Browsing by Subject "Sequence Homology, Amino Acid"

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    A C-terminal motif found in the beta2-adrenergic receptor, P2Y1 receptor and cystic fibrosis transmembrane conductance regulator determines binding to the Na+/H+ exchanger regulatory factor family of PDZ proteins.
    (Proc Natl Acad Sci U S A, 1998-07-21) Hall, RA; Ostedgaard, LS; Premont, RT; Blitzer, JT; Rahman, N; Welsh, MJ; Lefkowitz, RJ
    The Na+/H+ exchanger regulatory factor (NHERF) binds to the tail of the beta2-adrenergic receptor and plays a role in adrenergic regulation of Na+/H+ exchange. NHERF contains two PDZ domains, the first of which is required for its interaction with the beta2 receptor. Mutagenesis studies of the beta2 receptor tail revealed that the optimal C-terminal motif for binding to the first PDZ domain of NHERF is D-S/T-x-L, a motif distinct from those recognized by other PDZ domains. The first PDZ domain of NHERF-2, a protein that is 52% identical to NHERF and also known as E3KARP, SIP-1, and TKA-1, exhibits binding preferences very similar to those of the first PDZ domain of NHERF. The delineation of the preferred binding motif for the first PDZ domain of the NHERF family of proteins allows for predictions for other proteins that may interact with NHERF or NHERF-2. For example, as would be predicted from the beta2 receptor tail mutagenesis studies, NHERF binds to the tail of the purinergic P2Y1 receptor, a seven-transmembrane receptor with an intracellular C-terminal tail ending in D-T-S-L. NHERF also binds to the tail of the cystic fibrosis transmembrane conductance regulator, which ends in D-T-R-L. Because the preferred binding motif of the first PDZ domain of the NHERF family of proteins is found at the C termini of a variety of intracellular proteins, NHERF and NHERF-2 may be multifunctional adaptor proteins involved in many previously unsuspected aspects of intracellular signaling.
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    A novel human endogenous retroviral protein inhibits cell-cell fusion.
    (Scientific reports, 2013-01) Sugimoto, Jun; Sugimoto, Makiko; Bernstein, Helene; Jinno, Yoshihiro; Schust, Danny
    While common in viral infections and neoplasia, spontaneous cell-cell fusion, or syncytialization, is quite restricted in healthy tissues. Such fusion is essential to human placental development, where interactions between trophoblast-specific human endogenous retroviral (HERV) envelope proteins, called syncytins, and their widely-distributed cell surface receptors are centrally involved. We have identified the first host cell-encoded protein that inhibits cell fusion in mammals. Like the syncytins, this protein, called suppressyn, is HERV-derived, placenta-specific and well-conserved over simian evolution. In vitro, suppressyn binds to the syn1 receptor and inhibits syn1-, but not syn2-mediated trophoblast syncytialization. Suppressyn knock-down promotes cell-cell fusion in trophoblast cells and cell-associated and secreted suppressyn binds to the syn1 receptor, ASCT2. Identification of the first host cell-encoded inhibitor of mammalian cell fusion may encourage improved understanding of cell fusion mechanisms, of placental morphogenesis and of diseases resulting from abnormal cell fusion.
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    Discovery of the Elusive UDP-Diacylglucosamine Hydrolase in the Lipid A Biosynthetic Pathway in Chlamydia trachomatis.
    (MBio, 2016-03-22) Young, Hayley E; Zhao, Jinshi; Barker, Jeffrey R; Guan, Ziqiang; Valdivia, Raphael H; Zhou, Pei
    Constitutive biosynthesis of lipid A via the Raetz pathway is essential for the viability and fitness of Gram-negative bacteria, includingChlamydia trachomatis Although nearly all of the enzymes in the lipid A biosynthetic pathway are highly conserved across Gram-negative bacteria, the cleavage of the pyrophosphate group of UDP-2,3-diacyl-GlcN (UDP-DAGn) to form lipid X is carried out by two unrelated enzymes: LpxH in beta- and gammaproteobacteria and LpxI in alphaproteobacteria. The intracellular pathogenC. trachomatislacks an ortholog for either of these two enzymes, and yet, it synthesizes lipid A and exhibits conservation of genes encoding other lipid A enzymes. Employing a complementation screen against aC. trachomatisgenomic library using a conditional-lethallpxHmutantEscherichia colistrain, we have identified an open reading frame (Ct461, renamedlpxG) encoding a previously uncharacterized enzyme that complements the UDP-DAGn hydrolase function inE. coliand catalyzes the conversion of UDP-DAGn to lipid Xin vitro LpxG shows little sequence similarity to either LpxH or LpxI, highlighting LpxG as the founding member of a third class of UDP-DAGn hydrolases. Overexpression of LpxG results in toxic accumulation of lipid X and profoundly reduces the infectivity ofC. trachomatis, validating LpxG as the long-sought-after UDP-DAGn pyrophosphatase in this prominent human pathogen. The complementation approach presented here overcomes the lack of suitable genetic tools forC. trachomatisand should be broadly applicable for the functional characterization of other essentialC. trachomatisgenes.IMPORTANCEChlamydia trachomatisis a leading cause of infectious blindness and sexually transmitted disease. Due to the lack of robust genetic tools, the functions of manyChlamydiagenes remain uncharacterized, including the essential gene encoding the UDP-DAGn pyrophosphatase activity for the biosynthesis of lipid A, the membrane anchor of lipooligosaccharide and the predominant lipid species of the outer leaflet of the bacterial outer membrane. We designed a complementation screen against theC. trachomatisgenomic library using a conditional-lethal mutant ofE. coliand identified the missing essential gene in the lipid A biosynthetic pathway, which we designatedlpxG We show that LpxG is a member of the calcineurin-like phosphatases and displays robust UDP-DAGn pyrophosphatase activityin vitro Overexpression of LpxG inC. trachomatisleads to the accumulation of the predicted lipid intermediate and reduces bacterial infectivity, validating thein vivofunction of LpxG and highlighting the importance of regulated lipid A biosynthesis inC. trachomatis.
