Добірка наукової літератури з теми "Complexe adapteur AP-1"

Clathrin-associated adaptor protein (AP) complexes are major structural components of clathrin-coated vesicles, functioning in clathrin coat assembly and cargo selection. We have carried out a systematic biochemical and genetic characterization of AP complexes inSaccharomyces cerevisiae. Using coimmunoprecipitation, the subunit composition of two complexes, AP-1 and AP-2R, has been defined. These results allow assignment of the 13 potential AP subunits encoded in the yeast genome to three AP complexes. As assessed by in vitro binding assays and coimmunoprecipitation, only AP-1 interacts with clathrin. Individual or combined disruption of AP-1 subunit genes in cells expressing a temperature-sensitive clathrin heavy chain results in accentuated growth and α-factor pheromone maturation defects, providing further evidence that AP-1 is a clathrin adaptor complex. However, in cells expressing wild-type clathrin, the same AP subunit deletions have no effect on growth or α-factor maturation. Furthermore, gel filtration chromatography revealed normal elution patterns of clathrin-coated vesicles in cells lacking AP-1. Similarly, combined deletion of genes encoding the β subunits of the three AP complexes did not produce defects in clathrin-dependent sorting in the endocytic and vacuolar pathways or alterations in gel filtration profiles of clathrin-coated vesicles. We conclude that AP complexes are dispensable for clathrin function in S. cerevisiae under normal conditions. Our results suggest that alternative factors assume key roles in stimulating clathrin coat assembly and cargo selection during clathrin-mediated vesicle formation in yeast.

Mutational analyses have revealed many genes that are required for proper biogenesis of lysosomes and lysosome-related organelles. The proteins encoded by these genes assemble into five distinct complexes (AP-3, BLOC-1-3, and HOPS) that either sort membrane proteins or interact with SNAREs. Several of these seemingly distinct complexes cause similar phenotypic defects when they are rendered defective by mutation, but the underlying cellular mechanism is not understood. Here, we show that the BLOC-1 complex resides on microvesicles that also contain AP-3 subunits and membrane proteins that are known AP-3 cargoes. Mouse mutants that cause BLOC-1 or AP-3 deficiencies affected the targeting of LAMP1, phosphatidylinositol-4-kinase type II alpha, and VAMP7-TI. VAMP7-TI is an R-SNARE involved in vesicle fusion with late endosomes/lysosomes, and its cellular levels were selectively decreased in cells that were either AP-3- or BLOC-1–deficient. Furthermore, BLOC-1 deficiency selectively altered the subcellular distribution of VAMP7-TI cognate SNAREs. These results indicate that the BLOC-1 and AP-3 protein complexes affect the targeting of SNARE and non-SNARE AP-3 cargoes and suggest a function of the BLOC-1 complex in membrane protein sorting.

Most epithelial cells contain two AP-1 clathrin adaptor complexes. AP-1A is ubiquitously expressed and involved in transport between the TGN and endosomes. AP-1B is expressed only in epithelia and mediates the polarized targeting of membrane proteins to the basolateral surface. Both AP-1 complexes are heterotetramers and differ only in their 50-kD μ1A or μ1B subunits. Here, we show that AP-1A and AP-1B, together with their respective cargoes, define physically and functionally distinct membrane domains in the perinuclear region. Expression of AP-1B (but not AP-1A) enhanced the recruitment of at least two subunits of the exocyst complex (Sec8 and Exo70) required for basolateral transport. By immunofluorescence and cell fractionation, the exocyst subunits were found to selectively associate with AP-1B–containing membranes that were both distinct from AP-1A–positive TGN elements and more closely apposed to transferrin receptor–positive recycling endosomes. Thus, despite the similarity of the two AP-1 complexes, AP-1A and AP-1B exhibit great specificity for endosomal transport versus cell polarity.

