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1.
Mol Cell ; 2018 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-30415949

RESUMO

Current models of SIRT1 enzymatic regulation primarily consider the effects of fluctuating levels of its co-substrate NAD+, which binds to the stably folded catalytic domain. By contrast, the roles of the sizeable disordered N- and C-terminal regions of SIRT1 are largely unexplored. Here we identify an insulin-responsive sensor in the SIRT1 N-terminal region (NTR), comprising an acidic cluster (AC) and a 3-helix bundle (3HB), controlling deacetylase activity. The allosteric assistor DBC1 removes a distal N-terminal shield from the 3-helix bundle, permitting PACS-2 to engage the acidic cluster and the transiently exposed helix 3 of the 3-helix bundle, disrupting its structure and inhibiting catalysis. The SIRT1 activator (STAC) SRT1720 binds and stabilizes the 3-helix bundle, protecting SIRT1 from inhibition by PACS-2. Identification of the SIRT1 insulin-responsive sensor and its engagement by the DBC1 and PACS-2 regulatory hub provides important insight into the roles of disordered regions in enzyme regulation and the mode by which STACs promote metabolic fitness.

2.
PeerJ ; 6: e5602, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30405966

RESUMO

Colorectal cancer ranks third among the most commonly diagnosed cancers in the United States. Current therapies have a range of side effects, and the development of a reliable animal model to speed the discovery of safe effective preventative therapies would be of great value. A cross-sectional study in a large Appalachian population recently showed an association between low circulating levels of perfluorooctane sulfonate (PFOS) and a reduced prevalence of colorectal cancer. A study using APCmin (C57BL/6J-ApcMin/J) mice prone to familial adenomatous polyposis found PFOS was protective when exposure occurred during tumor development. To test the possible benefit of PFOS on spontaneous colorectal cancer, we developed a mouse model utilizing primary patient colorectal cancer implants into NSG (NOD.Cg-PrkdcscidIl2rgtm1Wjl /Sz) mice. Study goals included: (1) to assess potential factors supporting the successful use of colorectal cancer from heterogeneous tumors for PDX studies; and, (2) evaluate PFOS as a therapy in tumor matched pairs of mice randomized to receive PFOS or vehicle. The time in days for mice to grow primary tumors to 5 mm took almost 2 months (mean = 53.3, se = 5.7, range = 17-136). Age of mice at implantation, patient age, gender and race appeared to have no discernable effect on engraftment rates. Engraftment rates for low and high-grade patient tumors were similar. PFOS appeared to reduce tumor size dramatically in one group of tumors, those from the right ascending colon. That is, by 5 weeks of treatment in two mice, PFOS had eliminated their 52.4 mm3 and 124.6 mm3 masses completely, an effect that was sustained for 10 weeks of treatment; in contrast, their corresponding matched vehicle control mice had tumors that grew to 472.7 mm3 and 340.1 mm3 in size respectively during the same period. In a third xenograft mouse, the tumor growth was dramatically blunted although not eliminated, and compared favorably to their matched vehicle controls over the same period. These preliminary findings suggested that this mouse model may be advantageous for testing compounds of potential value in the treatment of colorectal cancer, and PFOS may have utility in selected cases.

4.
Am J Hum Genet ; 102(5): 995-1007, 2018 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-29656858

RESUMO

Developmental and epileptic encephalopathies (DEEs) represent a large clinical and genetic heterogeneous group of neurodevelopmental diseases. The identification of pathogenic genetic variants in DEEs remains crucial for deciphering this complex group and for accurately caring for affected individuals (clinical diagnosis, genetic counseling, impacting medical, precision therapy, clinical trials, etc.). Whole-exome sequencing and intensive data sharing identified a recurrent de novo PACS2 heterozygous missense variant in 14 unrelated individuals. Their phenotype was characterized by epilepsy, global developmental delay with or without autism, common cerebellar dysgenesis, and facial dysmorphism. Mixed focal and generalized epilepsy occurred in the neonatal period, controlled with difficulty in the first year, but many improved in early childhood. PACS2 is an important PACS1 paralog and encodes a multifunctional sorting protein involved in nuclear gene expression and pathway traffic regulation. Both proteins harbor cargo(furin)-binding regions (FBRs) that bind cargo proteins, sorting adaptors, and cellular kinase. Compared to the defined PACS1 recurrent variant series, individuals with PACS2 variant have more consistently neonatal/early-infantile-onset epilepsy that can be challenging to control. Cerebellar abnormalities may be similar but PACS2 individuals exhibit a pattern of clear dysgenesis ranging from mild to severe. Functional studies demonstrated that the PACS2 recurrent variant reduces the ability of the predicted autoregulatory domain to modulate the interaction between the PACS2 FBR and client proteins, which may disturb cellular function. These findings support the causality of this recurrent de novo PACS2 heterozygous missense in DEEs with facial dysmorphim and cerebellar dysgenesis.

