the development of new efficient strategy to prevent and treat hypertension to avoid the development of cardiovascular disease. PMID:22666552

  6. Molecular characterization of human thyroid hormone receptor β isoform 4.

    PubMed

    Moriyama, Kenji; Yamamoto, Hiroyuki; Futawaka, Kumi; Atake, Asami; Kasahara, Masato; Tagami, Tetsuya

    2016-01-01

    Thyroid hormone exerts a pleiotropic effect on development, differentiation, and metabolism through thyroid hormone receptor (TR). A novel thyroid hormone receptor β isoform (TRβ4) was cloned using PCR from a human pituitary cDNA library as a template. We report here the characterization of TRβ4 from a molecular basis. Temporal expression of TRβ4 during the fetal period is abundant in the brain and kidney, comparable with the adult pattern. Western blot analysis revealed that TRs are ubiquitination labile proteins, while TRβ1 is potentially stable. TRβ1, peroxisome proliferator-activated receptors (PPAR), and vitamin D receptor (VDR), which belong to class II transcription factors that function via the formation of heterodimeric complexes with retinoid X receptor (RXR), were suppressed by TRβ4 in a dose-dependent manner. Thus, TRβ4 exhibits ligand-independent transcriptional silencing, possibly as a substitute for dimerized RXR. In this study, TRβ1 and TRβ4 transcripts were detected in several cell lines. Quantitative RT-PCR assay showed that the expression of TRβ4 in human embryonic carcinoma cells of the testis was suppressed by sex hormone in a reciprocal manner to TRβ1. In contrast, TRβ4 was expressed under a high dose of triiodothyronine (T3) in a reciprocal manner to TRβ1. Finally, in transiently transfected NIH-3T3 cells, green fluorescence protein (GFP)-tagged TRβ4 was mostly nuclear in both the absence and the presence of T3. By mutating defined regions of both TRβs, we found that both TRβ1 and TRβ4 had altered nuclear/cytoplasmic distribution as compared with wild-type, and different to T3 and the nuclear receptor corepressor (NCoR). Thus, site-specific DNA binding is not essential for maintaining TRβs within the nucleus. PMID:26513165

  7. Orthologous myosin isoforms and scaling of shortening velocity with body size in mouse, rat, rabbit and human muscles

    PubMed Central

    Pellegrino, M A; Canepari, M; Rossi, R; D'Antona, G; Reggiani, C; Bottinelli, R

    2003-01-01

    Maximum shortening velocity (V0) was determined in single fibres dissected from hind limb skeletal muscles of rabbit and mouse and classified according to their myosin heavy chain (MHC) isoform composition. The values for rabbit and mouse V0 were compared with the values previously obtained in man and rat under identical experimental conditions. Significant differences in V0 were found between fibres containing corresponding myosin isoforms in different species: as a general rule for each isoform V0 decreased with body mass. Myosin isoform distributions of soleus and tibialis anterior were analysed in mouse, rat, rabbit and man: the proportion of slow myosin generally increased with increasing body size. The diversity between V0 of corresponding myosin isoforms and the different myosin isoform composition of corresponding muscles determine the scaling of shortening velocity of whole muscles with body size, which is essential for optimisation of locomotion. The speed of actin translocation (Vf) in in vitro motility assay was determined with myosins extracted from single muscle fibres of all four species: significant differences were found between myosin isoforms in each species and between corresponding myosin isoforms in different species. The values of V0 and Vf determined for each myosin isoform were significantly correlated, strongly supporting the view that the myosin isoform expressed is the major determinant of maximum shortening velocity in muscle fibres. PMID:12562996

  8. Overexpression of CD45RA isoforms in carriers of the C77G mutation leads to hyporeactivity of CD4+CD25highFoxp3+ regulatory T cells.

    PubMed

    Pokoyski, C; Lienen, T; Rother, S; Schock, E; Plege-Fleck, A; Geffers, R; Schwinzer, R

    2015-12-01

    Disorders in regulatory T-cell (T(reg)) function can result in the breakdown of immunological self-tolerance. Thus, the identification of mechanisms controlling the activity of T(reg) is of great relevance. We used T(reg) from individuals carrying the C77G polymorphism as models to study the role of CD45 molecules in humans. C77G prevents splicing of CD45 exon A thereby leading to an aberrant expression pattern of CD45 isoforms in affected individuals. Resting and in vitro expanded/activated CD4(+)CD25(high)Foxp3(+) T(reg) from carriers of C77G strongly expressed CD45RA isoforms whereas these isoforms were almost absent in cells from individuals with wild-type CD45. C77G T(reg) showed diminished upregulation of activation markers, lower phosphorylation of p56(lck)(Y505) and a reduced proliferative potential when stimulated with anti-TcR or anti-TcR plus CD28 mAb suggesting decreased responsiveness to activating stimuli. In addition, the capacity to suppress proliferation of conventional CD4(+) T cells was impaired in C77G T(reg). Furthermore, microarray studies revealed distinct gene expression patterns in T(reg) from C77G carriers. These data suggest that the changes in CD45 isoform combination resulting from the C77G mutation alter the responsiveness of T(reg) to TcR-mediated signaling. Targeting CD45 isoform expression might be a useful approach to modulate T(reg) function. PMID:26355564

  9. Altered α-synuclein, parkin, and synphilin isoform levels in multiple system atrophy brains.

    PubMed

    Brudek, Tomasz; Winge, Kristian; Rasmussen, Nadja Bredo; Bahl, Justyna Maria Czarna; Tanassi, Julia; Agander, Tina Klitmøller; Hyde, Thomas M; Pakkenberg, Bente

    2016-01-01

    Together with Parkinson's disease (PD) and dementia with Lewy bodies, multiple system atrophy (MSA) is a member of a diverse group of neurodegenerative disorders termed α-synucleinopathies. Previously, it has been shown that α-synuclein, parkin, and synphilin-1 display disease-specific transcription patterns in frontal cortex in PD, dementia with Lewy bodies, and MSA, and thus may mediate the development of α-synucleinopathies. In this study, the differential expression of α-synuclein isoforms on transcriptional and translational levels was ascertained in MSA patients in comparison with PD cases and normal controls using isoform-specific primers and exon-specific antibodies in substantia nigra, striatum, cerebellar cortex, and nucleus dentatus. These regions are severely affected by α-synuclein pathology and neurodegeneration. Furthermore, we have also investigated transcript levels for parkin and synphilin-1 isoforms. In MSA brains, α-synuclein140 and α-synuclein 112 isoform levels were significantly increased, whereas levels of the α-synuclein 126 isoform were decreased in the substantia nigra, striatum, cerebellar cortex, and nucleus dentatus versus controls. Moreover, in MSA cases, we showed increased levels of parkin isoforms lacking the N-terminal ubiquitin-like domain and an aggregation-prone synphilin-1A isoform that causes neuronal toxicity in MSA. In PD brains, parkin transcript variant 3, 7, and 11 were significantly and specifically over-expressed in the striatum and cerebellar cortex, together with synphilin-1A and 1C. The changes of isoform expression profiles in neurodegenerative diseases suggest alterations in the regulation of transcription and/or splicing events, leading to regional/cellular events that may be important for the highly increased aggregation of α-synuclein in the brain. We report differential expression of α-synuclein, parkin, and synphilin-1 isoforms in multiple system atrophy (MSA) versus Parkinson's disease and normal

  10. Computational approaches for isoform detection and estimation: good and bad news

    PubMed Central

    2014-01-01

    Background The main goal of the whole transcriptome analysis is to correctly identify all expressed transcripts within a specific cell/tissue - at a particular stage and condition - to determine their structures and to measure their abundances. RNA-seq data promise to allow identification and quantification of transcriptome at unprecedented level of resolution, accuracy and low cost. Several computational methods have been proposed to achieve such purposes. However, it is still not clear which promises are already met and which challenges are still open and require further methodological developments. Results We carried out a simulation study to assess the performance of 5 widely used tools, such as: CEM, Cufflinks, iReckon, RSEM, and SLIDE. All of them have been used with default parameters. In particular, we considered the effect of the following three different scenarios: the availability of complete annotation, incomplete annotation, and no annotation at all. Moreover, comparisons were carried out using the methods in three different modes of action. In the first mode, the methods were forced to only deal with those isoforms that are present in the annotation; in the second mode, they were allowed to detect novel isoforms using the annotation as guide; in the third mode, they were operating in fully data driven way (although with the support of the alignment on the reference genome). In the latter modality, precision and recall are quite poor. On the contrary, results are better with the support of the annotation, even though it is not complete. Finally, abundance estimation error often shows a very skewed distribution. The performance strongly depends on the true real abundance of the isoforms. Lowly (and sometimes also moderately) expressed isoforms are poorly detected and estimated. In particular, lowly expressed isoforms are identified mainly if they are provided in the original annotation as potential isoforms. Conclusions Both detection and quantification

  11. Modeled microgravity-induced protein kinase C isoform expression in human lymphocytes

    NASA Technical Reports Server (NTRS)

    Sundaresan, A.; Risin, D.; Pellis, N. R.