Activation of the epidermal growth factor receptor (EGFR) by EGF results in binding of clathrin adaptor protein complex AP-2 to the receptor cytoplasmic tail. The transient interaction with AP-2 is thought to be responsible for the selective recruitment of the EGFR into coated pits during endocytosis. In this study we found that EGF-induced EGFR/AP-2 association, measured by co-immunoprecipitation, persists after receptor internalization. Double-label immunofluorescence of EGF-treated A-431 and COS-1 cells revealed the presence of AP-2, clathrin and eps15, another component of the plasma membrane coated pits, in the large perinuclear endosomes loaded with EGFRs. By optical sectioning and image deconvolution, the immunoreactivities were seen to be distributed within vesicular and tubular elements of these endosomes. In addition, these compartments contained the transferrin receptors and a EEA.1 protein, markers of early endosomes. Furthermore, Golgi clathrin adaptor complex AP-1 was found in EGFR-containing endosomes and EGFR immunoprecipitates in A-431 cells. The direct interaction of the EGFR with micro1 as well as micro2 subunits of AP-1 and AP-2, correspondingly, was shown using the yeast two-hybrid assay. Brefeldin A, a drug that releases AP-1 from the trans-Golgi membranes, had no effect on AP-1 association with endosomes and its co-precipitation with EGFR. Taken together, the data suggest that endosomal EGFR-AP complexes make up a significant portion of the total amount of these complexes detectable by co-immunoprecipitation. It can be proposed that APs are capable of binding to the endosomal membrane via a mechanism that requires AP interaction with the intracellular tails of multimeric receptors like activated EGFR, which in turn allows recruitment of clathrin and eps15. The hypothesis that the competition between adaptor complexes for binding to the receptor tails in endosomes may regulate of the sorting of receptors is discussed.

Expression of the epithelial cell–specific heterotetrameric adaptor complex AP-1B is required for the polarized distribution of many membrane proteins to the basolateral surface of LLC-PK1 kidney cells. AP-1B is distinguished from the ubiquitously expressed AP-1A by exchange of its single 50-kD μ subunit, μ1A, being replaced by the closely related μ1B. Here we show that this substitution is sufficient to couple basolateral plasma membrane proteins, such as a low-density lipoprotein receptor (LDLR), to the AP-1B complex and to clathrin. The interaction between LDLR and AP-1B is likely to occur in the trans-Golgi network (TGN), as was suggested by the localization of functional, epitope-tagged μ1 by immunofluorescence and immunoelectron microscopy. Tagged AP-1A and AP-1B complexes were found in the perinuclear region close to the Golgi complex and recycling endosomes, often in clathrin-coated buds and vesicles. Yet, AP-1A and AP-1B localized to different subdomains of the TGN, with only AP-1A colocalizing with furin, a membrane protein that uses AP-1 to recycle between the TGN and endosomes. We conclude that AP-1B functions by interacting with its cargo molecules and clathrin in the TGN, where it acts to sort basolateral proteins from proteins destined for the apical surface and from those selected by AP-1A for transport to endosomes and lysosomes.

The four members of the AP (adaptor protein) family are heterotetrameric cytosolic complexes that are involved in the intracellular trafficking of cargo proteins between different organelles. They interact with motifs present in the cytoplasmic tails of their specific cargo proteins at different intracellular locations. While AP-1, AP-2 and AP-3 have been investigated extensively, very few studies have focused on the fourth member, AP-4. In the present study, we report on the intracellular localization of AP-4 in the MDCK (Madin–Darby canine kidney) and MelJuSo cell lines after immunogold labelling of ultrathin cryosections. We find that AP-4 is localized mainly in the Golgi complex, as well as on endosomes and transport vesicles. Interestingly, we show for the first time that AP-4 is localized with the clathrin coat machinery in the Golgi complex and in the endocytic pathway. Furthermore, we find that AP-4 is localized with the CI-MPR (cation-independent mannose 6-phosphate receptor), but not with the transferrin receptor, LAMP-2 (lysosomal-associated membrane protein-2) or invariant chain. The difference in morphology between CI-MPR/AP-4-positive vesicles and CI-MPR/AP-1-positive vesicles raises the possibility that AP-4 acts at a location different from that of AP-1 in the intracellular trafficking pathway of CI-MPR.

