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1.
Mol Cell ; 79(1): 68-83.e7, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32533918

RESUMO

BAX is a pro-apoptotic protein that transforms from a cytosolic monomer into a toxic oligomer that permeabilizes the mitochondrial outer membrane. How BAX monomers assemble into a higher-order conformation, and the structural determinants essential to membrane permeabilization, remain a mechanistic mystery. A key hurdle has been the inability to generate a homogeneous BAX oligomer (BAXO) for analysis. Here, we report the production and characterization of a full-length BAXO that recapitulates physiologic BAX activation. Multidisciplinary studies revealed striking conformational consequences of oligomerization and insight into the macromolecular structure of oligomeric BAX. Importantly, BAXO enabled the assignment of specific roles to particular residues and α helices that mediate individual steps of the BAX activation pathway, including unexpected functionalities of BAX α6 and α9 in driving membrane disruption. Our results provide the first glimpse of a full-length and functional BAXO, revealing structural requirements for the elusive execution phase of mitochondrial apoptosis.


Assuntos
Apoptose , Mitocôndrias/patologia , Membranas Mitocondriais/metabolismo , Multimerização Proteica , Proteína X Associada a bcl-2/química , Proteína X Associada a bcl-2/metabolismo , Animais , Transporte Biológico , Permeabilidade da Membrana Celular , Citosol/metabolismo , Humanos , Camundongos , Mitocôndrias/metabolismo , Modelos Moleculares , Conformação Proteica , Proteínas Proto-Oncogênicas c-fos
2.
Mol Cell ; 69(5): 729-743.e7, 2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29499131

RESUMO

MCL-1 is a BCL-2 family protein implicated in the development and chemoresistance of human cancer. Unlike its anti-apoptotic homologs, Mcl-1 deletion has profound physiologic consequences, indicative of a broader role in homeostasis. We report that the BCL-2 homology 3 (BH3) α helix of MCL-1 can directly engage very long-chain acyl-CoA dehydrogenase (VLCAD), a key enzyme of the mitochondrial fatty acid ß-oxidation (FAO) pathway. Proteomic analysis confirmed that the mitochondrial matrix isoform of MCL-1 (MCL-1Matrix) interacts with VLCAD. Mcl-1 deletion, or eliminating MCL-1Matrix alone, selectively deregulated long-chain FAO, causing increased flux through the pathway in response to nutrient deprivation. Transient elevation in MCL-1 upon serum withdrawal, a striking increase in MCL-1 BH3/VLCAD interaction upon palmitic acid titration, and direct modulation of enzymatic activity by the MCL-1 BH3 α helix are consistent with dynamic regulation. Thus, the MCL-1 BH3 interaction with VLCAD revealed a separable, gain-of-function role for MCL-1 in the regulation of lipid metabolism.


Assuntos
Acil-CoA Desidrogenase de Cadeia Longa/metabolismo , Metabolismo dos Lipídeos/fisiologia , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Ácido Palmítico/metabolismo , Acil-CoA Desidrogenase de Cadeia Longa/genética , Animais , Linhagem Celular , Camundongos , Camundongos Knockout , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Oxirredução , Estrutura Secundária de Proteína
3.
Pharm Res ; 28(11): 2808-19, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21833796

RESUMO

PURPOSE: To create a new class of mitochondria-penetrating peptides (MPPs) that would facilitate drug delivery into the organelle through the inclusion of delocalized lipophilic cations (DLCs) in the peptide sequence. METHODS: We synthesized two novel amino acids featuring DLCs and incorporated them into peptides. Systematic studies were conducted to compare peptides containing these residues to those with natural cationic amino acids. Diastereomers were compared to determine the most advantageous arrangement for these peptides. Peptide lipophilicity, cellular uptake and mitochondrial specificity were compared for a variety of peptides. RESULTS: Synthetic DLC residues were found to increase mitochondrial localization of MPPs due to higher overall hydrophobicity. MPP stereochemistry was important for cellular uptake rather than subcellular localization. This study reaffirmed the importance of uniform overall charge distribution for mitochondrial specificity. CONCLUSIONS: DLCs can be incorporated into synthetic peptides and facilitate mitochondrial drug delivery. Lipophilicity and charge distribution must be carefully balanced to ensure localization within mitochondria.