    2004-01-01

    In long-term space travel, the crew is exposed to microgravity and radiation that invoke potential hazards to the immune system. T cell activation is a critical step in the immune response. Receptor-mediated signaling is inhibited in both microgravity and modeled microgravity (MMG) as reflected by diminished DNA synthesis in peripheral blood lymphocytes and their locomotion through gelled type I collagen. Direct activation of protein kinase C (PKC) bypassing cell surface events using the phorbol ester PMA rescues MMG-inhibited lymphocyte activation and locomotion, whereas the calcium ionophore ionomycin had no rescue effect. Thus calcium-independent PKC isoforms may be affected in MMG-induced locomotion inhibition and rescue. Both calcium-dependent isoforms and calcium-independent PKC isoforms were investigated to assess their expression in lymphocytes in 1 g and MMG culture. Human lymphocytes were cultured and harvested at 24, 48, 72, and 96 h, and serial samples were assessed for locomotion by using type I collagen and expression of PKC isoforms. Expression of PKC-alpha, -delta, and -epsilon was assessed by RT-PCR, flow cytometry, and immunoblotting. Results indicated that PKC isoforms delta and epsilon were downregulated by >50% at the transcriptional and translational levels in MMG-cultured lymphocytes compared with 1-g controls. Events upstream of PKC, such as phosphorylation of phospholipase Cgamma in MMG, revealed accumulation of inactive enzyme. Depressed calcium-independent PKC isoforms may be a consequence of an upstream lesion in the signal transduction pathway. The differential response among calcium-dependent and calcium-independent isoforms may actually result from MMG intrusion events earlier than PKC, but after ligand-receptor interaction.

  12. Drosophila TRPA1 isoforms detect UV light via photochemical production of H2O2

    PubMed Central

    Guntur, Ananya R.; Gu, Pengyu; Takle, Kendra; Chen, Jingyi; Xiang, Yang; Yang, Chung-Hui

    2015-01-01

    The transient receptor potential A1 (TRPA1) channel is an evolutionarily conserved detector of temperature and irritant chemicals. Here, we show that two specific isoforms of TRPA1 in Drosophila are H2O2 sensitive and that they can detect strong UV light via sensing light-induced production of H2O2. We found that ectopic expression of these H2O2-sensitive Drosophila TRPA1 (dTRPA1) isoforms conferred UV sensitivity to light-insensitive HEK293 cells and Drosophila neurons, whereas expressing the H2O2-insensitive isoform did not. Curiously, when expressed in one specific group of motor neurons in adult flies, the H2O2-sensitive dTRPA1 isoforms were as competent as the blue light-gated channelrhodopsin-2 in triggering motor output in response to light. We found that the corpus cardiacum (CC) cells, a group of neuroendocrine cells that produce the adipokinetic hormone (AKH) in the larval ring gland endogenously express these H2O2-sensitive dTRPA1 isoforms and that they are UV sensitive. Sensitivity of CC cells required dTRPA1 and H2O2 production but not conventional phototransduction molecules. Our results suggest that specific isoforms of dTRPA1 can sense UV light via photochemical production of H2O2. We speculate that UV sensitivity conferred by these isoforms in CC cells may allow young larvae to activate stress response—a function of CC cells—when they encounter strong UV, an aversive stimulus for young larvae. PMID:26443856

  13. NADPH-diaphorase activity and nitric oxide synthase isoforms in the trophoblast of Calomys callosus

    PubMed Central

    MORAES, NECI; ZAGO, DOUGLAS; GAGIOTI, SONIA; HOSHIDA, MARA SANDRA; BEVILACQUA, ESTELA

    2001-01-01

    The pattern of expression of a variety of placental nitric oxide synthase isoforms has contributed to elucidating the regulatory mechanisms of nitric oxide (NO) synthesis during gestation. The maintenance of vascular tone, attenuation of vasoconstriction, prevention of platelet and leukocyte adhesion to the trophoblast surface, and possible participation in uterine blood flow seem to be the main functions of NO generated at the fetal-maternal interface in humans and mice. Extending this knowledge to other rodent species commonly used as laboratory animals, in this study we focus on NADPH-diaphorase activity and the distribution of nitric oxide synthase isoforms (NOS) in the trophoblast cells of Calomys callosus during different phases of pregnancy. NADPH-diaphorase activity was evaluated cytochemically and the presence of NOS isoforms detected by immunohistochemistry. These techniques were performed on pre- and postimplantation embryos in situ and in vitro, as well as in placentae on d 14 and 18 of pregnancy. Neither NADPH-diaphorase activity nor inducible or endothelial NOS isoforms were found in pre-implanting embryos except after culturing for at least 48 h, when some of the embryonic cells were positive for the diaphorase reaction. On d 6·5 of pregnancy, trophoblast cells showed intense diaphorase activity both in situ and under in vitro conditions. A positive reaction was also found in the different placental trophoblast cells on d 14 and 18 of pregnancy. The inducible NOS (iNOS) isoform, but not the endothelial isoform, was immunodetected in trophoblast cells from the placenta and from postimplantation embryos in situ and under in vitro conditions. These results strongly suggest the production of NO by the iNOS isoform in the trophoblast of Calomys callosus after embryo implantation. The data also emphasise a possible role for the trophoblast in producing and releasing cytotoxic molecules at the fetal-maternal interface. PMID:11327206

  14. Development and validation of MRM methods to quantify protein isoforms of polyphenol oxidase in loquat fruits.

    PubMed

    Martínez-Márquez, Ascensión; Morante-Carriel, Jaime; Sellés-Marchart, Susana; Martínez-Esteso, María José; Pineda-Lucas, José Luis; Luque, Ignacio; Bru-Martínez, Roque

    2013-12-01

    Multiple reaction monitoring (MRM) is emerging as a promising technique for the detection and quantification of protein biomarkers in complex biological samples. Compared to Western blotting or enzyme assays, its high sensitivity, specificity, accuracy, assay speed, and sample throughput represent a clear advantage for being the approach of choice for the analysis of proteins. MRM assays are capable of detecting and quantifying proteolytic peptides differing in mass unique to particular proteins, that is, proteotypic peptides, through which different protein isoforms can be distinguished. We have focused on polyphenol oxidase (PPO), a plant conspicuous enzyme encoded by a multigenic family in loquat (Eriobotrya japonica Lindl.) and other related species. PPO is responsible for both the protection of plants from biotic stress as a feeding deterrent for herbivore insects and the enzymatic browning of fruits and vegetables. The latter makes fruit more attractive to seed dispersal agents but is also a major cause of important economic losses in agriculture and food industry. An adequate management of PPO at plant breeding level would maximize the benefits and minimize the disadvantages of this enzyme, but it would require a precise knowledge of the biological role played by each isoform in the plant. Thus, for the functional study of the PPOs, we have cloned and overexpressed fragments of three PPO isoforms from loquat to develop MRM-based methods for the quantification of each isoform. The method was developed using an ion trap instrument and validated in a QQQ instrument. It resulted in the selection of at least two peptides for each isoform that can be monitored by at least three transitions. A combination of SDS-PAGE and MRM lead to detect two out of three monitored isoforms in different gel bands corresponding to different processing stages of PPO. The method was applied to determine the amount of the PPO2 isoform in protein extracts from fruit samples using

  15. Modeled Microgravity-Induced Protein Kinase C Isoform Expression in Human Lymphocytes

    NASA Technical Reports Server (NTRS)

    Sundaresan, A.; Risin, D.; Pellis, N. R.

    2003-01-01

    In long-term space travel, the crew is exposed to microgravity and radiation that invoke potential hazards to the immune system. T cell activation is a critical step in the immune response. Receptor-mediated signaling is inhibited both in microgravity and modeled microgravity (MMG) as reflected in diminished DNA synthess in peripheral blood lymphocytes and their locomotion through gelled type 1 collagen. Direct activation of Protein Kinase C (PKC) bypassing cell surface events using the phorbol ester PMA rescues MMG-inhibited lymphocyte activation and locomotion, whereas calcium ionophore ionomycin had no rescue effect. Thus calcium-independent PKC isoforms may be affected in MMG-induced locomotion inhibition and rescue. Both calcium-dependent isoforms and calcium-independent PKC isoforms were investigated to assess their expression in lymphocytes in 19 and MMG-culture. Human lymphocytes were cultured and harvested at 24, 48, 72 and 96 hours and serial samples assessed for locomotion using type I collagen and expression of PKC isoforms. Expression of PKC-alpha, -delta and -epsilon was assessed by RT-PCR, flow cytometry and immunoblotting. Results indicated that PKC isoforms delta and epsilon were down-regulated by more than 50% at the transcriptional and translational levels in MMG-cultured lymphocytes compared with 19 controls. Events upstream of PKC such as phosphorylation of Phospholipase C(gamma) (PLC-gamma) in MMG, revealed accumulation of inactive enzyme. Depressed Ca++ -independent PKC isoforms may be a consequence of an upstream lesion in the signal transduction pathway. The differential response among calcium-dependent and calcium-independent isoforms may actually result from MMG intrusion events earlier than, but after ligand-receptor interaction. Keywords: Signal transduction, locomotion, immunity

  16. Characterization of a saporin isoform with lower ribosome-inhibiting activity.

    PubMed Central

    Fabbrini, M S; Rappocciolo, E; Carpani, D; Solinas, M; Valsasina, B; Breme, U; Cavallaro, U; Nykjaer, A; Rovida, E; Legname, G; Soria, M R