Adaptor protein complexes (AP) are major components of the cytoplasmic coat found on clathrin-coated vesicles. Here, we report the molecular and functional characterization of Dictyostelium clathrin-associated AP-1 complex, which in mammalian cells, participates mainly in budding of clathrin-coated vesicles from the trans-Golgi network (TGN). The γ-adaptin AP-1 subunit was cloned and shown to belong to a Golgi-localized 300-kDa protein complex. Time-lapse analysis of cells expressing γ-adaptin tagged with the green-fluorescent protein demonstrates the dynamics of AP-1–coated structures leaving the Golgi apparatus and rarely moving toward the TGN. Targeted disruption of the AP-1 medium chain results in viable cells displaying a severe growth defect and a delayed developmental cycle compared with parental cells. Lysosomal enzymes are constitutively secreted as precursors, suggesting that protein transport between the TGN and lysosomes is defective. Although endocytic protein markers are correctly localized to endosomal compartments, morphological and ultrastructural studies reveal the absence of large endosomal vacuoles and an increased number of small vacuoles. In addition, the function of the contractile vacuole complex (CV), an osmoregulatory organelle is impaired and some CV components are not correctly targeted.

Adaptor protein (AP) complexes are essential components for the formation of coated vesicles and the recognition of cargo proteins for intracellular transport. Each AP complex exposes two appendage domains with that function to bind regulatory accessory proteins in the cytosol. Secondary structure predictions, sequence alignments and CD spectroscopy were used to relate the β-appendages of all human AP complexes to the previously published crystal structure of AP-2. The results suggested that the β-appendages of AP-1, AP-2 and AP-3 have similar structures, consisting of two subdomains, whereas that of AP-4 lacks the inner subdomain. Pull-down and overlay assays showed partial overlap in the binding specificities of the β-appendages of AP-1 and AP-2, whereas the corresponding domain of AP-3 displayed a unique binding pattern. That AP-4 may have a truncated, non-functional domain was indicated by its apparent inability to bind any proteins from cytosol. Of several novel β-appendage-binding proteins detected, one that had affinity exclusively for AP-2 was identified as sorting nexin 9 (SNX9). SNX9, which contains a phox and an Src homology 3 domain, was found in large complexes and was at least partially associated with AP-2 in the cytosol. SNX9 may function to assist AP-2 in its role at the plasma membrane.

L'infection par le virus de l'Hépatite E (HEV) est un problème majeur de santé publique qui toucherait 100 millions de personnes et tuerait 100 000 personnes chaque année dans le monde. Le HEV est la première cause d'hépatite aigüe dans le monde. En France, la séroprévalence s'élève à 22,4%. Ce virus se transmet par voie féco-orale ou par la consommation de viande contaminée mal cuite. Au cours de ma thèse, je me suis intéressé plus particulièrement à la protéine de capside ORF2, qui est l'unité structurale des particules virales et un acteur central du cycle infectieux du HEV. La protéine ORF2 est une protéine de 660 acides aminés qui possède un peptide signal N-terminal et trois sites potentiels de N-glycosylation. Au cours de son cycle infectieux, le HEV produit au moins trois formes de sa protéine de capside : (i) la forme ORF2g (pour glycosylée) et (ii) la forme ORF2c (pour clivée), abréviées ORF2g/c, qui sont des formes glycosylées et massivement sécrétées dans les surnageants de culture ou le sérum des patients infectés, et (iii) la forme ORF2i (pour infectieuse), qui n'est pas glycosylée et qui est associée aux particules virales.Dans le cadre de mes travaux de thèse, j'ai étudié les mécanismes de maturation protéolytique et de trafic intracellulaire de la protéine ORF2. Plus