5.
J Cell Sci ; 130(11): 1865-1876, 2017 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-28476937

RESUMO

Vertebrate proteins that fulfill multiple and seemingly disparate functions are increasingly recognized as vital solutions to maintaining homeostasis in the face of the complex cell and tissue physiology of higher metazoans. However, the molecular adaptations that underpin this increased functionality remain elusive. In this Commentary, we review the PACS proteins - which first appeared in lower metazoans as protein traffic modulators and evolved in vertebrates to integrate cytoplasmic protein traffic and interorganellar communication with nuclear gene expression - as examples of protein adaptation 'caught in the act'. Vertebrate PACS-1 and PACS-2 increased their functional density and roles as metabolic switches by acquiring phosphorylation sites and nuclear trafficking signals within disordered regions of the proteins. These findings illustrate one mechanism by which vertebrates accommodate their complex cell physiology with a limited set of proteins. We will also highlight how pathogenic viruses exploit the PACS sorting pathways as well as recent studies on PACS genes with mutations or altered expression that result in diverse diseases. These discoveries suggest that investigation of the evolving PACS protein family provides a rich opportunity for insight into vertebrate cell and organ homeostasis.


Assuntos
Homeostase/genética , Neoplasias/genética , Doenças Neurodegenerativas/genética , Obesidade/genética , Proteínas de Transporte Vesicular/genética , Adaptação Biológica , Animais , Apoptose , Transporte Biológico , Sinalização do Cálcio , Sequência Conservada , Regulação da Expressão Gênica , Humanos , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Obesidade/metabolismo , Obesidade/patologia , Filogenia , Ligante Indutor de Apoptose Relacionado a TNF/genética , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Proteínas de Transporte Vesicular/metabolismo
6.
Toxicology ; 383: 1-12, 2017 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-28342779

RESUMO

Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used for the alleviation of pain and inflammation, but these drugs are also associated with a suite of negative side effects. Gastrointestinal (GI) toxicity is particularly concerning since it affects an estimated 70% of individuals taking NSAIDs routinely, and evidence suggests the majority of toxicity is occurring in the small intestine. Traditionally, NSAID-induced GI toxicity has been associated with indiscriminate inhibition of cyclooxygenase isoforms, but other mechanisms, including inhibition of cell migration, intestinal restitution, and wound healing, are likely to contribute to toxicity. Previous efforts demonstrated that treatment of cultured intestinal epithelial cells (IEC) with NSAIDs inhibits expression and activity of calpain proteases, but the effects of specific inhibition of calpain expression in vitro or the effects of NSAIDs on intestinal cell migration in vivo remain to be determined. Accordingly, we examined the effect of suppression of calpain protease expression with siRNA on cell migration in cultured IECs and evaluated the effects of NSAID treatment on epithelial cell migration and calpain protease expression in rat duodenum. Our results show that calpain siRNA inhibits protease expression and slows migration in cultured IECs. Additionally, NSAID treatment of rats slowed migration up the villus axis and suppressed calpain expression in duodenal epithelial cells. Our results are supportive of the hypothesis that suppression of calpain expression leading to slowing of cell migration is a potential mechanism through which NSAIDs cause GI toxicity.


Assuntos
Anti-Inflamatórios não Esteroides/toxicidade , Calpaína/antagonistas & inibidores , Células Epiteliais/efeitos dos fármacos , Animais , Calpaína/genética , Calpaína/metabolismo , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Duodeno/efeitos dos fármacos , Duodeno/metabolismo , Duodeno/patologia , Células Epiteliais/fisiologia , Indometacina/toxicidade , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Masculino , Nitrobenzenos/toxicidade , RNA Interferente Pequeno/genética , Ratos Wistar , Sulfonamidas/toxicidade
7.
BMC Cancer ; 16(1): 942, 2016 12 08.
Artigo em Inglês | MEDLINE | ID: mdl-27927180