    1997-01-01

    We have expressed in Escherichia coli five isoforms of saporin, a single-chain ribosome-inactivating protein (RIP). Translation inhibition activities of the purified recombinant polypeptides in vitro were compared with those of recombinant dianthin 30, a less potent and closely related RIP, and of ricin A chain. Dianthin 30, and a saporin isoform encoded by a cDNA from leaf tissue (SAP-C), both had about one order of magnitude lower activity in translation inhibition assays than all other isoforms of saporin tested. We recently demonstrated that saporin extracted from seeds of Saponaria officinalis binds to alpha2-macroglobulin receptor (alpha2MR; also termed low density lipoprotein-receptor-related-protein), indicating a general mechanism of interaction of plant RIPs with the alpha2MR system [Cavallaro, Nykjaer, Nielsen and Soria (1995) Eur. J. Biochem. 232, 165-171]. Here we report that SAP-C bound to alpha2MR equally well as native saporin. However, the same isoform had about ten times lower cytotoxicity than the other saporin isoforms towards different cell lines. This indicates that the lower cell-killing ability of the SAP-C isoform is presumably due to its altered interaction with the protein synthesis machinery of target cells. Since saporin binding to the alpha2MR is competed by heparin, we also tested in cell-killing experiments Chinese hamster ovary cell lines defective for expression of either heparan sulphates or proteoglycans. No differences were observed in cytotoxicity using native saporin or the recombinant isoforms. Therefore saporin binding to the cell surface should not be mediated by interaction with proteoglycans, as is the case for other alpha2MR ligands. PMID:9148741

  17. PI3K isoform-selective inhibitors: next-generation targeted cancer therapies

    PubMed Central

    Wang, Xiang; Ding, Jian; Meng, Ling-hua

    2015-01-01

    The pivotal roles of phosphatidylinositol 3-kinases (PI3Ks) in human cancers have inspired active development of small molecules to inhibit these lipid kinases. However, the first-generation pan-PI3K and dual-PI3K/mTOR inhibitors have encountered problems in clinical trials, with limited efficacies as a monotherapeutic agent as well as a relatively high rate of side effects. It is increasingly recognized that different PI3K isoforms play non-redundant roles in particular tumor types, which has prompted the development of isoform-selective inhibitors for pre-selected patients with the aim for improving efficacy while decreasing undesirable side effects. The success of PI3K isoform-selective inhibitors is represented by CAL101 (Idelalisib), a first-in-class PI3Kδ-selective small-molecule inhibitor that has been approved by the FDA for the treatment of chronic lymphocytic leukemia, indolent B-cell non-Hodgkin's lymphoma and relapsed small lymphocytic lymphoma. Inhibitors targeting other PI3K isoforms are also being extensively developed. This review focuses on the recent progress in development of PI3K isoform-selective inhibitors for cancer therapy. A deeper understanding of the action modes of novel PI3K isoform-selective inhibitors will provide valuable information to further validate the concept of targeting specific PI3K isoforms, while the identification of biomarkers to stratify patients who are likely to benefit from the therapy will be essential for the success of these agents. PMID:26364801

  18. PI3K isoform-selective inhibitors: next-generation targeted cancer therapies.

    PubMed

    Wang, Xiang; Ding, Jian; Meng, Ling-hua

    2015-10-01

    The pivotal roles of phosphatidylinositol 3-kinases (PI3Ks) in human cancers have inspired active development of small molecules to inhibit these lipid kinases. However, the first-generation pan-PI3K and dual-PI3K/mTOR inhibitors have encountered problems in clinical trials, with limited efficacies as a monotherapeutic agent as well as a relatively high rate of side effects. It is increasingly recognized that different PI3K isoforms play non-redundant roles in particular tumor types, which has prompted the development of isoform-selective inhibitors for pre-selected patients with the aim for improving efficacy while decreasing undesirable side effects. The success of PI3K isoform-selective inhibitors is represented by CAL101 (Idelalisib), a first-in-class PI3Kδ-selective small-molecule inhibitor that has been approved by the FDA for the treatment of chronic lymphocytic leukemia, indolent B-cell non-Hodgkin's lymphoma and relapsed small lymphocytic lymphoma. Inhibitors targeting other PI3K isoforms are also being extensively developed. This review focuses on the recent progress in development of PI3K isoform-selective inhibitors for cancer therapy. A deeper understanding of the action modes of novel PI3K isoform-selective inhibitors will provide valuable information to further validate the concept of targeting specific PI3K isoforms, while the identification of biomarkers to stratify patients who are likely to benefit from the therapy will be essential for the success of these agents. PMID:26364801

  19. Development and characterization of human monoclonal antibodies that neutralize multiple TGFβ isoforms

    PubMed Central

    Bedinger, Daniel; Lao, Llewelyn; Khan, Shireen; Lee, Steve; Takeuchi, Toshihiko; Mirza, Amer M.

    2016-01-01

    ABSTRACT Transforming growth factor (TGF)β levels are elevated in, and drive the progression of, numerous disease states such as advanced metastatic cancer and systemic and ocular fibrosis. There are 3 main isoforms, TGFβ1, 2, and 3. As multiple TGFβ isoforms are involved in disease processes, maximal therapeutic efficacy may require neutralization of 2 or more of the TGFβ isoforms. Fully human antibody phage display libraries were used to discover a number of antibodies that bind and neutralize various combinations of TGFβ1, 2 or 3. The primary panning did not yield any uniformly potent pan-isoform neutralizing antibodies; therefore, an antibody that displayed potent TGFβ 1, 2 inhibition, but more modest affinity versus TGFβ3, was affinity matured by shuffling with a light chain sub-library and further screening. This process yielded a high affinity pan-isoform neutralizing clone. Antibodies were analyzed and compared by binding affinity, as well as receptor and epitope competition by surface plasmon resonance methods. The antibodies were also shown to neutralize TGFβ effects in vitro in 3 assays: 1) interleukin (IL)-4 induced HT-2 cell proliferation; 2) TGFβ-mediated IL-11 release by A549 cells; and 3) decreasing SMAD2 phosphorylation in Detroit 562 cells. The antibodies’ potency in these in vitro assays correlated well with their isoform-specific affinities. Furthermore, the ability of the affinity-matured clone to decrease tumor burden in a Detroit 562 xenograft study was superior to that of the parent clone. This affinity-matured antibody acts as a very potent inhibitor of all 3 main isoforms of TGFβ and may have utility for therapeutic intervention in human disease. PMID:26563652

  20. Identification of a Novel Human LAP1 Isoform That Is Regulated by Protein Phosphorylation

    PubMed Central

    Santos, Mariana; Domingues, Sara C.; Costa, Patrícia; Muller, Thorsten; Galozzi, Sara; Marcus, Katrin; da Cruz e Silva, Edgar F.; da Cruz e Silva, Odete A.; Rebelo, Sandra

    2014-01-01

    Lamina associated polypeptide 1 (LAP1) is an integral protein of the inner nuclear membrane that is ubiquitously expressed. LAP1 binds to lamins and chromatin, probably contributing to the maintenance of the nuclear envelope architecture. Moreover, LAP1 also interacts with torsinA and emerin, proteins involved in DYT1 dystonia and X-linked Emery-Dreifuss muscular dystrophy disorder, respectively. Given its relevance to human pathological conditions, it is important to better understand the functional diversity of LAP1 proteins. In rat, the LAP1 gene (TOR1AIP1) undergoes alternative splicing to originate three LAP1 isoforms (LAP1A, B and C). However, it remains unclear if the same occurs with the human TOR1AIP1 gene, since only the LAP1B isoform had thus far been identified in human cells. In silico analysis suggested that, across different species, potential new LAP1 isoforms could be generated by alternative splicing. Using shRNA to induce LAP1 knockdown and HPLC-mass spectrometry analysis the presence of two isoforms in human cells was described and validated: LAP1B and LAP1C; the latter is putatively N-terminal truncated. LAP1B and LAP1C expression profiles appear to be dependent on the specific tissues analyzed and in cultured cells LAP1C was the major isoform detected. Moreover, LAP1B and LAP1C expression increased during neuronal maturation, suggesting that LAP1 is relevant in this process. Both isoforms were found to be post-translationally modified by phosphorylation and methionine oxidation and two LAP1B/LAP1C residues were shown to be dephosphorylated by PP1. This study permitted the identification of the novel human LAP1C isoform and partially unraveled the molecular basis of LAP1 regulation. PMID:25461922

  1. Physiological Content and Intrinsic Activities of 10 Cytochrome P450 Isoforms in Human Normal Liver Microsomes.

    PubMed

    Zhang, Hai-Feng; Wang, Huan-Huan; Gao, Na; Wei, Jun-Ying; Tian, Xin; Zhao, Yan; Fang, Yan; Zhou, Jun; Wen, Qiang; Gao, Jie; Zhang, Yang-Jun; Qian, Xiao-Hong; Qiao, Hai-Ling

    2016-07-01

    Due to a lack of physiologic cytochrome P450 (P450) isoform content, P450 activity is typically only determined at the microsomal level (per milligram of microsomal protein) and not at the isoform level (per picomole of P450 isoform), which could result in the misunderstanding of variations in P450 activity between individuals and further hinder development of personalized medicine. We found that there were large variations in protein content, mRNA levels, and intrinsic activities of the 10 P450s in 100 human liver samples, in which CYP2E1 and CYP2C9 showed the highest expression levels. P450 gene polymorphisms had different effects on activity at two levels: CYP3A5*3 and CYP2A6*9 alleles conferred increased activity at the isoform level but decreased activity at the microsomal level; CYP2C9*3 had no effect at the isoform level but decreased activity at the microsomal level. The different effects at each level stem from the different effects of each polymorphism on the resulting P450 protein. Individuals with CYP2A6*1/*4, CYP2A6*1/*9, CYP2C9*1/*3, CYP2D6 100C>T TT, CYP2E1 7632T>A AA, CYP3A5*1*3, and CYP3A5*3*3 genotypes had significantly lower protein content, whereas CYP2D6 1661G>C mutants had a higher protein content. In conclusion, we first offered the physiologic data of 10 P450 isoform contents and found that some single nucleotide polymorphisms had obvious effects on P450 expression in human normal livers. The effects of gene polymorphisms on intrinsic P450 activity at the isoform level were quite different from those at the microsomal level, which might be due to changes in P450 protein content. PMID:27189963

  2. The ability of apolipoprotein E fragments to promote intraneuronal accumulation of amyloid beta peptide 42 is both isoform and size-specific.