précisément, j'ai d'abord montré que la protéine ORF2 était importée dans le noyau des cellules infectées par le biais d'un mécanisme dépendant de l'Importine-alpha1, ceci grâce à un motif riche en résidus arginine situé à l'extrémité N-terminale de l'ORF2 et nommé ARM. La protéine ORF2 est ensuite exportée vers le cytoplasme par un mécanisme dépendant de l'exportine CRM1, ceci grâce à trois sites d'export nucléaire (NES9, NES10 et NES12) que nous avons identifiés dans la séquence de l'ORF2. J'ai également montré que le trafic nucléo-cytoplasmique de l'ORF2 régule probablement l'expression de certains gènes de l'immunité antivirale aux temps précoces de l'infection.Dans un second temps, j'ai participé à l'identification et à la caractérisation des usines virales du HEV. En ce sens, nous avons montré que les protéine virales ORF1, ORF2 et ORF3, ainsi que l'ARN viral sont enrichis dans des structures vésiculaires et tubulaires localisées dans les régions périnucléaires des cellules infectées. Nous avons montré que ces structures sont également enrichies en marqueurs du compartiment endosomal de recyclage (ERC), comme Rab11 et CD71, indiquant que les usines virales du HEV dérivent probablement de l'ERC. En réalisant des expériences d'extinction de Rab11 avec des ARN interférents, nous avons confirmé l'importance de l'ERC dans la production des particules virales du HEV.Dans un troisième temps, j'ai démontré que la protéine ORF2i, qui est associée aux membranes de la voie de sécrétion, est adressée aux usines virales par un mécanisme faisant intervenir le complexe adapteur AP-1.Dans un quatrième temps, j'ai caractérisé les mécanismes de maturation protéolytique des formes ORF2g/c et ORF2i. J'ai montré que la furine, une proproprotéine convertase de la voie de sécrétion, est impliquée dans la maturation protéolytique des glycoprotéines ORF2g/c. J'ai également montré que la préséniline, qui est la sous-unité catalytique du macro-complexe Gamma-sécrétase, est impliquée dans la maturation protéolytique de la forme infectieuse ORF2i. De manière intéressante, j'ai montré que l'inhibition pharmacologique de la préséniline réduit drastiquement l'infectiosité virale dans des lignées d'hépatocarcinome humain et dans des hépatocytes primaires humains. Ces données suggèrent que l'inhibition pharmacologique de la préséniline représente une stratégie antivirale prometteuse.En conclusion, les résultats obtenus dans le cadre de ma thèse permettent de mieux comprendre les mécanismes d'adressage subcellulaire et de maturation protéolytique de la protéine de capside ORF2, et ouvrent la voie au développement de nouvelles thérapies pour lutter contre le HEV
Hepatitis E virus (HEV) infection is a major public health problem, affecting an estimated 100 million people and killing 100,000 every year worldwide. HEV is the leading cause of acute hepatitis worldwide. In France, seroprevalence is 22.4%. This virus is transmitted via the feco-oral route, or by eating contaminated undercooked meat. During my thesis, I focused on the ORF2 capsid protein, which is the structural unit of viral particles and a central player in the HEV lifecycle. ORF2 is a 660 amino acid protein with an N-terminal signal peptide and three potential N-glycosylation sites. During its lifecycle, HEV produces at least three forms of its capsid protein: (i) the ORF2g (for glycosylated) form and (ii) the ORF2c (for cleaved) form, abbreviated ORF2g/c, which are glycosylated forms and massively secreted in culture supernatants or serum from infected patients, and (iii) the ORF2i (for infectious) form, which is not glycosylated and is associated with viral particles.As part of my thesis work, I studied the mechanisms of proteolytic maturation and intracellular trafficking of the ORF2 protein. More specifically, I first showed that ORF2 is imported into the nucleus of infected cells via an Importin-alpha1-dependent mechanism, thanks to an arginine-rich motif located at the