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1.
Autophagy ; 17(11): 3725-3739, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-33783314

RESUMO

MOAP1 (modulator of apoptosis 1) is a BAX-binding protein tightly regulated by the ubiquitin-proteasome system. Apoptotic stimuli stabilize MOAP1 protein and facilitate its interaction with BAX to promote apoptosis. Here we show that in contrast to being resistant to apoptotic stimuli, MOAP1-deficient cells are hypersensitive to cell death mediated by starvation rendered by EBSS treatment. MOAP1-deficient cells exhibited impairment in macroautophagy/autophagy signaling induced by EBSS. Mechanistic analysis revealed that MOAP1-deficient cells had no notable defect in the recruitment of the pre-autophagosomal phosphatidylinositol-3-phosphate (PtdIns3P)-binding proteins, ZFYVE1/DFCP1 and WIPI2, nor in the LC3 lipidation mechanism regulated by the ATG12-ATG5-ATG16L1 complex upon EBSS treatment. Interestingly, MOAP1 is required for facilitating efficient closure of phagophore in the EBSS-treated cells. Analysis of LC3-positive membrane structures using Halo-tagged LC3 autophagosome completion assay showed that predominantly unclosed phagophore rather than closed autophagosome was present in the EBSS-treated MOAP1-deficient cells. The autophagy substrate SQSTM1/p62, which is normally contained within the enclosed autophagosome under EBSS condition, was also highly sensitive to degradation by proteinase K in the absence of MOAP1. MOAP1 binds LC3 and the binding is critically dependent on a LC3-interacting region (LIR) motif detected at its N-terminal region. Re-expression of MOAP1, but not its LC3-binding defective mutant, MOAP1-LIR, in the MOAP1-deficient cells, restored EBSS-induced autophagy. Together, these observations suggest that MOAP1 serves a distinct role in facilitating autophagy through interacting with LC3 to promote efficient phagophore closure during starvation.Abbreviations: CQ: Chloroquine; EBSS: Earle's Balanced Salt Solution; GABARAP: Gamma-Amino Butyric Acid Receptor Associated Protein; IF: Immunofluorescence; IP: Immunoprecipitation; LAMP1: Lysosomal-Associated Membrane Protein 1; LIR: LC3-Interacting Region; MAP1LC3/LC3: Microtubule Associated Protein 1 Light Chain 3; MEF: Mouse Embryonic Fibroblast; MOAP1: Modulator of Apoptosis 1; PE: Phosphatidylethanolamine; PtdIns3K: class III PtdIns3K complex I; PtdIns3P: Phosphatidylinositol-3-phosphate; STX17: Syntaxin 17; ULK1: unc-51 like autophagy activating kinase 1.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Autofagossomos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Animais , Proteínas Reguladoras de Apoptose/fisiologia , Autofagossomos/fisiologia , Imunofluorescência , Células HEK293 , Células HeLa , Humanos , Imunoprecipitação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Proteínas Associadas aos Microtúbulos/fisiologia
2.
Cell Rep ; 16(1): 174-185, 2016 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-27320914

RESUMO

Fas apoptotic signaling regulates diverse physiological processes. Acute activation of Fas signaling triggers massive apoptosis in liver. Upon Fas receptor stimulation, the BH3-only protein Bid is cleaved into the active form, tBid. Subsequent tBid recruitment to mitochondria, which is facilitated by its receptor MTCH2 at the outer mitochondrial membrane (OMM), is a critical step for commitment to apoptosis via the effector proteins Bax or Bak. MOAP-1 is a Bax-binding protein enriched at the OMM. Here, we show that MOAP-1-deficient mice are resistant to Fas-induced hepatocellular apoptosis and lethality. In the absence of MOAP-1, mitochondrial accumulation of tBid is markedly impaired. MOAP-1 binds to MTCH2, and this interaction appears necessary for MTCH2 to engage tBid. These findings reveal a role for MOAP-1 in Fas signaling in the liver by promoting MTCH2-mediated tBid recruitment to mitochondria.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Apoptose , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Fígado/citologia , Fígado/metabolismo , Mitocôndrias/metabolismo , Receptor fas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Sequência de Aminoácidos , Animais , Proteínas Reguladoras de Apoptose/química , Proteínas Reguladoras de Apoptose/deficiência , Fibroblastos/citologia , Fibroblastos/metabolismo , Células HCT116 , Hepatócitos/citologia , Hepatócitos/metabolismo , Humanos , Camundongos Knockout , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Ligação Proteica
3.
Chembiochem ; 17(2): 155-8, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26556305