    PubMed

    Dafnis, Ioannis; Argyri, Letta; Sagnou, Marina; Tzinia, Athina; Tsilibary, Effie C; Stratikos, Efstratios; Chroni, Angeliki

    2016-01-01

    The apolipoprotein (apo) E4 isoform is the strongest risk factor for late-onset Alzheimer's disease (AD). ApoE4 is more susceptible to proteolysis than apoE2 and apoE3 isoforms and carboxyl-terminal truncated apoE4 forms have been found in AD patients' brain. We have previously shown that a specific apoE4 fragment, apoE4-165, promotes amyloid-peptide beta 42 (Aβ42) accumulation in human neuroblastoma SK-N-SH cells and increased intracellular reactive oxygen species formation, two events considered to occur early in AD pathogenesis. Here, we show that these effects are allele-dependent and absolutely require the apoE4 background. Furthermore, the exact length of the fragment is critical since longer or shorter length carboxyl-terminal truncated apoE4 forms do not elicit the same effects. Structural and thermodynamic analyses showed that apoE4-165 has a compact structure, in contrast to other carboxyl-terminal truncated apoE4 forms that are instead destabilized. Compared however to other allelic backgrounds, apoE4-165 is structurally distinct and less thermodynamically stable suggesting that the combination of a well-folded structure with structural plasticity is a unique characteristic of this fragment. Overall, our findings suggest that the ability of apoE fragments to promote Aβ42 intraneuronal accumulation is specific for both the apoE4 isoform and the particular structural and thermodynamic properties of the fragment. PMID:27476701

  3. Overexpression of Isoforms of Nitric Oxide Synthase 1 Adaptor Protein, Encoded by a Risk Gene for Schizophrenia, Alters Actin Dynamics and Synaptic Function

    PubMed Central

    Hernandez, Kristina; Swiatkowski, Przemyslaw; Patel, Mihir V.; Liang, Chen; Dudzinski, Natasha R.; Brzustowicz, Linda M.; Firestein, Bonnie L.

    2016-01-01

    Proper communication between neurons depends upon appropriate patterning of dendrites and correct distribution and structure of spines. Schizophrenia is a neuropsychiatric disorder characterized by alterations in dendrite branching and spine density. Nitric oxide synthase 1 adaptor protein (NOS1AP), a risk gene for schizophrenia, encodes proteins that are upregulated in the dorsolateral prefrontal cortex (DLPFC) of individuals with schizophrenia. To elucidate the effects of NOS1AP overexpression observed in individuals with schizophrenia, we investigated changes in actin dynamics and spine development when a long (NOS1AP-L) or short (NOS1AP-S) isoform of NOS1AP is overexpressed. Increased NOS1AP-L protein promotes the formation of immature spines when overexpressed in rat cortical neurons from day in vitro (DIV) 14 to DIV 17 and reduces the amplitude of miniature excitatory postsynaptic currents (mEPSCs). In contrast, increased NOS1AP-S protein increases the rate of actin polymerization and the number of immature and mature spines, which may be attributed to a decrease in total Rac1 expression and a reduction in the levels of active cofilin. The increase in the number of mature spines by overexpression of NOS1AP-S is accompanied by an increase in the frequency of mEPSCs. Our findings show that overexpression of NOS1AP-L or NOS1AP-S alters the actin cytoskeleton and synaptic function. However, the mechanisms by which these isoforms induce these changes are distinct. These results are important for understanding how increased expression of NOS1AP isoforms can influence spine development and synaptic function. PMID:26869880

  4. The ability of apolipoprotein E fragments to promote intraneuronal accumulation of amyloid beta peptide 42 is both isoform and size-specific

    PubMed Central

    Dafnis, Ioannis; Argyri, Letta; Sagnou, Marina; Tzinia, Athina; Tsilibary, Effie C.; Stratikos, Efstratios; Chroni, Angeliki

    2016-01-01

    The apolipoprotein (apo) E4 isoform is the strongest risk factor for late-onset Alzheimer’s disease (AD). ApoE4 is more susceptible to proteolysis than apoE2 and apoE3 isoforms and carboxyl-terminal truncated apoE4 forms have been found in AD patients’ brain. We have previously shown that a specific apoE4 fragment, apoE4-165, promotes amyloid-peptide beta 42 (Aβ42) accumulation in human neuroblastoma SK-N-SH cells and increased intracellular reactive oxygen species formation, two events considered to occur early in AD pathogenesis. Here, we show that these effects are allele-dependent and absolutely require the apoE4 background. Furthermore, the exact length of the fragment is critical since longer or shorter length carboxyl-terminal truncated apoE4 forms do not elicit the same effects. Structural and thermodynamic analyses showed that apoE4-165 has a compact structure, in contrast to other carboxyl-terminal truncated apoE4 forms that are instead destabilized. Compared however to other allelic backgrounds, apoE4-165 is structurally distinct and less thermodynamically stable suggesting that the combination of a well-folded structure with structural plasticity is a unique characteristic of this fragment. Overall, our findings suggest that the ability of apoE fragments to promote Aβ42 intraneuronal accumulation is specific for both the apoE4 isoform and the particular structural and thermodynamic properties of the fragment. PMID:27476701

  5. Overexpression of Isoforms of Nitric Oxide Synthase 1 Adaptor Protein, Encoded by a Risk Gene for Schizophrenia, Alters Actin Dynamics and Synaptic Function.

    PubMed

    Hernandez, Kristina; Swiatkowski, Przemyslaw; Patel, Mihir V; Liang, Chen; Dudzinski, Natasha R; Brzustowicz, Linda M; Firestein, Bonnie L

    2016-01-01

    Proper communication between neurons depends upon appropriate patterning of dendrites and correct distribution and structure of spines. Schizophrenia is a neuropsychiatric disorder characterized by alterations in dendrite branching and spine density. Nitric oxide synthase 1 adaptor protein (NOS1AP), a risk gene for schizophrenia, encodes proteins that are upregulated in the dorsolateral prefrontal cortex (DLPFC) of individuals with schizophrenia. To elucidate the effects of NOS1AP overexpression observed in individuals with schizophrenia, we investigated changes in actin dynamics and spine development when a long (NOS1AP-L) or short (NOS1AP-S) isoform of NOS1AP is overexpressed. Increased NOS1AP-L protein promotes the formation of immature spines when overexpressed in rat cortical neurons from day in vitro (DIV) 14 to DIV 17 and reduces the amplitude of miniature excitatory postsynaptic currents (mEPSCs). In contrast, increased NOS1AP-S protein increases the rate of actin polymerization and the number of immature and mature spines, which may be attributed to a decrease in total Rac1 expression and a reduction in the levels of active cofilin. The increase in the number of mature spines by overexpression of NOS1AP-S is accompanied by an increase in the frequency of mEPSCs. Our findings show that overexpression of NOS1AP-L or NOS1AP-S alters the actin cytoskeleton and synaptic function. However, the mechanisms by which these isoforms induce these changes are distinct. These results are important for understanding how increased expression of NOS1AP isoforms can influence spine development and synaptic function. PMID:26869880

  6. A mechanism for branchial acid excretion in marine fish: identification of multiple Na+/H+ antiporter (NHE) isoforms in gills of two seawater teleosts.

    PubMed

    Claiborne, J B; Blackston, C R; Choe, K P; Dawson, D C; Harris, S P; Mackenzie, L A; Morrison-Shetlar, A I

    1999-02-01

    Both Na+/H+ exchange and the electrogenic extrusion of H+ via an H+-ATPase have been postulated to drive acid excretion across the branchial epithelium of fishes. While the H+-ATPase/Na+ channel system appears to be the predominant mechanism in some freshwater species, it may play a reduced role in seawater and brackish-water animals, where high external Na+ concentrations may thermodynamically favor Na+/H+ exchange driven by a Na+/H+ antiporter (NHE). In this study, we used molecular and immunological methods to assess the role of NHE isoforms in the branchial epithelium of the marine long-horned sculpin (Myoxocephalus octodecimspinosus) and the euryhaline killifish (Fundulus heteroclitus). Northern blot analysis of RNA probed with the human NHE-1 BamHI fragment suggested the presence of homologous gill NHE mRNA in sculpin. RT-PCR on gill RNA isolated from sculpin recovering from metabolic acidosis provided evidence for two distinct NHE isoforms; one with 76 % amino acid homology to mammalian NHE-2, and another 92 % homologous to trout erythrocytic beta-NHE. Killifish also have transcripts with 91 % homology to beta-NHE. Immunological detection using monoclonal antibodies for mammalian NHE-1 revealed a protein antigenically similar to this isoform in the gills of both species. Metabolic acidosis caused an approximately 30-fold decrease in expression of the NHE-1-like protein in sculpin. We speculate that beta-NHE in the gills plays the intracellular 'housekeeping' roles described for mammalian NHE-1. During systemic acidosis, apical gill NHE-2 (which is sensitive to external amiloride and low [Na+]) in parallel with a dramatic suppression of basolateral NHE-1 activity enhances net capdelta H+ transfers to the water. PMID:9882643

  7. The N-Terminal Peptides of the Three Human Isoforms of the Mitochondrial Voltage-Dependent Anion Channel Have Different Helical Propensities.