N-terminus of ORF2 and named ARM. The ORF2 protein is then exported to the cytoplasm via a CRM1 exportin-dependent mechanism, thanks to three nuclear export sites (NES9, NES10 and NES12) that we have identified in the ORF2 sequence. I have also shown that nucleocytoplasmic trafficking of ORF2 probably regulates the expression of certain antiviral immunity genes in the early stages of infection.Secondly, I participated in the identification and characterization of HEV viral factories. We have shown that the viral proteins ORF1, ORF2 and ORF3, as well as viral RNA, are enriched in vesicular and tubular structures located in the perinuclear regions of infected cells. We have shown that these structures are also enriched in markers of the endosomal recycling compartment (ERC), such as Rab11 and CD71, indicating that HEV viral factories probably derive from the ERC. By performing Rab11 silencing experiments with siRNAs, we confirmed the importance of the ERC in the production of HEV viral particles. Thirdly, I demonstrated that the ORF2i protein, which is associated with the membranes of the secretion pathway, is addressed to viral factories by a mechanism involving the AP-1 adaptor complex.In a fourth step, I characterized the proteolytic maturation mechanisms of the ORF2g/c and ORF2i forms. I showed that furin, a proproprotein convertase of the secretory pathway, is involved in the proteolytic maturation of ORF2g/c glycoproteins. I have also shown that presenilin, which is the catalytic subunit of the macro-complex Gamma-secretase, is involved in the proteolytic maturation of the ORF2i form. Interestingly, I have shown that pharmacological inhibition of presenilin drastically reduces viral infectivity in human hepatocarcinoma lines and in primary human hepatocytes. These data suggest that pharmacological inhibition of presenilin represents a promising antiviral strategy. In conclusion, the results obtained during my thesis provide a better understanding of the mechanisms of subcellular addressing and proteolytic maturation of the ORF2 capsid protein, and pave the way for the development of new therapies to combat HEV

La polarité épithéliale est un processus essentiel au cours du développement d'un organisme. Ici, nous nous focalisons sur le tissu épithélial intestinal et épidermal de C. elegans pour comprendre comment la cellule maintient sa polarité en définissant un pôle apical et un pôle basolatéral. Afin d'assurer la mise en place et le maintien de cette polarité, des protéines appelées déterminants de polarité interviennent. Parmi ces déterminants, le module PAR-3/PAR-6/aPKC et CDC-42 sont des acteurs majeurs pour spécifier la polarité apicale. Nous avons montré que le complexe adaptateur pour la clathrine AP-1, un régulateur clé du trafic intracellulaire remplit une fonction inattendue dans ce processus. En effet, nous avons confirmé le rôle d'AP-1 dans le tri basolatéral observé chez les mammifères, mais de façon intéressante nous avons démontré qu'AP-1 contrôle également le tri apical d'une protéine transmembranaire ainsi que la localisation asymétrique apicale de CDC-42 et PAR-6. En effet, l'inhibition d'AP-1 cause une délocalisation basolatérale de CDC-42 et PAR-6. La perte de fonction d'AP-1 induit une conversion de la membrane latérale en membrane apicale et la formation de lumières intestinales ectopiques. La perte de fonction du complexe AP-1 induit également une létalité embryonnaire qui peut s'expliquer par le phénotype identifié dans l'épiderme. Dans cet épithélium, AP-1 contrôle l'intégrité des jonctions cellulaire et notamment le tri apical de la E-cadhérine. Nos résultats démontrent une fonction essentielle d'AP-1 dans le tri apical, directement responsable du maintien de la polarité épithéliale.

Thesis (Ph. D.)--University of Missouri-Columbia, 2005.
The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file viewed on (November 7, 2006) Vita. Includes bibliographical references.

