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1.
Am J Pathol ; 179(5): 2254-65, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21945805

RESUMO

Podocalyxin (PC) is a polysialylated, anti-adhesin that is essential for maintaining foot process architecture and the integrity of the glomerular filtration barrier. We showed previously that PC is firmly attached to the actin cytoskeleton through ezrin, that in puromycin aminonucleoside (PAN)-mediated nephrosis the PC-ezrin-actin complex is disrupted, and that PC is uncoupled from actin. However, the precise mechanisms involved remained unknown. Here we show that detachment of PC from actin is regulated by phosphorylation of PC. PC is hyperphosphorylated at serines in PAN- and protamine sulfate (PS)-treated rat glomeruli. We determined that PC is a substrate of PKC and that the site of phosphorylation is Ser415, located within the juxtamembrane, ezrin-binding domain of the cytoplasmic tail of PC. Mutation of Ser415 to the phosphomimetic residues Glu (S415E) or Asp (S415D) interfered with direct binding of the PC cytoplasmic tail to ezrin in vitro. Moreover, stable expression of a phosphomimetic (S415E) PC mutant but not the WT or the phosphorylation-deficient (S415A) PC mutant, disrupted PC-ezrin-actin interaction, failed to activate RhoA, and the cytoskeletal linker, ezrin, remained inactive. Our data indicate that phosphorylation of PC at Ser415 prevents attachment of PC and ezrin to actin and highlights the strategic position of Ser415 and direct binding of PC to ezrin in regulating podocyte foot process architecture.


Assuntos
Citoesqueleto de Actina/metabolismo , Proteínas do Citoesqueleto/antagonistas & inibidores , Sialoglicoproteínas/metabolismo , Proteína rhoA de Ligação ao GTP/antagonistas & inibidores , Animais , Células Cultivadas , Glomérulos Renais/metabolismo , Masculino , Microscopia Confocal , Microscopia de Fluorescência , Nefrose/metabolismo , Fosforilação/fisiologia , Ligação Proteica , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Transfecção
2.
Neurobiol Dis ; 38(3): 386-94, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20206263

RESUMO

The presymptomatic phase of Parkinson's disease (PD) is now recognized as a prodromal phase, with compensatory mechanism masking its progression and non-motor early manifestations, such as depression, cognitive disturbances and apathy. Those mechanisms were thought to be strictly dopamine-mediated until recent advances have shed light upon involvement of putative outside-basal ganglia, i.e. cortical, structures. We took advantage of our progressive 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated macaque model to monitor whole genome transcriptional changes in several brain areas. Our data reveals that transcriptomic activity changes take place from early stages, suggesting very early compensatory mechanisms or pathological activity outside the basal ganglia, including the PFC. Specific transcriptomic changes occurring in the PFC of fully parkinsonian MPTP-treated macaques have been identified. Interestingly, a large part of these transcriptomic changes were also observed in human post-mortem samples of patients with neurodegenerative diseases analysed by quantitative PCR. These results suggest that the PFC is able to detect the progression of dopamine denervation even at very early time points. There are therefore mechanisms, within the PFC, leading to compensatory alterations and/or participating to pathophysiology of prodromal PD manifestations.


Assuntos
Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Transtornos Parkinsonianos/genética , Transtornos Parkinsonianos/metabolismo , Córtex Pré-Frontal/metabolismo , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Animais , Modelos Animais de Doenças , Feminino , Globo Pálido/metabolismo , Humanos , Macaca fascicularis , Masculino , Pessoa de Meia-Idade , Modelos Neurológicos , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , Putamen/metabolismo , RNA Mensageiro/metabolismo , Especificidade da Espécie , Tálamo/metabolismo
3.
BMC Evol Biol ; 8: 330, 2008 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-19068116

RESUMO

BACKGROUND: Brain-expressed genes that were created in primate lineage represent obvious candidates to investigate molecular mechanisms that contributed to neural reorganization and emergence of new behavioural functions in Homo sapiens. PMCHL1 arose from retroposition of a pro-melanin-concentrating hormone (PMCH) antisense mRNA on the ancestral human chromosome 5p14 when platyrrhines and catarrhines diverged. Mutations before divergence of hylobatidae led to creation of new exons and finally PMCHL1 duplicated in an ancestor of hominids to generate PMCHL2 at the human chromosome 5q13. A complex pattern of spliced and unspliced PMCHL RNAs were found in human brain and testis. RESULTS: Several novel spliced PMCHL transcripts have been characterized in human testis and fetal brain, identifying an additional exon and novel splice sites. Sequencing of PMCHL genes in several non-human primates allowed to carry out phylogenetic analyses revealing that the initial retroposition event took place within an intron of the brain cadherin (CDH12) gene, soon after platyrrhine/catarrhine divergence, i.e. 30-35 Mya, and was concomitant with the insertion of an AluSg element. Sequence analysis of the spliced PMCHL transcripts identified only short ORFs of less than 300 bp, with low (VMCH-p8 and protein variants) or no evolutionary conservation. Western blot analyses of human and macaque tissues expressing PMCHL RNA failed to reveal any protein corresponding to VMCH-p8 and protein variants encoded by spliced transcripts. CONCLUSION: Our present results improve our knowledge of the gene structure and the evolutionary history of the primate-specific chimeric PMCHL genes. These genes produce multiple spliced transcripts, bearing short, non-conserved and apparently non-translated ORFs that may function as mRNA-like non-coding RNAs.