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    Evolution of the sex-related locus and genomic features shared in microsporidia and fungi.
    (PLoS One, 2010-05-07) Lee, Soo Chan; Corradi, Nicolas; Doan, Sylvia; Dietrich, Fred S; Keeling, Patrick J; Heitman, Joseph
    BACKGROUND: Microsporidia are obligate intracellular, eukaryotic pathogens that infect a wide range of animals from nematodes to humans, and in some cases, protists. The preponderance of evidence as to the origin of the microsporidia reveals a close relationship with the fungi, either within the kingdom or as a sister group to it. Recent phylogenetic studies and gene order analysis suggest that microsporidia share a particularly close evolutionary relationship with the zygomycetes. METHODOLOGY/PRINCIPAL FINDINGS: Here we expanded this analysis and also examined a putative sex-locus for variability between microsporidian populations. Whole genome inspection reveals a unique syntenic gene pair (RPS9-RPL21) present in the vast majority of fungi and the microsporidians but not in other eukaryotic lineages. Two other unique gene fusions (glutamyl-prolyl tRNA synthetase and ubiquitin-ribosomal subunit S30) that are present in metazoans, choanoflagellates, and filasterean opisthokonts are unfused in the fungi and microsporidians. One locus previously found to be conserved in many microsporidian genomes is similar to the sex locus of zygomycetes in gene order and architecture. Both sex-related and sex loci harbor TPT, HMG, and RNA helicase genes forming a syntenic gene cluster. We sequenced and analyzed the sex-related locus in 11 different Encephalitozoon cuniculi isolates and the sibling species E. intestinalis (3 isolates) and E. hellem (1 isolate). There was no evidence for an idiomorphic sex-related locus in this Encephalitozoon species sample. According to sequence-based phylogenetic analyses, the TPT and RNA helicase genes flanking the HMG genes are paralogous rather than orthologous between zygomycetes and microsporidians. CONCLUSION/SIGNIFICANCE: The unique genomic hallmarks between microsporidia and fungi are independent of sequence based phylogenetic comparisons and further contribute to define the borders of the fungal kingdom and support the classification of microsporidia as unusual derived fungi. And the sex/sex-related loci appear to have been subject to frequent gene conversion and translocations in microsporidia and zygomycetes.
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    Functional evolution of mammalian odorant receptors.
    (2012) Adipietro, Kaylin Alexis
    The ability to detect small volatile molecules in the environment is mediated by the large repertoire of odorant receptors (ORs) in each species. The mammalian OR repertoire is an attractive model to study evolution because ORs have been subjected to rapid gene gains and losses between species, presumably caused by changes of the olfactory system to adapt to the environment. Despite the complicated history, clear orthologs—genes related via speciation—can still be identified even in distantly related species. Functional assessment of ORs in related species remains largely untested and sequence similarity is often used as a proxy for functional data. Here I describe the functional properties of primate and rodent ORs to determine how well evolutionary distance predicts functional characteristics. Using human and mouse ORs with previously identified ligands, we cloned 18 OR orthologs from chimpanzee and rhesus macaque and 17 mouse-rat orthologous pairs that are broadly representative of the OR repertoire. Using a heterologous expression system, we functionally characterized the responses of ORs to a wide panel of odors and found similar ligand selectivity but dramatic differences in response magnitude. 87% of human-primate orthologs and 94% of mouse-rat orthologs showed differences in receptor potency (EC50) and/or efficacy (dynamic range) to an individual ligand. Notably dN/dS ratio, an indication of selective pressure during evolution, does not predict functional similarities between orthologs. Additionally, we found that orthologs responded to a common ligand 82% of the time, while human OR paralogs of the same subfamily responded to the common ligand only 33% of the time. Our results suggest that while OR orthologs tend to show conserved ligand selectivity, their potency and/or efficacy dynamically change during evolution, even in closely related species. These functional changes in orthologs provide a platform for examining how the evolution of ORs can meet species-specific demands.