La glutathion S-Transférase Pl-1 (GSTP1-1), impliquée dans la conjugaison de composés électrophiles au glutathion, la cancérogénèse et le développement de résistances aux anticancéreux, reste peu étudiée dans 16' cas des leucémies humaines en ce qui concerne l'expression du gène ainsi que des voies de transduction du signal impliquées. Dans une étude précédente, notre équipe a montré l'importance du site AP-l en tant que régulateur du promoteur minimal de la GSTP1-l. Pour ce travail, nous montrons dans un premier temps que des inducteurs typiques d'AP-l comme l'ester de phorbol TPA ou les mét1!. Ux lourds n'induisent pas l'expression de l'ARNm de la GSTPl-l dans la lignée leucémique K562. Par contre, nous confirmons l'importance de c-Jun, c-Fos et de la voie de signalisation menant à l'induction de AP-l dans la régulation de l'expression de la GSTPl-l. La plupart des gènes régulés par AP-l étant aussi régulés par NF-KB, nous avons décidé de vérifier le rôle de ce facteur dans la régulation de la GSTPl-l dont l'ARNm est inductible au TNFa. Grâce à un outil informatique, nous avons pu découvrir la présence d'un site NF-KB qui fixe les sous-unités p50 et p65 de NF-KB en réponse à un traitement au TNFa. Des expériences de co-transfections nous ont permis de compléter nos études en prouvant que la voie de signalisation NF-KB est bien impliquée dans la régulation de la GSTP1-1. Finalement, nous avons démontré l'efficacité d'agents chimiopréventifs, grâce à l'utilisation de la curcumine dont nous démontrons ici la capacité d'inhiber l'expression de la GSTPl-l en bloquant la fixation d' AP-l et de NF-KB tout en induisant l'apoptose dans les cellules leucémiques K562. Nous avons élargi nos résultats vers à l'utilisation d'autres agents chimiopréventifs dont la capsaicine et l'émodine. En résumé, nos résultats montrent que la GSTPl-l est régulée par les facteurs de transcription AP-l et NF-KB et inhibée par des agents chimiopréventifs qui présentent un intérêt potentiel pour de nouvelles thérapies anti-cancéreuses.

Le complexe AP-1 est l'une des plaques tournantes majeures de la signalisation intracellulaire. C'est un facteur transcriptionnel homodimérique, formé de deux protéines Jun (c-Jun, junB, JunD) ou hétérodimérique, formé d'une protéine Jun et d'une protéine Fos (c-Fos, FosB, Frai , Fra2). Cette thèse a été focalisée sur le fonctionnement du complexe AP-1 dans les hépatocytes et sur ses possibles interactions avec NF-KB. Ces travaux ont abouti à la mise en évidence d'une différence qualitative de la composition du complexe AP-1 entre hépatocytes normaux et transformés. A l'état basal, le complexe AP-1 est composé essentiellement d'homodimères Jun (JunD et c¬Jun dans les hépatocytes normaux, JunD et JunB dans les hépatocytes transformés). La stimulation par les facteurs de croissance ou les promoteurs tumoraux induit la formation d'hétérodimères Fos/Jun. La protéine Frai est constamment recrutée dans les dimères en réponse à toutes les stimulations effectuées. Elle apparaît donc comme le partenaire privilégié des protéines Jun dans les hépatocytes. La protéine c-Fos n'apparaît dans les dimères AP-1 que dans les hépatocytes transformés stimulés. La différence de composition du complexe AP-1 entre hépatocytes normaux et transformés est associée à une différence d'activité fonctionnelle du complexe AP-1 (transactivation de promoteurs dépendants de AP-1) suggérant que le complexe AP-1 assure la régulation de gènes différents dans les deux types cellulaires. La présence de la protéine JunB exclusivement dans les dimères AP-1 des hépatocytes transformés soulève l'hypothèse d'un rôle possible de cet oncogène dans la transformation de ces cellules. Nous avons observé une dissociation entre d'une part l'augmentation des ARNm et la quantité des protéines correspondantes, et d'autre part entre l'augmentation d'une protéine donnée et son recrutement dans les dimères AP-1, impliquant l'importance de mécanismes post-transcriptionnels dans la régulation de AP-1. Enfin, cette thèse montre pour la première fois, l'existence d'une coopération fonctionnelle entre la protéine JunD et la protéine p65 de NF-KB. Cette coopération se traduit par une augmentation de la transactivation au niveau du site NF-KB aussi bien à l'état basal qu'après stimulation de la prolifération par l'EGF, l'HGF, ou le TNFa. Ces observations suggèrent un rôle important des facteurs AP-1 et NF-KB dans le contrôle de la prolifération hépatocytaire.