RESUMO

We report a rationally designed nanobody activation immunotherapeutic that selectively redirects anti-dinitrophenyl (anti-DNP) antibodies to the surface of HER2-positive breast cancer cells, resulting in their targeted destruction by antibody-dependent cellular cytotoxicity. As nanobodies are relatively easy to express, stable, can be humanized, and can be evolved to potently and selectively bind virtually any disease-relevant cell surface receptor, we anticipate broad utility of this therapeutic strategy.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Genes erbB-2 , Linhagem Celular Tumoral , Feminino , Genes erbB-2/efeitos dos fármacos , Humanos , Imunoterapia , Estrutura Molecular
4.
Chem Sci ; 5(6): 2311-2317, 2014 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-25379167

RESUMO

Here we report on the structure-based optimization of antibody-recruiting molecules targeting HIV gp120 (ARM-H). These studies have leveraged a combination of medicinal chemistry, biochemical and cellular assay analysis, and computation. Our findings have afforded an optimized analog of ARM-H, which is ~1000 fold more potent in gp120-binding and MT-2 antiviral assays than our previously reported derivative. Furthermore, computational analysis, taken together with experimental data, provides evidence that azaindole- and indole-based attachment inhibitors bind gp120 at an accessory hydrophobic pocket beneath the CD4-binding site and can also adopt multiple unique binding modes in interacting with gp120. These results are likely to prove highly enabling in the development of novel HIV attachment inhibitors, and more broadly, they suggest novel applications for ARMs as probes of conformationally flexible systems.

5.
ACS Chem Biol ; 8(11): 2404-11, 2013 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-24053626

RESUMO

The ability to profile the prevalence and functional activity of endogenous antibodies is of vast clinical and diagnostic importance. Serum antibodies are an important class of biomarkers and are also crucial elements of immune responses elicited by natural disease-causing agents as well as vaccines. In particular, materials for manipulating and/or enhancing immune responses toward disease-causing cells or viruses have exhibited significant promise for therapeutic applications. Antibody-recruiting molecules (ARMs), bifunctional organic molecules that redirect endogenous antibodies to pathological targets, thereby increasing their recognition and clearance by the immune system, have proven particularly interesting. Notably, although ARMs capable of hijacking antibodies against oligosaccharides and electron-poor aromatics have proven efficacious, systematic comparisons of the prevalence and effectiveness of natural anti-hapten antibody populations have not appeared in the literature. Herein we report head-to-head comparisons of three chemically simple antigens, which are known ligands for endogenous antibodies. Thus, we have chemically synthesized bifunctional molecules containing 2,4-dinitrophenyl (DNP), phosphorylcholine (PC), and rhamnose. We have then used a combination of ELISA, flow cytometry, and cell-viability assays to compare these antigens in terms of their abilities both to recruit natural antibody from human serum and also to direct serum-dependent cytotoxicity against target cells. These studies have revealed rhamnose to be the most efficacious of the synthetic antigens examined. Furthermore, analysis of 122 individual serum samples has afforded comprehensive insights into population-wide prevalence and isotype distributions of distinct anti-hapten antibody populations. In addition to providing a general platform for comparing and studying anti-hapten antibodies, these studies serve as a useful starting point for the optimization of antibody-recruiting molecules and other synthetic strategies for modulating human immunity.


Assuntos
Anticorpos/metabolismo , Antígenos/metabolismo , Indústria Farmacêutica , Fatores Imunológicos/síntese química , Animais , Anticorpos/química , Ligação Competitiva , Células CHO , Sobrevivência Celular , Cricetulus , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Fatores Imunológicos/química , Fatores Imunológicos/metabolismo , Estrutura Molecular , Peso Molecular , Fenilacetatos/metabolismo , Fosforilcolina/metabolismo , Ligação Proteica , Ramnose/metabolismo
6.
Org Lett ; 14(17): 4544-7, 2012 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-22917221

RESUMO

The carbocyclic core of the phomoidrides has been synthesized efficiently and in high yield. Key steps include a phenolic oxidation/intramolecular Diels-Alder sequence, tandem radical cyclization, and a late-stage Wharton fragmentation of a densely functionalized isotwistane skeleton.