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    Generation of high curvature membranes mediated by direct endophilin bilayer interactions.
    (J Cell Biol, 2001-10-15) Farsad, K; Ringstad, N; Takei, K; Floyd, SR; Rose, K; De Camilli, P
    Endophilin 1 is a presynaptically enriched protein which binds the GTPase dynamin and the polyphosphoinositide phosphatase synptojanin. Perturbation of endophilin function in cell-free systems and in a living synapse has implicated endophilin in endocytic vesicle budding (Ringstad, N., H. Gad, P. Low, G. Di Paolo, L. Brodin, O. Shupliakov, and P. De Camilli. 1999. Neuron. 24:143-154; Schmidt, A., M. Wolde, C. Thiele, W. Fest, H. Kratzin, A.V. Podtelejnikov, W. Witke, W.B. Huttner, and H.D. Soling. 1999. Nature. 401:133-141; Gad, H., N. Ringstad, P. Low, O. Kjaerulff, J. Gustafsson, M. Wenk, G. Di Paolo, Y. Nemoto, J. Crun, M.H. Ellisman, et al. 2000. Neuron. 27:301-312). Here, we show that purified endophilin can directly bind and evaginate lipid bilayers into narrow tubules similar in diameter to the neck of a clathrin-coated bud, providing new insight into the mechanisms through which endophilin may participate in membrane deformation and vesicle budding. This property of endophilin is independent of its putative lysophosphatydic acid acyl transferase activity, is mediated by its NH2-terminal region, and requires an amino acid stretch homologous to a corresponding region in amphiphysin, a protein previously shown to have similar effects on lipid bilayers (Takei, K., V.I. Slepnev, V. Haucke, and P. De Camilli. 1999. Nat. Cell Biol. 1:33-39). Endophilin cooligomerizes with dynamin rings on lipid tubules and inhibits dynamin's GTP-dependent vesiculating activity. Endophilin B, a protein with homology to endophilin 1, partially localizes to the Golgi complex and also deforms lipid bilayers into tubules, underscoring a potential role of endophilin family members in diverse tubulovesicular membrane-trafficking events in the cell.
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    Selective expression of insulin-like growth factor II in the songbird brain.
    (J Neurosci, 1997-09-15) Holzenberger, M; Jarvis, ED; Chong, C; Grossman, M; Nottebohm, F; Scharff, C
    Neuronal replacement occurs in the forebrain of juvenile and adult songbirds. To address the molecular processes that govern this replacement, we cloned the zebra finch insulin-like growth factor II (IGF-II) cDNA, a factor known to regulate neuronal development and survival in other systems, and examined its expression pattern by in situ hybridization and immunocytochemistry in juvenile and adult songbird brains. The highest levels of IGF-II mRNA expression occurred in three nuclei of the song system: in the high vocal center (HVC), in the medial magnocellular nucleus of the neostriatum (mMAN), which projects to HVC, and to a lesser extent in the robust nucleus of the archistriatum (RA), which receives projections from HVC. IGF-II mRNA expression was developmentally regulated in zebra finches. In canary HVC, monthly changes in IGF-II mRNA expression covaried with previously reported monthly differences in neuron incorporation. Combining retrograde tracers with in situ hybridization and immunocytochemistry, we determined that the HVC neurons that project to area X synthesize the IGF-II mRNA, whereas the adjacent RA-projecting neurons accumulate the IGF-II peptide. Our findings raise the possibility that within HVC IGF-II acts as a paracrine signal between nonreplaceable area X-projecting neurons and replaceable RA-projecting neurons, a mode of action that is compatible with the involvement of IGF-II with the replacement of neurons. Additional roles for IGF-II expression in songbird brain are likely, because expression also occurs in some brain areas outside the song system, among them the cerebellar Purkinje cells in which neurogenesis is not known to occur.
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    Site-specific retinoic acid production in the brain of adult songbirds.
    (Neuron, 2000-08) Denisenko-Nehrbass, NI; Jarvis, E; Scharff, C; Nottebohm, F; Mello, CV
    The song system of songbirds, a set of brain nuclei necessary for song learning and production, has distinctive morphological and functional properties. Utilizing differential display, we searched for molecular components involved in song system regulation. We identified a cDNA (zRalDH) that encodes a class 1 aldehyde dehydrogenase. zRalDH was highly expressed in various song nuclei and synthesized retinoic acid efficiently. Brain areas expressing zRalDH generated retinoic acid. Within song nucleus HVC, only projection neurons not undergoing adult neurogenesis expressed zRalDH. Blocking zRalDH activity in the HVC of juveniles interfered with normal song development. Our results provide conclusive evidence for localized retinoic acid synthesis in an adult vertebrate brain and indicate that the retinoic acid-generating system plays a significant role in the maturation of a learned behavior.