    PubMed

    Guardiani, Carlo; Scorciapino, Mariano Andrea; Amodeo, Giuseppe Federico; Grdadolnik, Joze; Pappalardo, Giuseppe; De Pinto, Vito; Ceccarelli, Matteo; Casu, Mariano

    2015-09-15

    The voltage-dependent anion channel (VDAC) is the main mitochondrial porin allowing the exchange of ions and metabolites between the cytosol and the mitochondrion. In addition, VDAC was found to actively interact with proteins playing a fundamental role in the regulation of apoptosis and being of central interest in cancer research. VDAC is a large transmembrane β-barrel channel, whose N-terminal helical fragment adheres to the channel interior, partially closing the pore. This fragment is considered to play a key role in protein stability and function as well as in the interaction with apoptosis-related proteins. Three VDAC isoforms are differently expressed in higher eukaryotes, for which distinct and complementary roles are proposed. In this work, the folding propensity of their N-terminal fragments has been compared. By using multiple spectroscopic techniques, and complementing the experimental results with theoretical computer-assisted approaches, we have characterized their conformational equilibrium. Significant differences were found in the intrinsic helical propensity of the three peptides, decreasing in the following order: hVDAC2 > hVDAC3 > hVDAC1. In light of the models proposed in the literature to explain voltage gating, selectivity, and permeability, as well as interactions with functionally related proteins, our results suggest that the different chemicophysical properties of the N-terminal domain are possibly correlated to different functions for the three isoforms. The overall emerging picture is that a similar transmembrane water accessible conduit has been equipped with not identical domains, whose differences can modulate the functional roles of the three VDAC isoforms. PMID:26303511

  8. Distinct responses to reduplicated chromosomes require distinct Mad2 responses

    PubMed Central

    Stormo, Benjamin M; Fox, Donald T

    2016-01-01

    Duplicating chromosomes once each cell cycle produces sister chromatid pairs, which separate accurately at anaphase. In contrast, reduplicating chromosomes without separation frequently produces polytene chromosomes, a barrier to accurate mitosis. Chromosome reduplication occurs in many contexts, including: polytene tissue development, polytene tumors, and following treatment with mitosis-blocking chemotherapeutics. However, mechanisms responding to or resolving polyteny during mitosis are poorly understood. Here, using Drosophila, we uncover two distinct reduplicated chromosome responses. First, when reduplicated polytene chromosomes persist into metaphase, an anaphase delay prevents tissue malformation and apoptosis. Second, reduplicated polytene chromosomes can also separate prior to metaphase through a spindle-independent mechanism termed Separation-Into-Recent-Sisters (SIRS). Both reduplication responses require the spindle assembly checkpoint protein Mad2. While Mad2 delays anaphase separation of metaphase polytene chromosomes, Mad2’s control of overall mitotic timing ensures efficient SIRS. Our results pinpoint mechanisms enabling continued proliferation after genome reduplication, a finding with implications for cancer progression and prevention. DOI: http://dx.doi.org/10.7554/eLife.15204.001 PMID:27159240

  9. Distinct responses to reduplicated chromosomes require distinct Mad2 responses.

    PubMed

    Stormo, Benjamin M; Fox, Donald T

    2016-01-01

    Duplicating chromosomes once each cell cycle produces sister chromatid pairs, which separate accurately at anaphase. In contrast, reduplicating chromosomes without separation frequently produces polytene chromosomes, a barrier to accurate mitosis. Chromosome reduplication occurs in many contexts, including: polytene tissue development, polytene tumors, and following treatment with mitosis-blocking chemotherapeutics. However, mechanisms responding to or resolving polyteny during mitosis are poorly understood. Here, using Drosophila, we uncover two distinct reduplicated chromosome responses. First, when reduplicated polytene chromosomes persist into metaphase, an anaphase delay prevents tissue malformation and apoptosis. Second, reduplicated polytene chromosomes can also separate prior to metaphase through a spindle-independent mechanism termed Separation-Into-Recent-Sisters (SIRS). Both reduplication responses require the spindle assembly checkpoint protein Mad2. While Mad2 delays anaphase separation of metaphase polytene chromosomes, Mad2's control of overall mitotic timing ensures efficient SIRS. Our results pinpoint mechanisms enabling continued proliferation after genome reduplication, a finding with implications for cancer progression and prevention. PMID:27159240

  10. Dichotomy of short and long thymic stromal lymphopoietin isoforms in inflammatory disorders of the bowel and skin

    PubMed Central

    Fornasa, Giulia; Tsilingiri, Katerina; Caprioli, Flavio; Botti, Fiorenzo; Mapelli, Marina; Meller, Stephan; Kislat, Andreas; Homey, Bernhard; Di Sabatino, Antonio; Sonzogni, Angelica; Viale, Giuseppe; Diaferia, Giuseppe; Gori, Alessandro; Longhi, Renato; Penna, Giuseppe; Rescigno, Maria

    2015-01-01

    Background Thymic stromal lymphopoietin (TSLP) is a cytokine with pleiotropic functions in the immune system. It has been associated with allergic reactions in the skin and lungs but also homeostatic tolerogenic responses in the thymus and gut. Objective In human subjects TSLP is present in 2 isoforms, short and long. Here we wanted to investigate the differential expression of the TSLP isoforms and discern their biological implications under homeostatic or inflammatory conditions. Methods We evaluated the expression of TSLPs in tissues from healthy subjects, patients with ulcerative colitis, patients with celiac disease, and patients with atopic dermatitis and on epithelial cells and keratinocytes under steady-state conditions or after stimulation. We then tested the immune activity of TSLP isoforms both in vitro and in vivo. Results We showed that TSLP isoforms are responsible for 2 opposite immune functions. The short isoform is expressed under steady-state conditions and exerts anti-inflammatory activities by affecting the capacity of PBMCs and dendritic cells to produce inflammatory cytokines. Moreover, the short isoform TSLP ameliorates experimental colitis in mice and prevents endotoxin shock. The long isoform of TSLP is proinflammatory and is only expressed during inflammation. The isoforms are differentially regulated by pathogenic bacteria, such as Salmonella species and adhesive-invasive Escherichia coli. Conclusions We have solved the dilemma of TSLP being both homeostatic and inflammatory. The TSLP isoform ratio is altered during several inflammatory disorders, with strong implications in disease treatment and prevention. Indeed, targeting of the long isoform of TSLP at the C-terminal portion, which is common to both isoforms, might lead to unwanted side effects caused by neutralization of the homeostatic short isoform. PMID:26014813

  11. Observations on the Role of TcdE Isoforms in Clostridium difficile Toxin Secretion

    PubMed Central

    Fitzwater, Leah; Nichols, Rebekah

    2015-01-01

    ABSTRACT Clostridium difficile is a major nosocomial pathogen and the principal causative agent of antibiotic-associated diarrhea. The toxigenic C. difficile strains that cause disease secrete virulence factors, toxin A and toxin B, that cause colonic injury and inflammation. C. difficile toxins have no export signature and are secreted by an unusual mechanism that involves TcdE, a holin-like protein. We isolated a TcdE mutant of the epidemic R20291 strain with impaired toxin secretion, which was restored by complementation with functional TcdE. In the TcdE open reading frame (ORF), we identified three possible translation start sites; each translated isoform may play a specific role in TcdE-controlled toxin release. We created plasmid constructs that express only one of the three TcdE isoforms and complemented the TcdE mutant with these isoforms. Western blot analysis of the complemented strains demonstrated that TcdE is translated efficiently from the start codon at the 25th and 27th positions in the predicted ORF, producing proteins with 142 amino acids (TcdE142) and 140 amino acids (TcdE140), respectively. TcdE166 was not detected when expressed from its own ribosomal binding site (RBS). The effects of all three TcdE isoforms on C. difficile cell viability and toxin release were determined. Among the three isoforms, overexpression of TcdE166 and TcdE142 had a profound effect on cell viability compared to the TcdE140 isoform. Similarly, TcdE166 and TcdE142 facilitated toxin release more efficiently than did TcdE140. The importance of these variations among TcdE isoforms and their role in toxin release are discussed. IMPORTANCE C. difficile is a nosocomial pathogen that has become the most prevalent cause of antibiotic-associated diarrhea in North America and in several countries in Europe. Most strains of C. difficile produce two high-molecular-weight toxins that are regarded as the primary virulence factors. The mechanism by which these large toxins are

  12. Sodium Kinetics of Na,K-ATPase α Isoforms in Intact Transfected HeLa Cells

    PubMed Central

    Zahler, Raphael; Zhang, Zhong-Ting; Manor, Mira; Boron, Walter F.