RESUMO

BACKGROUND: Colorectal cancer is the second most common cause of cancer deaths for both men and women, and the third most common cause of cancer in the U.S. Toxicity of current chemotherapeutic agents for colorectal cancer, and emergence of drug resistance underscore the need to develop new, potentially less toxic alternatives. Our recent cross-sectional study in a large Appalachian population, showed a strong, inverse, dose-response association of serum perfluorooctane sulfonate (PFOS) levels to prevalent colorectal cancer, suggesting PFOS may have therapeutic potential in the prevention and/or treatment of colorectal cancer. In these preliminary studies using a mouse model of familial colorectal cancer, the APCmin mouse, and exposures comparable to those reported in human populations, we assess the efficacy of PFOS for reducing tumor burden, and evaluate potential dose-response effects. METHODS: At 5-6 weeks of age, APCmin mice were randomized to receive 0, 20, 250 mg PFOS/kg (females) or 0, 10, 50 and 200 mg PFOS/kg (males) via their drinking water. At 15 weeks of age, gastrointestinal tumors were counted and scored and blood PFOS levels measured. RESULTS: PFOS exposure was associated with a significant, dose-response reduction in total tumor number in both male and female mice. This inverse dose-response effect of PFOS exposure was particularly pronounced for larger tumors (r2 for linear trend = 0.44 for males, p's <0.001). CONCLUSIONS: The current study in a mouse model of familial adenomatous polyposis offers the first experimental evidence that chronic exposure to PFOS in drinking water can reduce formation of gastrointestinal tumors, and that these reductions are both significant and dose-dependent. If confirmed in further studies, these promising findings could lead to new therapeutic strategies for familial colorectal cancer, and suggest that PFOS testing in both preventive and therapeutic models for human colorectal cancer is warranted.


Assuntos
Proteína da Polipose Adenomatosa do Colo/fisiologia , Polipose Adenomatosa do Colo/tratamento farmacológico , Ácidos Alcanossulfônicos/administração & dosagem , Modelos Animais de Doenças , Fluorcarbonetos/administração & dosagem , Polipose Adenomatosa do Colo/genética , Polipose Adenomatosa do Colo/patologia , Administração Oral , Ácidos Alcanossulfônicos/farmacologia , Animais , Relação Dose-Resposta a Droga , Feminino , Fluorcarbonetos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL
8.
Biochem Pharmacol ; 98(4): 614-28, 2015 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-26549367

RESUMO

Clinical use of non-steroidal anti-inflammatory drugs (NSAIDs) is well known to cause gastrointestinal ulcer formation via several mechanisms that include inhibiting epithelial cell migration and mucosal restitution. The drug-affected signaling pathways that contribute to inhibition of migration by NSAIDs are poorly understood, though previous studies have shown that NSAIDs depolarize membrane potential and suppress expression of calpain proteases and voltage-gated potassium (Kv) channel subunits. Kv channels play significant roles in cell migration and are targets of NSAID activity in white blood cells, but the specific functional effects of NSAID-induced changes in Kv channel expression, particularly on cell migration, are unknown in intestinal epithelial cells. Accordingly, we investigated the effects of NSAIDs on expression of Kv1.3, 1.4, and 1.6 in vitro and/or in vivo and evaluated the functional significance of loss of Kv subunit expression. Indomethacin or NS-398 reduced total and plasma membrane protein expression of Kv1.3 in cultured intestinal epithelial cells (IEC-6). Additionally, depolarization of membrane potential with margatoxin (MgTx), 40mM K(+), or silencing of Kv channel expression with siRNA significantly reduced IEC-6 cell migration and disrupted calpain activity. Furthermore, in rat small intestinal epithelia, indomethacin and NS-398 had significant, yet distinct, effects on gene and protein expression of Kv1.3, 1.4, or 1.6, suggesting that these may be clinically relevant targets. Our results show that inhibition of epithelial cell migration by NSAIDs is associated with decreased expression of Kv channel subunits, and provide a mechanism through which NSAIDs inhibit cell migration and may contribute to NSAID-induced gastrointestinal (GI) toxicity.


Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Calpaína/antagonistas & inibidores , Movimento Celular/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Canais de Potássio de Abertura Dependente da Tensão da Membrana/antagonistas & inibidores , Animais , Calpaína/metabolismo , Linhagem Celular , Movimento Celular/fisiologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Canal de Potássio Kv1.3/antagonistas & inibidores , Canal de Potássio Kv1.3/metabolismo , Canal de Potássio Kv1.4/antagonistas & inibidores , Canal de Potássio Kv1.4/metabolismo , Canal de Potássio Kv1.6/antagonistas & inibidores , Canal de Potássio Kv1.6/metabolismo , Potenciais da Membrana/fisiologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Ratos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia
9.
Nat Commun ; 6: 7518, 2015 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-26108729

RESUMO

The metalloproteinase ADAM17 activates ErbB signalling by releasing ligands from the cell surface, a key step underlying epithelial development, growth and tumour progression. However, mechanisms acutely controlling ADAM17 cell-surface availability to modulate the extent of ErbB ligand release are poorly understood. Here, through a functional genome-wide siRNA screen, we identify the sorting protein PACS-2 as a regulator of ADAM17 trafficking and ErbB signalling. PACS-2 loss reduces ADAM17 cell-surface levels and ADAM17-dependent ErbB ligand shedding, without apparent effects on related proteases. PACS-2 co-localizes with ADAM17 on early endosomes and PACS-2 knockdown decreases the recycling and stability of internalized ADAM17. Hence, PACS-2 sustains ADAM17 cell-surface activity by diverting ADAM17 away from degradative pathways. Interestingly, Pacs2-deficient mice display significantly reduced levels of phosphorylated EGFR and intestinal proliferation. We suggest that this mechanism controlling ADAM17 cell-surface availability and EGFR signalling may play a role in intestinal homeostasis, with potential implications for cancer biology.


Assuntos
Proteínas ADAM/metabolismo , Proteínas Oncogênicas v-erbB/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas ADAM/genética , Proteína ADAM17 , Animais , Linhagem Celular Tumoral , Regulação Enzimológica da Expressão Gênica , Estudo de Associação Genômica Ampla , Humanos , Camundongos , Proteínas Oncogênicas v-erbB/genética , Transdução de Sinais/fisiologia , Proteínas de Transporte Vesicular/genética
10.
Cell Rep ; 8(5): 1545-57, 2014 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-25159152

RESUMO

SIRT1 regulates the DNA damage response by deacetylating p53, thereby repressing p53 transcriptional output. Here, we demonstrate that the sorting protein PACS-2 regulates SIRT1-mediated deacetylation of p53 to modulate the DNA damage response. PACS-2 knockdown cells failed to efficiently undergo p53-induced cell-cycle arrest in response to DNA damage. Accordingly, p53 acetylation was reduced both in PACS-2 knockdown cells and thymocytes from Pacs-2(-/-) mice, thereby blunting induction of the cyclin-dependent kinase inhibitor p21 (CDKN1A). The SIRT1 inhibitor EX-527 or SIRT1 knockdown restored p53 acetylation and p21 induction as well as p21-dependent cell-cycle arrest in PACS-2 knockdown cells. Trafficking studies revealed that cytoplasmic PACS-2 shuttled to the nucleus, where it interacted with SIRT1 and repressed SIRT1-mediated p53 deacetylation. Correspondingly, in vitro assays demonstrated that PACS-2 directly inhibited SIRT1-catalyzed p53 deacetylation. Together, these findings identify PACS-2 as an in vivo mediator of the SIRT1-p53-p21 axis that modulates the DNA damage response.


Assuntos
Ciclo Celular , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Sirtuína 1/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Acetilação , Transporte Ativo do Núcleo Celular , Animais , Carbazóis/farmacologia , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Dano ao DNA , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Ligação Proteica , Sirtuína 1/antagonistas & inibidores , Sirtuína 1/genética , Timócitos/metabolismo , Proteínas de Transporte Vesicular/genética
11.
Retrovirology ; 10: 135, 2013 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-24229420

RESUMO

BACKGROUND: HIV-1 Nef is a viral accessory protein critical for AIDS progression. Nef lacks intrinsic catalytic activity and binds multiple host cell signaling proteins, including Hck and other Src-family tyrosine kinases. Nef binding induces constitutive Hck activation that may contribute to HIV pathogenesis by promoting viral infectivity, replication and downregulation of cell-surface MHC-I molecules. In this study, we developed a yeast-based phenotypic screen to identify small molecules that inhibit the Nef-Hck complex. RESULTS: Nef-Hck interaction was faithfully reconstituted in yeast cells, resulting in kinase activation and growth arrest. Yeast cells expressing the Nef-Hck complex were used to screen a library of small heterocyclic compounds for their ability to rescue growth inhibition. The screen identified a dihydrobenzo-1,4-dioxin-substituted analog of 2-quinoxalinyl-3-aminobenzene-sulfonamide (DQBS) as a potent inhibitor of Nef-dependent HIV-1 replication and MHC-I downregulation in T-cells. Docking studies predicted direct binding of DQBS to Nef which was confirmed in differential scanning fluorimetry assays with recombinant purified Nef protein. DQBS also potently inhibited the replication of HIV-1 NL4-3 chimeras expressing Nef alleles representative of all M-group HIV-1 clades. CONCLUSIONS: Our findings demonstrate the utility of a yeast-based growth reversion assay for the identification of small molecule Nef antagonists. Inhibitors of Nef function discovered with this assay, such as DQBS, may complement the activity of current antiretroviral therapies by enabling immune recognition of HIV-infected cells through the rescue of cell surface MHC-I.