Assuntos
Anidridos Maleicos/síntese química , Cristalografia por Raios X , Ciclização , Anidridos Maleicos/química , Anidridos Maleicos/farmacologia , Conformação Molecular , Estrutura Molecular , Oxirredução
7.
Tetrahedron ; 66(33): 6647-6655, 2010 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-20733933

RESUMO

Described is the construction of the N-methylwelwitindolinone C core via an efficient strategy that employs a sequential rhodium carbenoid-mediated O-H insertion, Claisen rearrangement and transannular [3+2] nitrone cycloaddition.

8.
Proc Natl Acad Sci U S A ; 107(11): 5018-23, 2010 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-20194754

RESUMO

Loss of the E3 ubiquitin ligase Parkin causes early onset Parkinson's disease, a neurodegenerative disorder of unknown etiology. Parkin has been linked to multiple cellular processes including protein degradation, mitochondrial homeostasis, and autophagy; however, its precise role in pathogenesis is unclear. Recent evidence suggests that Parkin is recruited to damaged mitochondria, possibly affecting mitochondrial fission and/or fusion, to mediate their autophagic turnover. The precise mechanism of recruitment and the ubiquitination target are unclear. Here we show in Drosophila cells that PINK1 is required to recruit Parkin to dysfunctional mitochondria and promote their degradation. Furthermore, PINK1 and Parkin mediate the ubiquitination of the profusion factor Mfn on the outer surface of mitochondria. Loss of Drosophila PINK1 or parkin causes an increase in Mfn abundance in vivo and concomitant elongation of mitochondria. These findings provide a molecular mechanism by which the PINK1/Parkin pathway affects mitochondrial fission/fusion as suggested by previous genetic interaction studies. We hypothesize that Mfn ubiquitination may provide a mechanism by which terminally damaged mitochondria are labeled and sequestered for degradation by autophagy.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Proteínas de Membrana/metabolismo , Mitocôndrias/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Ubiquitinação , Animais , Autofagia , Drosophila melanogaster/citologia , Técnicas de Silenciamento de Genes , Mutação/genética , Transporte Proteico , Ubiquitina-Proteína Ligases
9.
Nat Neurosci ; 12(9): 1129-35, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19684592

RESUMO

Mutations in PINK1 and PARK2 cause autosomal recessive parkinsonism, a neurodegenerative disorder that is characterized by the loss of dopaminergic neurons. To discover potential therapeutic pathways, we identified factors that genetically interact with Drosophila park and Pink1. We found that overexpression of the translation inhibitor Thor (4E-BP) can suppress all of the pathologic phenotypes, including degeneration of dopaminergic neurons in Drosophila. 4E-BP is activated in vivo by the TOR inhibitor rapamycin, which could potently suppress pathology in Pink1 and park mutants. Rapamycin also ameliorated mitochondrial defects in cells from individuals with PARK2 mutations. Recently, 4E-BP was shown to be inhibited by the most common cause of parkinsonism, dominant mutations in LRRK2. We also found that loss of the Drosophila LRRK2 homolog activated 4E-BP and was also able to suppress Pink1 and park pathology. Thus, in conjunction with recent findings, our results suggest that pharmacologic stimulation of 4E-BP activity may represent a viable therapeutic approach for multiple forms of parkinsonism.


Assuntos
Dopamina/metabolismo , Proteínas de Drosophila/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neurônios/fisiologia , Fármacos Neuroprotetores/farmacologia , Fatores de Iniciação de Peptídeos/metabolismo , Sirolimo/farmacologia , Animais , Animais Geneticamente Modificados , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Drosophila , Proteínas de Drosophila/genética , Fibroblastos/efeitos dos fármacos , Fibroblastos/fisiologia , Glutationa Transferase/metabolismo , Humanos , Locomoção/efeitos dos fármacos , Locomoção/fisiologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/fisiologia , Músculos/efeitos dos fármacos , Músculos/ultraestrutura , Degeneração Neural/tratamento farmacológico , Degeneração Neural/fisiopatologia , Neurônios/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/genética , Ubiquitina-Proteína Ligases/metabolismo
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