O Vírus da Imunodeficiência Humana (HIV) é o agente etiológico da Síndrome da Imunodeficiência Adquirida (AIDS). A AIDS é uma doença de distribuição mundial, e estima-se que existam atualmente pelo menos 36,9 milhões de pessoas infectadas com o vírus. Durante o seu ciclo replicativo, o HIV promove diversas alterações na fisiologia da célula hospedeira a fim de promover sua sobrevivência e potencializar a replicação. A rápida progressão da infecção pelo HIV-1 em humanos e em modelos animais está intimamente ligada à função da proteína acessória Nef. Dentre as diversas ações de Nef está a regulação negativa de proteínas importantes na resposta imunológica, como o receptor CD4. Sabe-se que esta ação resulta da indução da degradação de CD4 em lisossomos, mas os mecanismos moleculares envolvidos ainda são totalmente elucidados. Nef forma um complexo tripartite com a cauda citosólica de CD4 e a proteína adaptadora 2 (AP-2), em vesículas revestidas por clatrina nascentes, induzindo a internalização e degradação lisossomal de CD4. Pesquisas anteriores demonstraram que o direcionamento de CD4 aos lisossomos por Nef envolve a entrada do receptor na via dos corpos multivesiculares (MVBs), por um mecanismo atípico, pois, embora não necessite da ubiquitinação de carga, depende da ação de proteínas que compõem os ESCRTs (Endosomal Sorting Complexes Required for Transport) e da ação de Alix, uma proteína acessória da maquinaria ESCRT. Já foi reportado que Nef interage com subunidades dos complexos AP-1, AP-2, AP-3 e Nef não parece interagir com subunidades de AP-4 e AP-5. Entretanto, o papel da interação de Nef com AP-1 e AP-3 na regulação negativa de CD4 ainda não está totalmente elucidado. Ademais, AP-1, AP-2 e AP-3 são potencialmente heterogêneos devido à existência de isoformas múltiplas das subunidades codificadas por diferentes genes. Todavia, existem poucos estudos para demonstrar se as diferentes combinações de isoformas dos APs são formadas e se possuem propriedades funcionais distintas. O presente trabalho procurou identificar e caracterizar fatores celulares envolvidos na regulação do tráfego intracelular de proteínas no processo de regulação negativa de CD4 induzido por Nef. Mais especificamente, este estudo buscou caracterizar a participação do complexo AP-1 na modulação negativa de CD4 por Nef de HIV-1, através do estudo funcional das duas isoformas de ?-adaptina, subunidades de AP-1. Utilizando a técnica de Pull-down demonstramos que Nef é capaz de interagir com ?2. Além disso, nossos dados de Imunoblot indicaram que a proteína ?2-adaptina, e não ?1-adaptina, é necessária no processo de degradação lisossomal de CD4 por Nef e que esta participação é conservada para degradação de CD4 por Nef de diferentes cepas virais. Ademais, por citometria de fluxo, o silenciamento de ?2, e não de ?1, compromete a diminuição dos níveis de CD4 por Nef da membrana plasmática. A análise por imunofluorêsncia indireta também revelou que a diminuição dos níveis de ?2 impede a redistribuição de CD4 por Nef para regiões perinucleares, acarretando no acúmulo de CD4, retirados por Nef da membrana plasmática, em endossomos primários. A depleção de ?1A, outra subunidade de AP-1, acarretou na diminuição dos níveis celulares de ?2 e ?1, bem como, no comprometimento da eficiente degradação de CD4 por Nef. Além disso, foi possível observar que, ao perturbar a maquinaria ESCRT via super-expressão de HRS (uma subunidade do complexo ESCRT-0), ocorreu um acumulo de ?2 em endossomos dilatados contendo HRS-GFP, nos quais também detectou-se CD4 que foi internalizado por Nef. Em conjunto, os resultados indicam que ?2-adaptina é uma importante molécula para o direcionamento de CD4 por Nef para a via ESCRT/MVB, mostrando ser uma proteína relevante no sistema endo-lisossomal. Ademais, os resultados indicaram que as isoformas ?-adaptinas não só possuem funções distintas, mas também parecem compor complexos AP-1 com diferentes funções celulares, já que apenas a variante AP-1 contendo ?2, mas não ?1, participa da regulação negativa de CD4 por Nef. Estes estudos contribuem para o melhor entendimento dos mecanismos moleculares envolvidos na atividade de Nef, que poderão também ajudar na melhor compreensão da patogênese do HIV e da síndrome relacionada. Em adição, este trabalho contribui para o entendimento de processos fundamentais da regulação do tráfego de proteínas transmembrana no sistema endo-lisossomal.