Assuntos
Hormônios Hipotalâmicos/genética , Precursores de Proteínas/genética , Splicing de RNA , RNA não Traduzido/genética , Adulto , Sequência de Aminoácidos , Animais , Encéfalo/metabolismo , Cromossomos Humanos Par 5/genética , Evolução Molecular , Éxons , Humanos , Íntrons , Macaca/genética , Masculino , Dados de Sequência Molecular , Filogenia , Primatas , RNA não Traduzido/metabolismo , Alinhamento de Sequência , Testículo/metabolismo
4.
Sci Adv ; 1(10): e1501150, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26702449

RESUMO

Relics of ancient infections are abundant in eukaryote genomes, but little is known about how they evolve when they confer a functional benefit on their host. We show here, for the first time, that the virus-like particles shown to protect Venturia canescens eggs against host immunity are derived from a nudivirus genome incorporated by the parasitic wasp into its own genetic material. Nudivirus hijacking was also at the origin of protective particles from braconid wasps. However, we show here that the viral genes produce "liposomes" that wrap and deliver V. canescens virulence proteins, whereas the particles are used as gene transfer agents in braconid wasps. Our findings indicate that virus domestication has occurred repeatedly during parasitic wasp evolution but with different evolutionary trajectories after endogenization, resulting in different virulence molecule delivery strategies.

5.
Nat Commun ; 3: 895, 2012 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-22692538

RESUMO

In several Hymenoptera, sexual fate is determined by the allelic composition at the complementary sex-determiner locus, a sex-determination mechanism that can strongly affect population dynamics. To date, the molecular identification of complementary sex determiner has only been achieved in the honeybee, where the complementary sex-determiner gene was reported to have arisen from duplication of the feminizer gene. Strikingly, the complementary sex-determiner gene was also proposed to be unique to the honeybee lineage. Here we identify feminizer and complementary sex-determiner orthologues in bumble bees and ants. We further demonstrate that the duplication of feminizer that produced complementary sex determiner occurred before the divergence of Aculeata species (~120 Myr ago). Finally, we provide evidence that the two genes evolved concertedly through gene conversion, complementary sex-determiner evolution being additionally shaped by mosaic patterns of selection. Thus, the complementary sex-determiner gene likely represents the molecular basis for single locus-complementary sex determination in the Aculeata infra-order, and possibly, in the entire Hymenoptera order.


Assuntos
Formigas/crescimento & desenvolvimento , Abelhas/crescimento & desenvolvimento , Processos de Determinação Sexual/fisiologia , Animais , Formigas/genética , Formigas/metabolismo , Abelhas/genética , Abelhas/metabolismo , Evolução Molecular , Feminino , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Masculino , Processos de Determinação Sexual/genética
6.
J Cell Physiol ; 210(2): 479-88, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17111367

RESUMO

Mutations in the gene encoding ClC-5 lead to X-linked hypercalciuric nephrolithiasis (XLHN), characterized by proteinuria, hypercalciuria, and phosphaturia. In renal proximal tubule cells, ClC-5 was identified as an important player in endocytosis, which ensures reabsorption of filtered protein. However, the recent finding that ClC-5 is a Cl(-)/H(+) antiporter and not a Cl(-) channel as long thought points to the lack of understanding of its functional role. Also, little biochemical data are available about ClC-5 and its post-translational modifications have not been investigated. Here, we examined the role of N-glycosylation of xClC-5 in the Xenopus oocyte expression system by comparing wild-type (WT) xClC-5 and N-glycosylation site mutants. We found that xClC-5 is N-glycosylated on asparagines 169 and 470, which are the only N-glycosylated sites. xClC-5 mutants have an increased susceptibility to polyubiquitination and proteasomal degradation; however, without a notable impact on the expression level. Using a cross-linking reagent, we showed that xClC-5 assembles into protein complexes, independent of its N-glycosylation. Voltage-clamp measurements showed a reduced conductance in the presence of tunicamycin and with xClC-5 N-glycosylation site mutants. Using immunocytochemistry, we localized xClC-5 mainly in intracellular compartments, and found that its cell surface pool is reduced in the absence of N-glycans. We further examined the plasma membrane retrieval of WT and mutant xClC-5 in the presence of Brefeldin A (BFA), and found that the non-glycosylated mutant was retrieved more than five times faster than the WT protein. We conclude that N-glycosylation enhances cell surface expression of xClC-5, increasing its plasma membrane transport activity.