    1997-01-01

    By participating in the regulation of ion and voltage gradients, the Na-K pump (i.e., Na,K-ATPase) influences many aspects of cellular physiology. Of the four α isoforms of the pump, α1 is ubiquitous, α2 is predominant in skeletal muscle, and α3 is found in neurons and the cardiac conduction system. To determine whether the isoforms have different intracellular Na+ affinities, we used the Na+-sensitive dye sodium-binding benzofuran isophthalate (SBFI) to measure pump-mediated Na+ efflux as a function of [Na+]i in human HeLa cells stably transfected with rat Na-K pump isoforms. We Na+-loaded the cells, and then monitored the time course of the decrease in [Na+]i after removing external Na+. All transfected rat α subunits were highly ouabain resistant: the α1 isoform is naturally resistant, whereas the α2 and α3 isoforms had been mutagenized to render them resistant. Thus, the Na+ efflux mediated by endogenous and transfected pumps could be separated by studying the cells at low (1 μM) and high (4 mM) ouabain concentrations. We found that the apparent Km for Na+ efflux attributable to the native human α1 isoform was 12 mM, which was similar to the Km of rat α1. The α2 and α3 isoforms had apparent Km's of 22 and 33 mM, respectively. The cells expressing α3 had a high resting [Na+]i. The maximal activity of native α1 in the α3-transfected cells was only ∼56% of native α1 activity in untransfected HeLa cells, suggesting that transfection with α3 led to a compensatory decrease in endogenous α1 pumps. We conclude that the apparent Km(Na+) for rat Na-K pump isoforms increases in the sequence α1 < α2 < α3. The α3 isoform may be suited for handling large Na+ loads in electrically active cells. PMID:9236212

  13. Functional Analysis of the Short Isoform of Orf Virus Protein OV20.0

    PubMed Central

    Tseng, Yeu-Yang; Lin, Fong-Yuan; Cheng, Sun-Fang; Chulakasian, Songkhla; Chou, Chia-Chi; Liu, Ya-Fen; Chang, Wei-Shan; Wong, Min-Liang

    2015-01-01

    ABSTRACT Orf virus (ORFV) OV20.0L is an ortholog of vaccinia virus (VACV) gene E3L. The function of VACV E3 protein as a virulence factor is well studied, but OV20.0 has received less attention. Here we show that like VACV E3L, OV20.0L encodes two proteins, a full-length protein and a shorter form (sh20). The shorter sh20 is an N-terminally truncated OV20.0 isoform generated when a downstream AUG codon is used for initiating translation. These isoforms differed in cellular localization, with full-length OV20.0 and sh20 found throughout the cell and predominantly in the cytoplasm, respectively. Nonetheless, both OV20.0 isoforms were able to bind double-stranded RNA (dsRNA)-activated protein kinase (PKR) and dsRNA. Moreover, both isoforms strongly inhibited PKR activation as shown by decreased phosphorylation of the translation initiation factor eIF2α subunit and protection of Sindbis virus infection against the activity of interferon (IFN). In spite of this apparent conservation of function in vitro, a recombinant ORFV that was able to express only the sh20 isoform was attenuated in a mouse model. IMPORTANCE The OV20.0 protein of orf virus (ORFV) has two isoforms and contributes to virulence, but the roles of the two forms are not known. This study shows that the shorter isoform (sh20) arises due to use of a downstream initiation codon and is amino-terminally truncated. The sh20 form also differs in expression kinetics and cellular localization from full-length OV20.0. Similar to the full-length isoform, sh20 is able to bind dsRNA and PKR, inactivate PKR, and thus act as an antagonist of the interferon response in vitro. In vivo, however, wild-type OV20.0 could not be replaced with sh20 alone without a loss of virulence, suggesting that the functions of the isoforms are not simply redundant. PMID:25694596

  14. [Preparation and properties of isocitrate lyase isoforms from the cotyledons of Glycine max L].

    PubMed

    Eprintsev, A T; Diachenko, E V; Lykova, T V; Kuen, C T H; Popov, V N

    2010-01-01

    A four-stage purification procedure including ammonium sulfate precipitation and ion exchange chromatography on DEAE cellulose has been elaborated for isolation of isocitrate lyase (EC 4.1.3.1) isoforms from the cotyledons of soybean Glycine max L. Electrophoretically homogeneous preparations of two forms of the enzyme with specific activity of 5.28 and 5.81 U/mg protein have been obtained. Comparison of physicochemical, kinetic, and regulation characteristics of the isoforms obtained revealed fundamental differences between them. Thus, the isoform that migrated quickly in PAAG had a much lower affinity to isocitrate (K(M) - 50 microM) than the slowly migrating form (K(M) - 16 microM). It has been shown that the conservation of activity of the isoforms obtained depends on the presence of divalent cations (Mn2+ and Mg2+) in the medium. It is suggested to use the isoforms of isocitrate lyase isolated from soybeans for the development of biosensors for biochemical and kinetic assays. PMID:20198926

  15. iReckon: simultaneous isoform discovery and abundance estimation from RNA-seq data.

    PubMed

    Mezlini, Aziz M; Smith, Eric J M; Fiume, Marc; Buske, Orion; Savich, Gleb L; Shah, Sohrab; Aparicio, Sam; Chiang, Derek Y; Goldenberg, Anna; Brudno, Michael

    2013-03-01

    High-throughput RNA sequencing (RNA-seq) promises to revolutionize our understanding of genes and their role in human disease by characterizing the RNA content of tissues and cells. The realization of this promise, however, is conditional on the development of effective computational methods for the identification and quantification of transcripts from incomplete and noisy data. In this article, we introduce iReckon, a method for simultaneous determination of the isoforms and estimation of their abundances. Our probabilistic approach incorporates multiple biological and technical phenomena, including novel isoforms, intron retention, unspliced pre-mRNA, PCR amplification biases, and multimapped reads. iReckon utilizes regularized expectation-maximization to accurately estimate the abundances of known and novel isoforms. Our results on simulated and real data demonstrate a superior ability to discover novel isoforms with a significantly reduced number of false-positive predictions, and our abundance accuracy prediction outmatches that of other state-of-the-art tools. Furthermore, we have applied iReckon to two cancer transcriptome data sets, a triple-negative breast cancer patient sample and the MCF7 breast cancer cell line, and show that iReckon is able to reconstruct the complex splicing changes that were not previously identified. QT-PCR validations of the isoforms detected in the MCF7 cell line confirmed all of iReckon's predictions and also showed strong agreement (r(2) = 0.94) with the predicted abundances. PMID:23204306

  16. iReckon: Simultaneous isoform discovery and abundance estimation from RNA-seq data

    PubMed Central

    Mezlini, Aziz M.; Smith, Eric J.M.; Fiume, Marc; Buske, Orion; Savich, Gleb L.; Shah, Sohrab; Aparicio, Sam; Chiang, Derek Y.; Goldenberg, Anna; Brudno, Michael

    2013-01-01

    High-throughput RNA sequencing (RNA-seq) promises to revolutionize our understanding of genes and their role in human disease by characterizing the RNA content of tissues and cells. The realization of this promise, however, is conditional on the development of effective computational methods for the identification and quantification of transcripts from incomplete and noisy data. In this article, we introduce iReckon, a method for simultaneous determination of the isoforms and estimation of their abundances. Our probabilistic approach incorporates multiple biological and technical phenomena, including novel isoforms, intron retention, unspliced pre-mRNA, PCR amplification biases, and multimapped reads. iReckon utilizes regularized expectation-maximization to accurately estimate the abundances of known and novel isoforms. Our results on simulated and real data demonstrate a superior ability to discover novel isoforms with a significantly reduced number of false-positive predictions, and our abundance accuracy prediction outmatches that of other state-of-the-art tools. Furthermore, we have applied iReckon to two cancer transcriptome data sets, a triple-negative breast cancer patient sample and the MCF7 breast cancer cell line, and show that iReckon is able to reconstruct the complex splicing changes that were not previously identified. QT-PCR validations of the isoforms detected in the MCF7 cell line confirmed all of iReckon's predictions and also showed strong agreement (r2 = 0.94) with the predicted abundances. PMID:23204306

  17. Differential Roles of Postsynaptic Density-93 Isoforms in Regulating Synaptic Transmission

    PubMed Central

    Krüger, Juliane M.; Favaro, Plinio D.; Liu, Mingna; Kitlińska, Agata; Huang, Xiaojie; Raabe, Monika; Akad, Derya S.; Liu, Yanling; Urlaub, Henning; Dong, Yan; Xu, Weifeng

    2013-01-01

    In the postsynaptic density of glutamatergic synapses, the discs large (DLG)-membrane-associated guanylate kinase (MAGUK) family of scaffolding proteins coordinates a multiplicity of signaling pathways to maintain and regulate synaptic transmission. Postsynaptic density-93 (PSD-93) is the most variable paralog in this family; it exists in six different N-terminal isoforms. Probably because of the structural and functional variability of these isoforms, the synaptic role of PSD-93 remains controversial. To accurately characterize the synaptic role of PSD-93, we quantified the expression of all six isoforms in the mouse hippocampus and examined them individually in hippocampal synapses. Using molecular manipulations, including overexpression, gene knockdown, PSD-93 knock-out mice combined with biochemical assays, and slice electrophysiology both in rat and mice, we demonstrate that PSD-93 is required at different developmental synaptic states to maintain the strength of excitatory synaptic transmission. This strength is differentially regulated by the six isoforms of PSD-93, including regulations of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor-active and inactive synapses, and activity-dependent modulations. Collectively, these results demonstrate that alternative combinations of N-terminal PSD-93 isoforms and DLG-MAGUK paralogs can fine-tune signaling scaffolds to adjust synaptic needs to regulate synaptic transmission. PMID:24068818

  18. Differential expression of VegT and Antipodean protein isoforms in Xenopus.

    PubMed

    Stennard, F; Zorn, A M; Ryan, K; Garrett, N; Gurdon, J B

    1999-08-01

    The VegT/Antipodean (Apod) gene is important for germ layer formation in Xenopus. To investigate the role of this gene at the protein level, as opposed to the RNA level, we have generated affinity purified polyclonal antibodies to Apod, and for comparison, to the other early T-box proteins Xbrachyury and Eomesodermin. An anti-VegT/Apod antibody reveals that there are two protein isoforms in Xenopus, one that we refer to as VegT and a smaller molecular weight isoform that we refer to as Apod. These isoforms have different N-terminal domains resulting from developmentally regulated alternative splicing of a primary transcript arising from a single VegT/Apod gene. VegT is maternally expressed. Its translation is blocked during oogenesis but the protein is present from the egg until gastrulation in the presumptive endoderm. There is no evidence for zygotic expression of this isoform. Conversely, the Apod protein isoform is expressed only after the onset of zygotic transcription in the presumptive mesoderm and is inducible by activin. We conclude that the developmental role of VegT/Apod is mediated by two different proteins, with entirely different patterns of expression and response to growth factors. PMID:10446268

  19. Nesprin-2 epsilon: A novel nesprin isoform expressed in human ovary and Ntera-2 cells

    SciTech Connect

    Lam, Le Thanh; Boehm, Sabrina V.; Roberts, Roland G.; Morris, Glenn E.