Assuntos
Aminoácidos/síntese química , Cátions/química , Peptídeos Penetradores de Células/síntese química , Sistemas de Liberação de Medicamentos , Mitocôndrias/química , Terapia de Alvo Molecular , Aminoácidos/análise , Aminoácidos/química , Aminoácidos/metabolismo , Cátions/análise , Cátions/síntese química , Cátions/metabolismo , Técnicas de Cultura de Células , Peptídeos Penetradores de Células/análise , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/metabolismo , Composição de Medicamentos/métodos , Células HeLa , Humanos , Interações Hidrofóbicas e Hidrofílicas , Mitocôndrias/metabolismo , Sensibilidade e Especificidade
4.
Nat Biotechnol ; 37(10): 1186-1197, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31427820

RESUMO

The clinical translation of cationic α-helical antimicrobial peptides (AMPs) has been hindered by structural instability, proteolytic degradation and in vivo toxicity from nonspecific membrane lysis. Although analyses of hydrophobic content and charge distribution have informed the design of synthetic AMPs with increased potency and reduced in vitro hemolysis, nonspecific membrane toxicity in vivo continues to impede AMP drug development. Here, we analyzed a 58-member library of stapled AMPs (StAMPs) based on magainin II and applied the insights from structure-function-toxicity measurements to devise an algorithm for the design of stable, protease-resistant, potent and nontoxic StAMP prototypes. We show that a lead double-stapled StAMP named Mag(i+4)1,15(A9K,B21A,N22K,S23K) can kill multidrug-resistant Gram-negative pathogens, such as colistin-resistant Acinetobacter baumannii in a mouse peritonitis-sepsis model, without observed hemolysis or renal injury in murine toxicity studies. Inputting the amino acid sequences alone, we further generated membrane-selective StAMPs of pleurocidin, CAP18 and esculentin, highlighting the generalizability of our design platform.


Assuntos
Peptídeos Catiônicos Antimicrobianos/síntese química , Bactérias/efeitos dos fármacos , Peritonite/tratamento farmacológico , Sepse/tratamento farmacológico , Animais , Antibacterianos , Linhagem Celular , Desenho de Fármacos , Farmacorresistência Bacteriana , Eritrócitos/efeitos dos fármacos , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Peritonite/microbiologia , Sepse/microbiologia
6.
PLoS One ; 8(4): e60253, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23585833

RESUMO

We have successfully delivered a reactive alkylating agent, chlorambucil (Cbl), to the mitochondria of mammalian cells. Here, we characterize the mechanism of cell death for mitochondria-targeted chlorambucil (mt-Cbl) in vitro and assess its efficacy in a xenograft mouse model of leukemia. Using a ρ° cell model, we show that mt-Cbl toxicity is not dependent on mitochondrial DNA damage. We also illustrate that re-targeting Cbl to mitochondria results in a shift in the cell death mechanism from apoptosis to necrosis, and that this behavior is a general feature of mitochondria-targeted Cbl. Despite the change in cell death mechanisms, we show that mt-Cbl is still effective in vivo and has an improved pharmacokinetic profile compared to the parent drug. These findings illustrate that mitochondrial rerouting changes the site of action of Cbl and also alters the cell death mechanism drastically without compromising in vivo efficacy. Thus, mitochondrial delivery allows the exploitation of Cbl as a promiscuous mitochondrial protein inhibitor with promising therapeutic potential.


Assuntos
Antineoplásicos Alquilantes/farmacologia , Peptídeos Penetradores de Células/química , Clorambucila/farmacologia , Sistemas de Liberação de Medicamentos/métodos , Leucemia/tratamento farmacológico , Mitocôndrias/efeitos dos fármacos , Animais , Antineoplásicos Alquilantes/síntese química , Apoptose/efeitos dos fármacos , Clorambucila/síntese química , Reagentes de Ligações Cruzadas/química , DNA Mitocondrial , Células HeLa , Humanos , Leucemia/metabolismo , Leucemia/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Necrose/patologia , Espécies Reativas de Oxigênio/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Chem Biol ; 18(4): 445-53, 2011 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-21513881

RESUMO

The difficulty of accessing the mitochondrial matrix has limited the targeting of therapeutics to this organelle. Here, we report, to our knowledge, the first successful delivery of an active DNA alkylating agent--chlorambucil--to mitochondria, and describe unexpected features that result from rerouting this drug within the cell. Mitochondrial targeting of this agent dramatically potentiates its activity, and promotes apoptotic cell death in a variety of cancer cell lines and patient samples. This retention of activity is observed even in cells with resistance to chlorambucil or disabled apoptotic triggering.


Assuntos
Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Clorambucila/metabolismo , Clorambucila/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Mitocôndrias/metabolismo , Neoplasias/metabolismo , Alquilação/efeitos dos fármacos , Transporte Biológico , Linhagem Celular Tumoral , Dano ao DNA , Células HeLa , Humanos , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Neoplasias/patologia
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