Assuntos
Fármacos Anti-HIV/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Proteínas Proto-Oncogênicas c-hck/antagonistas & inibidores , Quinoxalinas/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Sulfonamidas/farmacologia , Produtos do Gene nef do Vírus da Imunodeficiência Humana/antagonistas & inibidores , Fármacos Anti-HIV/isolamento & purificação , Humanos , Simulação de Acoplamento Molecular , Ligação Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-hck/genética , Quinoxalinas/isolamento & purificação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Sulfonamidas/isolamento & purificação , Produtos do Gene nef do Vírus da Imunodeficiência Humana/genética
12.
J Biol Chem ; 287(29): 24427-37, 2012 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-22645134

RESUMO

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis of liver cancer cell lines requires death receptor-5 (DR5)-dependent permeabilization of lysosomal membranes. Ligated DR5 triggers recruitment of the proapoptotic proteins Bim and Bax to lysosomes, releasing cathepsin B into the cytosol where it mediates mitochondria membrane permeabilization and activation of executioner caspases. Despite the requirement for lysosome membrane permeabilization during TRAIL-induced apoptosis, little is known about the mechanism that controls recruitment of Bim and Bax to lysosomal membranes. Here we report that TRAIL induces recruitment of the multifunctional sorting protein phosphofurin acidic cluster sorting protein-2 (PACS-2) to DR5-positive endosomes in Huh-7 cells where it forms an immunoprecipitatable complex with Bim and Bax on lysosomal membranes. shRNA-targeted knockdown of PACS-2 prevents recruitment of Bim or Bax to lysosomes, blunting the TRAIL-induced lysosome membrane permeabilization. Consistent with the reduced lysosome membrane permeabilization, shRNA knockdown of PACS-2 in Huh-7 cells reduced TRAIL-induced apoptosis and increased clonogenic cell survival. The determination that recombinant PACS-2 bound Bim but not Bax in vitro and that shRNA knockdown of Bim blocked Bax recruitment to lysosomes suggests that TRAIL/DR5 triggers endosomal PACS-2 to recruit Bim and Bax to lysosomes to release cathepsin B and induce apoptosis. Together, these findings provide insight into the lysosomal pathway of apoptosis.


Assuntos
Lisossomos/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Proteínas de Transporte Vesicular/metabolismo , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Proteína 11 Semelhante a Bcl-2 , Catepsina B/metabolismo , Linhagem Celular Tumoral , Imunofluorescência , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Imunoprecipitação , Lisossomos/efeitos dos fármacos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Microscopia Confocal , Transporte Proteico/efeitos dos fármacos , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Proteínas de Transporte Vesicular/genética , Proteína X Associada a bcl-2/metabolismo
13.
Mol Biol Cell ; 23(11): 2184-97, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22496420

RESUMO

The human immunodeficiency virus type 1 (HIV-1) accessory protein Nef directs virus escape from immune surveillance by subverting host cell intracellular signaling and membrane traffic to down-regulate cell-surface major histocompatibility complex class I (MHC-I). The interaction of Nef with the sorting proteins PACS-1 and PACS-2 mediates key signaling and trafficking steps required for Nef-mediated MHC-I down-regulation. Little is known, however, about the molecular basis underlying the Nef-PACS interaction. Here we identify the sites on Nef and the PACS proteins required for their interaction and describe the consequences of disrupting this interaction for Nef action. A previously unidentified cargo subsite on PACS-1 and PACS-2 interacted with a bipartite site on Nef formed by the EEEE(65) acidic cluster on the N-terminal domain and W(113) in the core domain. Mutation of these sites prevented the interaction between Nef and the PACS proteins on Rab5 (PACS-2 and PACS-1)- or Rab7 (PACS-1)-positive endosomes as determined by bimolecular fluorescence complementation and caused a Nef mutant defective in PACS binding to localize to distorted endosomal compartments. Consequently, disruption of the Nef-PACS interaction repressed Nef-induced MHC-I down-regulation in peripheral blood mononuclear cells. Our results provide insight into the molecular basis of Nef action and suggest new strategies to combat HIV-1.


