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1.
Nat Cell Biol ; 8(2): 124-36, 2006 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-16415858

RESUMEN

The recruitment of the small GTPase Arf6 and ARNO from cytosol to endosomal membranes is driven by V-ATPase-dependent intra-endosomal acidification. The molecular mechanism that mediates this pH-sensitive recruitment and its role are unknown. Here, we demonstrate that Arf6 interacts with the c-subunit, and ARNO with the a2-isoform of V-ATPase. The a2-isoform is targeted to early endosomes, interacts with ARNO in an intra-endosomal acidification-dependent manner, and disruption of this interaction results in reversible inhibition of endocytosis. Inhibition of endosomal acidification abrogates protein trafficking between early and late endosomal compartments. These data demonstrate the crucial role of early endosomal acidification and V-ATPase/ARNO/Arf6 interactions in the regulation of the endocytic degradative pathway. They also indicate that V-ATPase could modulate membrane trafficking by recruiting and interacting with ARNO and Arf6; characteristics that are consistent with the role of V-ATPase as an essential component of the endosomal pH-sensing machinery.


Asunto(s)
Factores de Ribosilacion-ADP/metabolismo , Endosomas/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Proteínas/metabolismo , ATPasas de Translocación de Protón Vacuolares/metabolismo , Factor 6 de Ribosilación del ADP , Cloruro de Amonio/farmacología , Animales , Carbonil Cianuro p-Trifluorometoxifenil Hidrazona/farmacología , Línea Celular , Dinaminas/genética , Dinaminas/metabolismo , Endocitosis/efectos de los fármacos , Endocitosis/fisiología , Endosomas/efectos de los fármacos , Células Epiteliales/metabolismo , Proteínas Activadoras de GTPasa/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Concentración de Iones de Hidrógeno/efectos de los fármacos , Isoenzimas/genética , Isoenzimas/metabolismo , Túbulos Renales Proximales/citología , Túbulos Renales Proximales/metabolismo , Macrólidos/farmacología , Ratones , Modelos Biológicos , Mutación/genética , Unión Proteica , Mapeo de Interacción de Proteínas , Transporte de Proteínas/fisiología , Albúmina Sérica Bovina/metabolismo , Transfección , ATPasas de Translocación de Protón Vacuolares/antagonistas & inhibidores , ATPasas de Translocación de Protón Vacuolares/genética
2.
Biochim Biophys Acta ; 1772(5): 570-9, 2007 May.
Artículo en Inglés | MEDLINE | ID: mdl-17382524

RESUMEN

The sarcoglycan complex in muscle consists of alpha-, beta-, gamma- and delta-sarcoglycan and is part of the larger dystrophin-glycoprotein complex (DGC), which is essential for maintaining muscle membrane integrity. Mutations in any of the four sarcoglycans cause limb-girdle muscular dystrophies (LGMD). In this report, we have identified a novel interaction between delta-sarcoglycan and the 16 kDa subunit c (16K) of vacuolar H(+)-ATPase. Co-expression studies in heterologous cell system revealed that 16K interacts specifically with delta-sarcoglycan and the highly related gamma-sarcoglycan through the transmembrane domains. In cultured C2C12 myotubes, 16K forms a complex with sarcoglycans at the plasma membrane. Loss of sarcoglycans in the sarcoglycan-deficient BIO14.6 hamster destabilizes the DGC and alters the localization of 16K at the sarcolemma. In addition, the steady state level of beta(1)-integrin is increased. Recent studies have shown that 16K also interacts directly with beta(1)-integrin and our data demonstrated that sarcoglycans, 16K and beta(1)-integrin were immunoprecipitated together in C2C12 myotubes. Since sarcoglycans have been proposed to participate in bi-directional signaling with integrins, our findings suggest that 16K might mediate the communication between sarcoglycans and integrins and play an important role in the pathogenesis of muscular dystrophy.


Asunto(s)
Sarcoglicanos/metabolismo , ATPasas de Translocación de Protón Vacuolares/metabolismo , Animales , Línea Celular , Membrana Celular/metabolismo , Chlorocebus aethiops , Cricetinae , Integrina beta1/metabolismo , Masculino , Ratones , Microscopía Inmunoelectrónica , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/ultraestructura , Músculo Esquelético/metabolismo , Unión Proteica , Subunidades de Proteína/metabolismo , Sarcolema/metabolismo , Sarcolema/ultraestructura , Transducción de Señal
3.
Cell Microbiol ; 8(6): 939-60, 2006 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-16681836

RESUMEN

After internalization into macrophages non-pathogenic mycobacteria are killed within phagosomes. Pathogenic mycobacteria can block phagosome maturation and grow inside phagosomes but under some conditions can also be killed by macrophages. Killing mechanisms are poorly understood, although phago-lysosome fusion and nitric oxide (NO) production are implicated. We initiated a systematic analysis addressing how macrophages kill 'non-pathogenic'Mycobacterium smegmatis. This system was dynamic, involving periods of initial killing, then bacterial multiplication, followed by two additional killing stages. NO synthesis represented the earliest killing factor but its synthesis stopped during the first killing period. Phagosome actin assembly and fusion with late endocytic organelles coincided with the first and last killing phase, while recycling of phagosome content and membrane coincided with bacterial growth. Phagosome acidification and acquisition of the vacuolar (V) ATPase followed a different pattern coincident with later killing phases. Moreover, V-ATPase localized to vesicles distinct from classical late endosomes and lysosomes. Map kinase p38 is a crucial regulator of all processes investigated, except NO synthesis, that facilitated the host for some functions while being usurped by live bacteria for others. A mathematical model argues that periodic high and low cellular killing activity is more effective than is a continuous process.


