RESUMO
Podocytes are highly differentiated and polarized epithelial cells located on the visceral side of the glomerulus. They form an indispensable component of the glomerular filter, the slit diaphragm, formed by several transmembrane proteins and adaptor molecules. Disruption of the slit diaphragm can lead to massive proteinuria and nephrotic syndrome in mice and humans. CD2AP is an adaptor protein that is important for the maintenance of the slit diaphragm. Together with its paralogue, CIN85, CD2AP belongs to a family of adaptor proteins that are primarily described as being involved in endocytosis and downregulation of receptor tyrosine kinase activity. We have shown that full-length CIN85 is upregulated in podocytes in the absence of CD2AP, whereas in wild-type cells, full-length CIN85 is not detectable. In this study, we show that full-length CIN85 is postranslationally modified by SUMOylation in wild-type podocytes. We can demonstrate that CIN85 is SUMOylated by SUMO-1, -2, and -3 and that SUMOylation is enhanced in the presence of CD2AP. Conversion of lysine 598 to arginine completely abolishes SUMOylation and leads to increased binding of CIN85 to nephrin. Our results indicate a novel role for CD2AP in regulating posttranslational modification of CIN85.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Podócitos/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Sumoilação , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Linhagem Celular , Proteínas do Citoesqueleto/genética , Deleção de Genes , Regulação da Expressão Gênica , Humanos , Camundongos , Proteínas de Neoplasias/genética , Proteínas do Tecido Nervoso/genética , FilogeniaRESUMO
Protein-protein interactions are essential for almost all cellular processes, hence understanding these processes mainly depends on the identification and characterization of the relevant protein-protein interactions. In the present paper, we introduce the concept of TRS (trans-SUMOylation), a new method developed to identify and verify protein-protein interactions in mammalian cells in vivo. TRS utilizes Ubc9-fusion proteins that trans-SUMOylate co-expressed interacting proteins. Using TRS, we analysed interactions of 65 protein pairs co-expressed in HEK (human embryonic kidney)-293 cells. We identified seven new and confirmed 16 known protein interactions, which were determined via endogenous SUMOylation sites of the binding partners or by using SUMOylation-site tags respectively. Four of the new protein interactions were confirmed by GST (glutathione transferase) pull-down and the p38α-Edr2 interaction was verified by co-localization analysis. Functionally, this p38α-Edr2 interaction could possibly be involved in the recruitment of p38α to the polycomb chromatin-remodelling complex to phosphorylate Bmi1. We also used TRS to characterize protein-interaction domains of the protein kinase pairs p38α-MK2 [MK is MAPK (mitogen-activated protein kinase)-activated protein kinase] and ERK3 (extracellular-signal-regulated kinase 3)-MK5 and of the p38α-p53 complex. The ability of TRS to monitor protein interactions in mammalian cells in vivo at levels similar to endogenous expression makes it an excellent new tool that can help in defining the protein interactome of mammalian cells.
Assuntos
Mapeamento de Interação de Proteínas/métodos , Sumoilação , Animais , Sítios de Ligação , Cromatina/metabolismo , Humanos , Mamíferos/genética , Mamíferos/metabolismo , Proteína Quinase 6 Ativada por Mitógeno/genética , Proteína Quinase 6 Ativada por Mitógeno/metabolismo , Fosforilação , Transdução de Sinais , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
BACKGROUND: Smooth pursuit eye movement dysfunctions are considered a biological indicator for vulnerability to schizophrenia. This study examines test-retest stability of specific eye movement variables such as velocity gain and different saccadic categories. METHODS: Smooth pursuit eye movements of 27 schizophrenic patients and 30 patients with major depression were examined three times during clinical treatment using high-resolution infrared oculography. Forty-one normal controls were retested after four weeks. RESULTS: Intraclass correlation coefficients as a measure for retest-stability were highly significant in each group for all time-points, except for anticipatory saccades in schizophrenics. No significant correlations were found between psychopathological status, neuroleptic medication and eye movement variables. CONCLUSIONS: Our results indicate that the most important measures of eye tracking performance in psychiatric patients are not significantly influenced by neuroleptic medication or clinical state and are stable across time.