Assuntos
Regulação para Baixo , Endossomos/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Produtos do Gene nef do Vírus da Imunodeficiência Humana/química , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismo , Sequência de Aminoácidos , Aminoácidos/metabolismo , Sítios de Ligação , Furina/metabolismo , Células HeLa , Humanos , Dados de Sequência Molecular , Mutação/genética , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Transporte Proteico , Relação Estrutura-Atividade , Proteínas de Transporte Vesicular/química
14.
Mol Biol Cell ; 21(19): 3279-92, 2010 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-20702582

RESUMO

HIV-1 Nef triggers down-regulation of cell-surface MHC-I by assembling a Src family kinase (SFK)-ZAP-70/Syk-PI3K cascade. Here, we report that chemical disruption of the Nef-SFK interaction with the small molecule inhibitor 2c blocks assembly of the multi-kinase complex and represses HIV-1-mediated MHC-I down-regulation in primary CD4(+) T-cells. 2c did not interfere with the PACS-2-dependent trafficking of Nef required for the Nef-SFK interaction or the AP-1 and PACS-1-dependent sequestering of internalized MHC-I, suggesting the inhibitor specifically interfered with the Nef-SFK interaction required for triggering MHC-I down-regulation. Transport studies revealed Nef directs a highly regulated program to down-regulate MHC-I in primary CD4(+) T-cells. During the first two days after infection, Nef assembles the 2c-sensitive multi-kinase complex to trigger down-regulation of cell-surface MHC-I. By three days postinfection Nef switches to a stoichiometric mode that prevents surface delivery of newly synthesized MHC-I. Pharmacologic inhibition of the multi-kinase cascade prevents the Nef-dependent block in MHC-I transport, suggesting the signaling and stoichiometric modes are causally linked. Together, these studies resolve the seemingly controversial models that describe Nef-induced MHC-I down-regulation and provide new insights into the mechanism of Nef action.


Assuntos
Regulação para Baixo/efeitos dos fármacos , HIV-1/efeitos dos fármacos , Antígenos de Histocompatibilidade Classe I/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismo , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/enzimologia , Linfócitos T CD4-Positivos/virologia , Linhagem Celular , Endocitose/efeitos dos fármacos , Humanos , Complexos Multienzimáticos/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Ligação Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Fator de Transcrição AP-1/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Quinases da Família src/metabolismo
15.
Mol Cell ; 34(4): 497-509, 2009 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-19481529

RESUMO

TRAIL selectively kills diseased cells in vivo, spurring interest in this death ligand as a potential therapeutic. However, many cancer cells are resistant to TRAIL, suggesting the mechanism mediating TRAIL-induced apoptosis is complex. Here we identify PACS-2 as an essential TRAIL effector, required for killing tumor cells in vitro and virally infected hepatocytes in vivo. PACS-2 is phosphorylated at Ser437 in vivo, and pharmacologic and genetic studies demonstrate Akt is an in vivo Ser437 kinase. Akt cooperates with 14-3-3 to regulate the homeostatic and apoptotic properties of PACS-2 that mediate TRAIL action. Phosphorylated Ser437 binds 14-3-3 with high affinity, which represses PACS-2 apoptotic activity and is required for PACS-2 to mediate trafficking of membrane cargo. TRAIL triggers dephosphorylation of Ser437, reprogramming PACS-2 to promote apoptosis. Together, these studies identify the phosphorylation state of PACS-2 Ser437 as a molecular switch that integrates cellular homeostasis with TRAIL-induced apoptosis.


Assuntos
Proteínas 14-3-3/metabolismo , Apoptose/fisiologia , Membrana Celular/metabolismo , Homeostase , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas 14-3-3/genética , Animais , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/genética , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Caspases/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Camundongos , Camundongos Knockout , Neoplasias/metabolismo , Neoplasias/patologia , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Serina/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/genética , Proteínas de Transporte Vesicular/genética
16.
J Biol Chem ; 283(17): 11772-84, 2008 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-18296443