    2011-08-26

    Highlights: {yields} A novel epsilon isoform of nesprin-2 has been discovered. {yields} This 120 kDa protein was predicted by bioinformatic analysis, but has not previously been observed. {yields} It is the main isoform expressed in a teratocarcinoma cell line and is also found in ovary. {yields} Like other nesprins, it is located at the nuclear envelope. {yields} We suggest it may have a role in very early development or in some ovary-specific function. -- Abstract: The nuclear envelope-associated cytoskeletal protein, nesprin-2, is encoded by a large gene containing several internal promoters that produce shorter isoforms. In a study of Ntera-2 teratocarcinoma cells, a novel isoform, nesprin-2-epsilon, was found to be the major mRNA and protein product of the nesprin-2 gene. Its existence was predicted by bioinformatic analysis, but this is the first direct demonstration of both the mRNA and the 120 kDa protein which is located at the nuclear envelope. In a panel of 21 adult and foetal human tissues, the nesprin-2-epsilon mRNA was strongly expressed in ovary but was a minor isoform elsewhere. The expression pattern suggests a possible link with very early development and a likely physiological role in ovary.

  20. Immunohistochemical localization of transforming growth factor beta isoforms in asbestos-related diseases.

    PubMed Central

    Jagirdar, J; Lee, T C; Reibman, J; Gold, L I; Aston, C; Bégin, R; Rom, W N

    1997-01-01

    Transforming growth factor beta (TGF-beta), a multifunctional cytokine and growth factor, plays a key role in scarring and fibrotic processes because of its ability to induce extracellular matrix proteins and modulate the growth and immune function of many cell types. These effects are important in inflammatory disorders with fibrosis and cancer. The asbestos-related diseases are characterized by fibrosis in the lower respiratory tract and pleura and increased occurrence of lung cancer and mesothelioma. We performed immunohistochemistry with isoform-specific antibodies to the three TGF-beta isoforms on 16 autopsy lungs from Quebec, Canada, asbestos miners and millers. There was increased immunolocalization of all three TGF-beta isoforms in the fibrotic lesions of asbestosis and pleural fibrosis. The hyperplastic type II pneumocytes contained all three isoforms. By contrast, there was differential spatial immunostaining for the TGF-beta isoforms in malignant mesothelioma, with TGF-beta 1 in the stroma but TGF-beta 2 in the tumor cells. These data are consistent with an important role for TGF-beta in accumulation of extracellular matrix and cell proliferation in asbestos-related diseases. Images Figure 1. Figure 2. Figure 3. Figure 4. Figure 5. Figure 6. Figure 7. Figure 8. Figure 9. PMID:9400723

  1. Quantitative Profiling of Drosophila melanogaster Dscam1 Isoforms Reveals No Changes in Splicing after Bacterial Exposure

    PubMed Central

    Kurtz, Joachim; Schmucker, Dietmar; Chen, Wei

    2014-01-01

    The hypervariable Dscam1 (Down syndrome cell adhesion molecule 1) gene can produce thousands of different ectodomain isoforms via mutually exclusive alternative splicing. Dscam1 appears to be involved in the immune response of some insects and crustaceans. It has been proposed that the diverse isoforms may be involved in the recognition of, or the defence against, diverse parasite epitopes, although evidence to support this is sparse. A prediction that can be generated from this hypothesis is that the gene expression of specific exons and/or isoforms is influenced by exposure to an immune elicitor. To test this hypothesis, we for the first time, use a long read RNA sequencing method to directly investigate the Dscam1 splicing pattern after exposing adult Drosophila melanogaster and a S2 cell line to live Escherichia coli. After bacterial exposure both models showed increased expression of immune-related genes, indicating that the immune system had been activated. However there were no changes in total Dscam1 mRNA expression. RNA sequencing further showed that there were no significant changes in individual exon expression and no changes in isoform splicing patterns in response to bacterial exposure. Therefore our studies do not support a change of D. melanogaster Dscam1 isoform diversity in response to live E. coli. Nevertheless, in future this approach could be used to identify potentially immune-related Dscam1 splicing regulation in other host species or in response to other pathogens. PMID:25310676

  2. Molecular evolution of cytochrome c oxidase: rate variation among subunit VIa isoforms.

    PubMed

    Schmidt, T R; Jaradat, S A; Goodman, M; Lomax, M I; Grossman, L I

    1997-06-01

    Cytochrome c oxidase (COX) consists of 13 subunits, 3 encoded in the mitochondrial genome and 10 in the nucleus. Little is known of the role of the nuclear-encoded subunits, some of which exhibit tissue-specific isoforms. Subunit VIa is unique in having tissue-specific isoforms in all mammalian species examined. We examined relative evolutionary rates for the COX6A heart (H) and liver (L) isoform genes along the length of the molecule, specifically in relation to the tissue-specific function(s) of the two isoforms. Nonsynonymous (amino acid replacement) substitutions in the COX6AH gene occurred more frequently than in the ubiquitously expressed COX6AL gene. Maximum-parsimony analysis and sequence divergences from reconstructed ancestral sequences revealed that after the ancestral COX6A gene duplicated to yield the genes for the H and L isoforms, the sequences encoding the mitochondrial matrix region of the COX VIa protein experienced an elevated rate of nonsynonymous substitutions relative to synonymous substitutions. This is expected for relaxed selective constraints after gene duplication followed by purifying selection to preserve the replacements with tissue-specific functions. PMID:9190060

  3. Two isoforms of aquaporin 2 responsive to hypertonic stress in the bottlenose dolphin.

    PubMed

    Suzuki, Miwa; Wakui, Hitomi; Itou, Takuya; Segawa, Takao; Inoshima, Yasuo; Maeda, Ken; Kikuchi, Kiyoshi

    2016-04-15

    This study investigated the expression of aquaporin 2 (AQP2) and its newly found alternatively spliced isoform (alternative AQP2) and the functions of these AQP2 isoforms in the cellular hyperosmotic tolerance in the bottlenose dolphin, ITALIC! Tursiops truncatus mRNA sequencing revealed that alternative AQP2 lacks the fourth exon and instead has a longer third exon that includes a part of the original third intron. The portion of the third intron, now part of the coding region of alternative AQP2, is highly conserved among many species of the order Cetacea but not among terrestrial mammals. Semi-quantitative PCR revealed that AQP2 was expressed only in the kidney, similar to terrestrial mammals. In contrast, alternative AQP2 was expressed in all organs examined, with strong expression in the kidney. In cultured renal cells, expression of both AQP2 isoforms was upregulated by the addition to the medium of NaCl but not by the addition of mannitol, indicating that the expression of both isoforms is induced by hypersalinity. Treatment with small interfering RNA for both isoforms resulted in a decrease in cell viability in hypertonic medium (500 mOsm kg(-1)) when compared with controls. These findings indicate that the expression of alternatively spliced AQP2 is ubiquitous in cetacean species, and it may be one of the molecules important for cellular osmotic tolerance throughout the body. PMID:26944501

  4. Cortisol differentially alters claudin isoforms in cultured puffer fish gill epithelia.

    PubMed

    Bui, Phuong; Bagherie-Lachidan, Mazdak; Kelly, Scott P

    2010-04-12

    A primary cultured gill epithelium from the puffer fish Tetraodon nigroviridis was developed to examine the corticosteroid regulation of claudin isoform mRNA abundance in fish gills. Preparations were composed of polygonal epithelial cells exhibiting concentric apical microridges and zonula occludens-1 immunoreactivity along cell margins. No evidence was found to indicate the presence of Na(+)-K(+)-ATPase-immunoreactive or mitochondria-rich cells in cultured preparations. Therefore, epithelia appear to be composed of gill pavement cells (PVCs) only. An RT-PCR profile of 12 salinity responsive gill claudin tight junction (TJ) proteins (Tncldn3a, -3c, -6, -8d, -10d, -10e, -11a, -23b, -27a, -27c, -32a, and -33b) revealed the absence of Tncldn6, -10d and -10e in cultured epithelia, suggesting that these isoforms are not associated with gill PVCs. Cortisol treatment of cultured epithelia dose-dependently increased or decreased mRNA abundance of select claudin isoforms. Transcript abundance of several claudin isoforms was unaffected by cortisol treatment. These data provide evidence for the cell specific distribution of claudins in fish gills and suggest that heterogeneous alterations in the abundance of select claudin isoforms contribute to the corticosteroid regulation of gill permeability. PMID:19969041

  5. The ZFHX1A Gene Is Differentially Autoregulated By Its Isoforms

    PubMed Central

    Manavella, Pablo A.; Roqueiro, Gonzalo; Darling, Douglas S.; Cabanillas, Ana M.