Asunto(s)
Macrófagos/microbiología , Macrófagos/fisiología , Viabilidad Microbiana , Mycobacterium smegmatis/fisiología , Actinas/análisis , Actinas/metabolismo , Animales , Muerte Celular , Línea Celular , Supervivencia Celular , Endosomas/fisiología , Inhibidores Enzimáticos/farmacología , Concentración de Iones de Hidrógeno , Lisosomas/enzimología , Lisosomas/fisiología , Macrólidos/farmacología , Macrófagos/citología , Ratones , Modelos Teóricos , Mycobacterium smegmatis/efectos de los fármacos , Mycobacterium smegmatis/patogenicidad , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo II/fisiología , Orgánulos/fisiología , Fagosomas/química , Fagosomas/fisiología , Proteínas Quinasas p38 Activadas por Mitógenos/fisiología
4.
Virology ; 313(2): 415-26, 2003 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-12954209

RESUMEN

The high risk forms of human papillomavirus (HPV) (primarily types 16 and 18) are the leading cause of cervical cancer worldwide. Infection results in expression of three oncoproteins, E5, E6, and E7, the latter two being of predominant importance in maintaining a transformed state of the host epithelial cell. While little is known about the role(s) of the HPV E5, the bovine papillomavirus type 1 (BPV1) E5 protein has been well characterized. A study of HPV16 E5 was performed, focusing on the protein's ability to self-interact, its ability to bind to the 16-kDa subunit of the vacuolar H(+)-ATPase (16K), and its cellular localization. As has been previously shown for BPV1 E5, we found that HPV16 E5 is also capable of self-interaction and binding to 16K. Further, we examined which portions of the HPV16 E5 protein were involved in these interactions using progressive deletions of putative transmembrane helices of the protein. All of the E5 deletion mutants tested bound to full-length E5 as well as to 16K, suggesting that these protein-protein interactions are based on hydrophobic interactions. The majority of E5 expressed in HEK 293-T7 cells was perinuclear but did not appear to localize to the cis/medial-Golgi, in contrast to previous reports for both HPV16 E5 and BPV1 E5.


Asunto(s)
Proteínas Oncogénicas Virales/metabolismo , Papillomaviridae/metabolismo , Línea Celular , Citoplasma/metabolismo , Dimerización , Técnica del Anticuerpo Fluorescente , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Mutación , Proteínas Oncogénicas Virales/biosíntesis , Proteínas Oncogénicas Virales/genética , Unión Proteica , Transfección , ATPasas de Translocación de Protón Vacuolares/química , ATPasas de Translocación de Protón Vacuolares/genética , ATPasas de Translocación de Protón Vacuolares/metabolismo
5.
Protein Expr Purif ; 30(1): 1-10, 2003 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-12821315

RESUMEN

The E5 proteins of human papillomavirus (HPV) are highly hydrophobic transmembrane proteins that display weak transforming activity. The HPV E5 proteins are localized largely to intracellular membranes, such as the Golgi apparatus and endoplasmic reticulum, but also appear in the plasma membrane. Infection with HPV16 is the cause of over 90% of human cervical cancers. HPV E5 is known to interact with growth factor receptors and gap junction proteins and is believed to play a role during the initiation of neoplasia. The structure of HPV E5 and the mechanism of its interactions with growth factor receptors remain largely unknown. In the present studies, the E5 protein of HPV16 was cloned into the pBAD/TOPO vector fused to an N-terminal thioredoxin leader and a C-terminal His-tag, and expressed in Escherichia coli. The identity of the protein was confirmed by immunoblotting using antibodies against a V5-epitope tag engineered into the protein. Due to formation of high molecular mass superaggregates of the protein, two chromatography steps were employed for its purification: (1) gel filtration chromatography to separate the superaggregated protein from other soluble proteins and (2) Ni-chelate affinity chromatography in the presence of detergent. The superaggregates of the E5-fusion protein were broken down to monomers and various oligomers by sonication in the presence of 0.2% SDS. The purified E5-fusion protein was then reconstituted into lipid vesicles and initial structural analysis of the protein was performed using circular dichroism spectroscopy.


Asunto(s)
Proteínas Oncogénicas Virales/química , Proteínas Oncogénicas Virales/aislamiento & purificación , Papillomaviridae/química , Cromatografía de Afinidad , Dicroismo Circular , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Liposomas/metabolismo , Proteínas Oncogénicas Virales/biosíntesis , Proteínas Oncogénicas Virales/genética , Estructura Secundaria de Proteína , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo
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