RESUMO

Human immunodeficiency virus, type 1, negative factor (Nef) initiates down-regulation of cell-surface major histocompatibility complex-I (MHC-I) by assembling an Src family kinase (SFK)-ZAP70/Syk-phosphoinositide 3-kinase (PI3K) cascade through the sequential actions of two sites, Nef EEEE(65) and PXXP(75). The internalized MHC-I molecules are then sequestered in endosomal compartments by a process requiring Nef Met(20). How Nef assembles the multikinase cascade to trigger the MHC-I down-regulation pathway is unknown. Here we report that EEEE(65)-dependent binding to the sorting protein PACS-2 targets Nef to the paranuclear region, enabling PXXP(75) to bind and activate a trans-Golgi network (TGN)-localized SFK. This SFK then phosphorylates ZAP-70 to recruit class I PI3K by interaction with the p85 C-terminal Src homology 2 domain. Using splenocytes and embryonic fibroblasts from PACS-2(-/-) mice, we confirm genetically that Nef requires PACS-2 to localize to the paranuclear region and assemble the multikinase cascade. Moreover, genetic loss of PACS-2 or inhibition of class I PI3K prevents Nef-mediated MHC-I down-regulation, demonstrating that short interfering RNA knockdown of PACS-2 phenocopies the gene knock-out. This PACS-2-dependent targeting pathway is not restricted to Nef, because PACS-2 is also required for trafficking of an endocytosed cation-independent mannose 6-phosphate receptor reporter from early endosomes to the TGN. Together, these results demonstrate PACS-2 is required for Nef action and sorting of itinerant membrane cargo in the TGN/endosomal system.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Regulação Viral da Expressão Gênica , Genes MHC Classe I , HIV-1/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismo , Animais , Endocitose , Endossomos/metabolismo , Células HeLa , Humanos , Camundongos , Camundongos Knockout , Modelos Biológicos , Fosforilação , Rede trans-Golgi/metabolismo
17.
Cell Host Microbe ; 1(2): 121-33, 2007 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-18005690

RESUMO

HIV-1 Nef, which is required for the efficient onset of AIDS, enhances viral replication and infectivity by exerting multiple effects on infected cells. Nef downregulates cell-surface MHC-I molecules by an uncharacterized PI3K pathway requiring the actions of two Nef motifs-EEEE(65) and PXXP(75). We report that the Nef EEEE(65) targeting motif enables Nef PXXP(75) to bind and activate a trans-Golgi network-localized Src family tyrosine kinase (SFK). The Nef/SFK complex then recruits and phosphorylates the tyrosine kinase ZAP-70, which binds class I PI3K to trigger MHC-I downregulation in primary CD4+ T cells. In promonocytic cells, Nef/SFK recruits the ZAP-70 homolog Syk to downregulate MHC-I, implicating this PI3K pathway in multiple HIV-1 reservoirs. Isoform-specific PI3K inhibitors repress MHC-I downregulation, identifying them as potential therapeutic agents to combat HIV-1. The discovery of this Nef-SFK-ZAP-70/Syk-PI3K signaling pathway explains the hierarchal role of the Nef motifs in effecting immunoevasion.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Membrana Celular/imunologia , HIV-1/fisiologia , Antígenos de Histocompatibilidade Classe I/imunologia , Complexo Principal de Histocompatibilidade , Fosfatidilinositol 3-Quinases/metabolismo , Proteína-Tirosina Quinase ZAP-70/metabolismo , Produtos do Gene nef do Vírus da Imunodeficiência Humana/fisiologia , Linfócitos T CD4-Positivos/virologia , Linhagem Celular , Regulação da Expressão Gênica/imunologia , Humanos , Fosfatidilinositol 3-Quinases/antagonistas & inibidores , RNA Interferente Pequeno/genética
18.
EMBO J ; 25(19): 4423-35, 2006 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-16977309

RESUMO

The cation-independent mannose-6-phosphate receptor (CI-MPR) follows a highly regulated sorting itinerary to deliver hydrolases from the trans-Golgi network (TGN) to lysosomes. Cycling of CI-MPR between the TGN and early endosomes is mediated by GGA3, which directs TGN export, and PACS-1, which directs endosome-to-TGN retrieval. Despite executing opposing sorting steps, GGA3 and PACS-1 bind to an overlapping CI-MPR trafficking motif and their sorting activity is controlled by the CK2 phosphorylation of their respective autoregulatory domains. However, how CK2 coordinates these opposing roles is unknown. We report a CK2-activated phosphorylation cascade controlling PACS-1- and GGA3-mediated CI-MPR sorting. PACS-1 links GGA3 to CK2, forming a multimeric complex required for CI-MPR sorting. PACS-1-bound CK2 stimulates GGA3 phosphorylation, releasing GGA3 from CI-MPR and early endosomes. Bound CK2 also phosphorylates PACS-1Ser(278), promoting binding of PACS-1 to CI-MPR to retrieve the receptor to the TGN. Our results identify a CK2-controlled cascade regulating hydrolase trafficking and sorting of itinerant proteins in the TGN/endosomal system.