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    Solution NMR studies of the plant peptide hormone CEP inform function.
    (FEBS letters, 2013-12) Bobay, Benjamin G; DiGennaro, Peter; Scholl, Elizabeth; Imin, Nijat; Djordjevic, Michael A; Mck Bird, David
    The C-terminally Encoded Peptide (CEP) family of regulatory peptides controls root development in vascular plants. Here, we present the first NMR structures of CEP. We show that root-knot nematode (RKN: Meloidogyne spp.) also encodes CEP, presumably to mimic plant CEP as part of their stereotypic, parasitic interaction with vascular plants. Molecular dynamics simulations of plant- and nematode-encoded CEP displaying known posttranslational modifications (PTM) provided insight into the structural effects of PTM and the conformational plasticity and rigidity of CEP. Potential mechanisms of action are discussed with respect to the structure and sampling of conformational space.
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    Sucrose Nonfermenting 1-Related Protein Kinase 1 Phosphorylates a Geminivirus Rep Protein to Impair Viral Replication and Infection.
    (Plant physiology, 2018-09) Shen, Wei; Bobay, Benjamin G; Greeley, Laura A; Reyes, Maria I; Rajabu, Cyprian A; Blackburn, R Kevin; Dallas, Mary Beth; Goshe, Michael B; Ascencio-Ibáñez, Jose T; Hanley-Bowdoin, Linda
    Geminiviruses are single-stranded DNA viruses that infect a wide variety of plants and cause severe crop losses worldwide. The geminivirus replication initiator protein (Rep) binds to the viral replication origin and catalyzes DNA cleavage and ligation to initiate rolling circle replication. In this study, we found that the Tomato golden mosaic virus (TGMV) Rep is phosphorylated at serine-97 by sucrose nonfermenting 1-related protein kinase 1 (SnRK1), a master regulator of plant energy homeostasis and metabolism. Phosphorylation of Rep or the phosphomimic S97D mutation impaired Rep binding to viral DNA. A TGMV DNA-A replicon containing the Rep S97D mutation replicated less efficiently in tobacco (Nicotiana tabacum) protoplasts than in wild-type or Rep phosphorylation-deficient replicons. The TGMV Rep-S97D mutant also was less infectious than the wild-type virus in Nicotiana benthamiana and was unable to infect tomato (Solanum lycopersicum). Nearly all geminivirus Rep proteins have a serine residue at the position equivalent to TGMV Rep serine-97. SnRK1 phosphorylated the equivalent serines in the Rep proteins of Tomato mottle virus and Tomato yellow leaf curl virus and reduced DNA binding, suggesting that SnRK1 plays a key role in combating geminivirus infection. These results established that SnRK1 phosphorylates Rep and interferes with geminivirus replication and infection, underscoring the emerging role for SnRK1 in the host defense response against plant pathogens.
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    The 70-kDa heat shock cognate protein (Hsc73) gene is enhanced by ovarian hormones in the ventromedial hypothalamus.
    (Proc Natl Acad Sci U S A, 1999-02-16) Krebs, CJ; Jarvis, ED; Pfaff, DW
    Estrogen (E) and progesterone (P) orchestrate many cellular responses involved in female reproductive physiology, including reproductive behaviors. E- and P-binding neurons important for lordosis behavior have been located within the ventromedial hypothalamus (VMH), and several hormone-responsive genes have been observed there as well. In attempts to identify additional E- and P-responsive genes in the VMH that may contribute to sexual behaviors, we used the differential display mRNA screening technique. One of the genes identified encodes the 73-kDa heat shock cognate protein (Hsc73). Quantitative in situ hybridization analysis of brains from naturally cycling female rats revealed a significant increase in Hsc73 mRNA in the VMH and arcuate nucleus of animals during proestrus compared with those at diestrus-1. To confirm that these increases were steroid hormone dependent, we compared vehicle-treated ovariectomized females with ovariectomized females treated with estradiol benzoate and P. Northern analysis and in situ hybridizations showed that the Hsc73 gene is enhanced by E and P in the pituitary and subregions of the VMH. Incidentally, by examining the primary amino acid sequence of rat, human, and chicken progesterone receptors, we noticed that putative Hsc73 binding sites are conserved across species with similar sites existing in the androgen and glucocorticoid receptors. Together these findings suggest a possible mechanism through which E could influence the activities of progesterone, androgen, and glucocorticoid receptors, by enhancing the expression of Hsc73 in cells where these proteins colocalize.