The Human Immunodeficiency Virus (HIV) is the etiologic agent of Acquired Immunodeficiency Syndrome (AIDS). AIDS is a disease which has a global distribution, and it is estimated that there are currently at least 36.9 million people infected with the virus. During the replication cycle, HIV promotes several changes in the physiology of the host cell to promote their survival and enhance replication. The fast progression of HIV-1 in humans and animal models is closely linked to the function of an accessory protein Nef. Among several actions of Nef, one is the most important is the down-regulation of proteins from the immune response, such as the CD4 receptor. It is known that this action causes CD4 degradation in lysosome, but the molecular mechanisms are still incompletely understood. Nef forms a tripartite complex with the cytosolic tail of the CD4 and adapter protein 2 (AP-2) in clathrin-coated vesicles, inducing CD4 internalization and lysosome degradation. Previous research has demonstrated that CD4 target to lysosomes by Nef involves targeting of this receptor to multivesicular bodies (MVBs) pathway by an atypical mechanism because, although not need charging ubiquitination, depends on the proteins from ESCRTs (Endosomal Sorting Complexes Required for Transport) machinery and the action of Alix, an accessory protein ESCRT machinery. It has been reported that Nef interacts with subunits of AP- 1, AP-2, AP-3 complexes and Nef does not appear to interact with AP-4 and AP-5 subunits. However, the role of Nef interaction with AP-1 or AP-3 in CD4 down-regulation is poorly understood. Furthermore, AP-1, AP-2 and AP-3 are potentially heterogeneous due to the existence of multiple subunits isoforms encoded by different genes. However, there are few studies to demonstrate if the different combinations of APs isoforms are form and if they have distinct functional properties. This study aim to identify and characterize cellular factors involved on CD4 down-modulation induced by Nef from HIV-1. More specifically, this study aimed to characterize the involvement of AP-1 complex in the down-regulation of CD4 by Nef HIV-1 through the functional study of the two isoforms of ?-adaptins, AP-1 subunits. By pull-down technique, we showed that Nef is able to interact with ?2. In addition, our data from immunoblots indicated that ?2- adaptin, not ?1-adaptin, is required in Nef-mediated targeting of CD4 to lysosomes and the ?2 participation in this process is conserved by Nef from different viral strains. Furthermore, by flow cytometry assay, ?2 depletion, but not ?1 depletion, compromises the reduction of surface CD4 levels induced by Nef. Immunofluorescence microscopy analysis also revealed that ?2 depletion impairs the redistribution of CD4 by Nef to juxtanuclear region, resulting in CD4 accumulation in primary endosomes. Knockdown of ?1A, another subunit of AP-1, resulted in decreased cellular levels of ?1 and ?2 and, compromising the efficient CD4 degradation by Nef. Moreover, upon artificially stabilizing ESCRT-I in early endosomes, via overexpression of HRS, internalized CD4 accumulates in enlarged HRS-GFP positive endosomes, where co-localize with ?2. Together, the results indicate that ?2-adaptin is a molecule that is essential for CD4 targeting by Nef to ESCRT/MVB pathway, being an important protein in the endo-lysosomal system. Furthermore, the results indicate that ?-adaptins isoforms not only have different functions, but also seem to compose AP-1 complex with distinct cell functions, and only the AP-1 variant comprising ?2, but not ?1, acts in the CD4 down-regulation induced by Nef. These studies contribute to a better understanding on the molecular mechanisms involved in Nef activities, which may also help to improve the understanding of the HIV pathogenesis and the related syndrome. In addition, this work contributes with the understanding of primordial process regulation on intracellular trafficking of transmembrane proteins.