Assuntos
Fatores de Ribosilação do ADP/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Caseína Quinase II/metabolismo , Receptor IGF Tipo 2/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Sequência de Aminoácidos , Animais , Bovinos , Ativação Enzimática , Células HeLa , Humanos , Dados de Sequência Molecular , Proteínas Mutantes/metabolismo , Fosforilação , Ligação Proteica , Transporte Proteico , Ratos , Receptor IGF Tipo 2/química , Suínos , Vírus Vaccinia/fisiologia , Proteínas de Transporte Vesicular/química , Rede trans-Golgi/metabolismo
19.
J Biol Chem ; 281(23): 16108-16, 2006 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-16601116

RESUMO

The folding and activation of furin occur through two pH- and compartment-specific autoproteolytic steps. In the endoplasmic reticulum (ER), profurin folds under the guidance of its prodomain and undergoes an autoproteolytic excision at the consensus furin site Arg-Thr-Lys-Arg107/ generating an enzymatically masked furin-propeptide complex competent for transport to late secretory compartments. In the mildly acidic environment of the trans-Golgi network/endosomal system, the bound propeptide is cleaved at the internal site 69HRGVTKR75/, unmasking active furin capable of cleaving substrates in trans. Here, by using cellular, biochemical, and modeling studies, we demonstrate that the conserved His69 is a pH sensor that regulates the compartment-specific cleavages of the propeptide. In the ER, unprotonated His69 stabilizes a solvent-accessible hydrophobic pocket necessary for autoproteolytic excision at Arg107. Profurin molecules unable to form the hydrophobic pocket, and hence, the furin-propeptide complex, are restricted to the ER by a PACS-2- and COPI-dependent mechanism. Once exposed to the acidic pH of the late secretory pathway, protonated His69 disrupts the hydrophobic pocket, resulting in exposure and cleavage of the internal cleavage site at Arg75 to unmask the enzyme. Together, our data explain the pH-regulated activation of furin and how this His-dependent regulatory mechanism is a model for other proteins.


Assuntos
Furina/fisiologia , Concentração de Íons de Hidrogênio , Peptídeos/química , Sequência de Aminoácidos , Sequência de Bases , Western Blotting , Linhagem Celular , Eletroforese em Gel de Campo Pulsado , Retículo Endoplasmático/metabolismo , Ativação Enzimática , Furina/química , Furina/metabolismo , Dados de Sequência Molecular , Peptídeos/metabolismo , RNA Interferente Pequeno , Homologia de Sequência de Aminoácidos
20.
EMBO J ; 24(4): 717-29, 2005 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-15692567

RESUMO

The endoplasmic reticulum (ER) and mitochondria form contacts that support communication between these two organelles, including synthesis and transfer of lipids, and the exchange of calcium, which regulates ER chaperones, mitochondrial ATP production, and apoptosis. Despite the fundamental roles for ER-mitochondria contacts, little is known about the molecules that regulate them. Here we report the identification of a multifunctional sorting protein, PACS-2, that integrates ER-mitochondria communication, ER homeostasis, and apoptosis. PACS-2 controls the apposition of mitochondria with the ER, as depletion of PACS-2 causes BAP31-dependent mitochondria fragmentation and uncoupling from the ER. PACS-2 also controls formation of ER lipid-synthesizing centers found on mitochondria-associated membranes and ER homeostasis. However, in response to apoptotic inducers, PACS-2 translocates Bid to mitochondria, which initiates a sequence of events including the formation of mitochondrial truncated Bid, the release of cytochrome c, and the activation of caspase-3, thereby causing cell death. Together, our results identify PACS-2 as a novel sorting protein that links the ER-mitochondria axis to ER homeostasis and the control of cell fate, and provide new insights into Bid action.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Apoptose , Proteínas de Transporte/metabolismo , Retículo Endoplasmático/metabolismo , Mitocôndrias/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3 , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Retículo Endoplasmático/ultraestrutura , Homeostase , Humanos , Microscopia Eletrônica , Mitocôndrias/ultraestrutura , Transporte Proteico , RNA Interferente Pequeno , Proteínas de Transporte Vesicular
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