Assuntos
Membrana Celular/metabolismo , Canais de Cloreto/metabolismo , Proteínas de Xenopus/metabolismo , Animais , Asparagina/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/genética , Canais de Cloreto/efeitos dos fármacos , Canais de Cloreto/genética , Cloretos/metabolismo , Feminino , Glicosilação/efeitos dos fármacos , Líquido Intracelular/efeitos dos fármacos , Líquido Intracelular/metabolismo , Substâncias Macromoleculares/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Mutação/genética , Oócitos/metabolismo , Técnicas de Patch-Clamp , Polissacarídeos/metabolismo , Polissacarídeos/farmacologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/genética , Fatores de Tempo , Proteínas de Xenopus/efeitos dos fármacos , Proteínas de Xenopus/genética , Xenopus laevis
7.
J Am Soc Nephrol ; 15(9): 2289-98, 2004 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-15339978

RESUMO

Podocalyxin (PC) is the major sialoglycoprotein expressed on the apical membrane of the podocyte. Previously it was shown that PC is connected to actin through the PC/NHERF2/ezrin complex, and this connection is disrupted in the nephrotic syndrome. For assessing whether expression of PC affects the organization of the actin cytoskeleton, MDCK cell lines stably expressing either full-length PC or a PC mutant lacking the NHERF binding site was established. It was found that full-length PC but not the PC mutant is connected to actin, induces redistribution of actin toward the apical membrane, and leads to increased RhoA activity. By immunofluorescence redistribution of RhoA and RhoGDI was observed in the presence of both full-length PC and the PC mutant. With the use of pulldown assays, PC and ezrin were found to interact directly and the ezrin binding site was mapped to the juxtamembrane region of PC's cytoplasmic tail. It is concluded that PC binds to ezrin both directly and indirectly. PC activates RhoA through NHERF and ezrin, leading to redistribution of actin filaments. These results suggest that in podocytes, PC may also regulate foot process architecture through RhoA.


Assuntos
Actinas/efeitos dos fármacos , Fosfoproteínas/fisiologia , Sialoglicoproteínas/farmacologia , Proteína rhoA de Ligação ao GTP/efeitos dos fármacos , Actinas/fisiologia , Animais , Linhagem Celular , Células Cultivadas , Proteínas do Citoesqueleto , Cães , Trocadores de Sódio-Hidrogênio , Proteína rhoA de Ligação ao GTP/fisiologia
8.
J Am Soc Nephrol ; 12(8): 1589-1598, 2001 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-11461930

RESUMO

During development, renal glomerular epithelial cells (podocytes) undergo extensive morphologic changes necessary for creation of the glomerular filtration apparatus. These changes include formation of interdigitating foot processes, replacement of tight junctions with slit diaphragms, and the concomitant opening of intercellular urinary spaces. It was postulated previously and confirmed recently that podocalyxin, a sialomucin, plays a major role in maintaining the urinary space open by virtue of the physicochemical properties of its highly negatively charged ectodomain. This study examined whether the highly conserved cytoplasmic tail of podocalyxin also contributes to the unique organization of podocytes by interacting with the cytoskeletal network found in their cell bodies and foot processes. By immunocytochemistry, it was shown that podocalyxin and the actin binding protein ezrin are co-expressed in podocytes and co-localize along the apical plasma membrane, where they form a co-immunoprecipitable complex. Selective detergent extraction followed by differential centrifugation revealed that some of the podocalyxin cosediments with actin filaments. Moreover, its sedimentation is dependent on polymerized actin and is mediated by complex formation with ezrin. Once formed, podocalyxin/ezrin complexes are very stable, because they are insensitive to actin depolymerization or inactivation of Rho kinase, which is known to be necessary for regulation of ezrin and to mediate Rho-dependent actin organization. These data indicate that in podocytes, podocalyxin is complexed with ezrin, which mediates its link to the actin cytoskeleton. Thus, in addition to its ectodomain, the cytoplasmic tail of podocalyxin also likely contributes to maintaining the unique podocyte morphology.


Assuntos
Actinas/fisiologia , Citoesqueleto/fisiologia , Glomérulos Renais/metabolismo , Fosfoproteínas/fisiologia , Sialoglicoproteínas/fisiologia , Animais , Células CHO , Linhagem Celular , Cricetinae , Proteínas do Citoesqueleto , Cães , Células Epiteliais/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Glomérulos Renais/citologia , Neuraminidase/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Ratos , Ratos Sprague-Dawley , Distribuição Tecidual , Quinases Associadas a rho
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