    2009-01-01

    The Zfhx1a gene expresses two different isoforms; the full length Zfhx1a-1 and a truncated isoform termed Zfhx1a-2 lacking the first exon. Deletion analysis of the Zfhx1a-1 promoter localized cell-specific repressors, and a proximal G-string that is critically required for transactivation. Transfection of Zfhx1a-1 cDNA, but not Zfhx1a -2, downregulates Zfhx1a-1 promoter activity. Mutation of an E2-box disrupted the binding of both Zfhx1a isoforms. Consistent with this, transfected Zfhx1a-1 does not regulate the transcriptional activity of the E-box mutated Zfhx1a-1 promoter. Competitive EMSAs and transfection assays show that Zfhx1a-2 can function as a dominant negative isoform since it is able to compete and displace Zfhx1a-1 from its binding site and overcome Zfhx1a-1 induced repression of the Zfhx1a-1 promoter in cells. Hence, the Zfhx1a-1 gene is autoregulated in part by negative feedback on its own promoter which is, in turn, modified by the availability of the negative dominant isoform Zfhx1a-2. PMID:17610840

  6. The ZFHX1A gene is differentially autoregulated by its isoforms.

    PubMed

    Manavella, Pablo A; Roqueiro, Gonzalo; Darling, Douglas S; Cabanillas, Ana M

    2007-08-31

    The Zfhx1a gene expresses two different isoforms; the full length Zfhx1a-1 and a truncated isoform termed Zfhx1a-2 lacking the first exon. Deletion analysis of the Zfhx1a-1 promoter localized cell-specific repressors, and a proximal G-string that is critically required for transactivation. Transfection of Zfhx1a-1 cDNA, but not Zfhx1a-2, downregulates Zfhx1a-1 promoter activity. Mutation of an E2-box disrupted the binding of both Zfhx1a isoforms. Consistent with this, transfected Zfhx1a-1 does not regulate the transcriptional activity of the E-box mutated Zfhx1a-1 promoter. Competitive EMSAs and transfection assays show that Zfhx1a-2 can function as a dominant negative isoform since it is able to compete and displace Zfhx1a-1 from its binding site and overcome Zfhx1a-1 induced repression of the Zfhx1a-1 promoter in cells. Hence, the Zfhx1a-1 gene is autoregulated in part by negative feedback on its own promoter which is, in turn, modified by the availability of the negative dominant isoform Zfhx1a-2. PMID:17610840

  7. An innovative approach for the characterization of the isoforms of a monoclonal antibody product

    PubMed Central

    Sundaram, Shanmuuga; Matathia, Alice; Qian, Jun; Zhang, Jingming; Hsieh, Ming-Ching; Liu, Tun; Crowley, Richard

    2011-01-01

    Protein biopharmaceuticals, such as monoclonal antibodies (mAbs) are widely used for the prevention and treatment of various diseases. The complex and lengthy upstream and downstream production methods of the antibodies make them susceptible to physical and chemical modifications. Several IgG1 immunoglobulins are used as medical agents for the treatment of colon, breast and head and neck cancers, and at least four to eight isoforms exist in the products. The regulatory agencies understand the complex nature of the antibody molecules and allow the manufactures to set their own specifications for lot release, provided the safety and efficacy of the products are established in animal models prior to clinical trials. During the manufacture of a mAb product, we observed lot-to-lot variability in the isoform content and, although the variability is within the set specifications for lot release, made attempts to gain mechanistic insight by isolating and characterizing the individual isoforms. Matrix-assisted laser desorption/ionization (MALDI) and liquid chromatography (LC)/mass spectrometry (MS)/MS analyses of the isolated isoforms indicate that this variability is caused by sialic acid content, as well as truncation of C-terminal lysine of the individual isoforms. Sialidase and carboxypeptidase treatment of the product confirm the observations made by MALDI and LC/MS/MS. PMID:22123057

  8. The N-terminal Set-β Protein Isoform Induces Neuronal Death.

    PubMed

    Trakhtenberg, Ephraim F; Morkin, Melina I; Patel, Karan H; Fernandez, Stephanie G; Sang, Alan; Shaw, Peter; Liu, Xiongfei; Wang, Yan; Mlacker, Gregory M; Gao, Han; Velmeshev, Dmitry; Dombrowski, Susan M; Vitek, Michael P; Goldberg, Jeffrey L

    2015-05-22

    Set-β protein plays different roles in neurons, but the diversity of Set-β neuronal isoforms and their functions have not been characterized. The expression and subcellular localization of Set-β are altered in Alzheimer disease, cleavage of Set-β leads to neuronal death after stroke, and the full-length Set-β regulates retinal ganglion cell (RGC) and hippocampal neuron axon growth and regeneration in a subcellular localization-dependent manner. Here we used various biochemical approaches to investigate Set-β isoforms and their role in the CNS, using the same type of neurons, RGCs, across studies. We found multiple alternatively spliced isoforms expressed from the Set locus in purified RGCs. Set transcripts containing the Set-β-specific exon were the most highly expressed isoforms. We also identified a novel, alternatively spliced Set-β transcript lacking the nuclear localization signal and demonstrated that the full-length (∼39-kDa) Set-β is localized predominantly in the nucleus, whereas a shorter (∼25-kDa) Set-β isoform is localized predominantly in the cytoplasm. Finally, we show that an N-terminal Set-β cleavage product can induce neuronal death. PMID:25833944

  9. A Subtle Alternative Splicing Event Gives Rise to a Widely Expressed Human RNase k Isoform

    PubMed Central

    Karousis, Evangelos D.; Sideris, Diamantis C.

    2014-01-01

    Subtle alternative splicing leads to the formation of RNA variants lacking or including a small number of nucleotides. To date, the impact of subtle alternative splicing phenomena on protein biosynthesis has been studied in frame-preserving incidents. On the contrary, mRNA isoforms derived from frame-shifting events were poorly studied and generally characterized as non-coding. This work provides evidence for a frame-shifting subtle alternative splicing event which results in the production of a novel protein isoform. We applied a combined molecular approach for the cloning and expression analysis of a human RNase κ transcript (RNase κ-02) which lacks four consecutive bases compared to the previously isolated RNase κ isoform. RNase κ-02 mRNA is expressed in all human cell lines tested end encodes the synthesis of a 134-amino-acid protein by utilizing an alternative initiation codon. The expression of RNase κ-02 in the cytoplasm of human cells was verified by Western blot and immunofluorescence analysis using a specific polyclonal antibody developed on the basis of the amino-acid sequence difference between the two protein isoforms. The results presented here show that subtle changes during mRNA splicing can lead to the expression of significantly altered protein isoforms. PMID:24797913

  10. Aryl hydrocarbon receptor nuclear translocator (ARNT) isoforms control lymphoid cancer cell proliferation through differentially regulating tumor suppressor p53 activity

    PubMed Central

    Fang, Gloria; Sarkar, Krishnakali; Mendez, Omayra; Wright, Casey W.

    2016-01-01

    The aryl hydrocarbon receptor nuclear translocator (ARNT) is involved in xenobiotic and hypoxic responses, and we previously showed that ARNT also regulates nuclear factor-κB (NF-κB) signaling by altering the DNA binding activity of the RelB subunit. However, our initial study of ARNT-mediated RelB modulation was based on simultaneous suppression of the two ARNT isoforms, isoform 1 and 3, and precluded the examination of their individual functions. We find here that while normal lymphocytes harbor equal levels of isoform 1 and 3, lymphoid malignancies exhibit a shift to higher levels of ARNT isoform 1. These elevated levels of ARNT isoform 1 are critical to the proliferation of these cancerous cells, as suppression of isoform 1 in a human multiple myeloma (MM) cell line, and an anaplastic large cell lymphoma (ALCL) cell line, triggered S-phase cell cycle arrest, spontaneous apoptosis, and sensitized cells to doxorubicin treatment. Furthermore, co-suppression of RelB or p53 with ARNT isoform 1 prevented cell cycle arrest and blocked doxorubicin induced apoptosis. Together our findings reveal that certain blood cancers rely on ARNT isoform 1 to potentiate proliferation by antagonizing RelB and p53-dependent cell cycle arrest and apoptosis. Significantly, our results identify ARNT isoform 1 as a potential target for anticancer therapies. PMID:26909609

  11. Biologically distinct subsets of nevi.

    PubMed

    Rogers, Tova; Marino, Maria L; Raciti, Patricia; Jain, Manu; Busam, Klaus J; Marchetti, Michael A; Marghoob, Ashfaq A

    2016-08-01

    Melanocytic nevi (MN) encompass a range of benign tumors with varying microscopic and macroscopic features. Their development is a multifactorial process under genetic and environmental influences. The clinical importance of MN lies in distinguishing them from melanoma and in recognizing their associations with melanoma risk and cancer syndromes. Historically, the distinction between the different types of MN, as well as between MN and melanoma, was based on clinical history, gross morphology, and histopathological features. While histopathology with clinical correlation remains the gold standard for differentiating and diagnosing melanocytic lesions, in some cases, this may not be possible. The use of dermoscopy has allowed for the assessment of subsurface skin structures and has contributed to the clinical evaluation and classification of MN. Genetic profiling, while still in its early stages, has the greatest potential to refine the classification of MN by clarifying their developmental processes, biological behaviors, and relationships to melanoma. Here we review the most salient clinical, dermoscopic, histopathological, and genetic features of different MN subgroups. PMID:27119653