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1.
Proc Natl Acad Sci U S A ; 114(29): 7629-7634, 2017 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-28673969

RESUMO

BAK and BAX are the essential effectors of apoptosis because without them a cell is resistant to most apoptotic stimuli. BAK and BAX undergo conformation changes to homooligomerize then permeabilize the mitochondrial outer membrane during apoptosis. How BCL-2 homology 3 (BH3)-only proteins bind to activate BAK and BAX is unclear. We report that BH3-only proteins bind inactive full-length BAK at mitochondria and then dissociate following exposure of the BAK BH3 and BH4 domains before BAK homodimerization. Using a functional obstructive labeling approach, we show that activation of BAK involves important interactions of BH3-only proteins with both the canonical hydrophobic binding groove (α2-5) and α6 at the rear of BAK, with interaction at α6 promoting an open groove to receive a BH3-only protein. Once activated, how BAK homodimers multimerize to form the putative apoptotic pore is unknown. Obstructive labeling of BAK beyond the BH3 domain and hydrophobic groove did not inhibit multimerization and mitochondrial damage, indicating that critical protein-protein interfaces in BAK self-association are limited to the α2-5 homodimerization domain.


Assuntos
Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/química , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Animais , Apoptose , Sítios de Ligação , Linhagem Celular , Citocromos c/metabolismo , Dissulfetos/química , Epitopos/química , Fibroblastos/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Ligação Proteica , Domínios Proteicos , Mapeamento de Interação de Proteínas , Multimerização Proteica , Proteína X Associada a bcl-2/metabolismo
2.
Genome Res ; 20(12): 1629-38, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21051460

RESUMO

More than 25 loci have been linked to type 1 diabetes (T1D) in the nonobese diabetic (NOD) mouse, but identification of the underlying genes remains challenging. We describe here the positional cloning of a T1D susceptibility locus, Idd11, located on mouse chromosome 4. Sequence analysis of a series of congenic NOD mouse strains over a critical 6.9-kb interval in these mice and in 25 inbred strains identified several haplotypes, including a unique NOD haplotype, associated with varying levels of T1D susceptibility. Haplotype diversity within this interval between congenic NOD mouse strains was due to a recombination hotspot that generated four crossover breakpoints, including one with a complex conversion tract. The Idd11 haplotype and recombination hotspot are located within a predicted gene of unknown function, which exhibits decreased expression in relevant tissues of NOD mice. Notably, it was the recombination hotspot that aided our mapping of Idd11 and confirms that recombination hotspots can create genetic variation affecting a common polygenic disease. This finding has implications for human genetic association studies, which may be affected by the approximately 33,000 estimated hotspots in the genome.


Assuntos
Troca Genética/genética , Diabetes Mellitus Tipo 1/genética , Predisposição Genética para Doença/genética , Variação Genética , Animais , Sequência de Bases , Mapeamento Cromossômico , Biologia Computacional , Haplótipos/genética , Camundongos , Camundongos Endogâmicos NOD , Dados de Sequência Molecular , Análise de Sequência de DNA
3.
Cell Death Differ ; 30(3): 632-646, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36171332

RESUMO

Intrinsic apoptosis is principally governed by the BCL-2 family of proteins, but some non-BCL-2 proteins are also critical to control this process. To identify novel apoptosis regulators, we performed a genome-wide CRISPR-Cas9 library screen, and it identified the mitochondrial E3 ubiquitin ligase MARCHF5/MITOL/RNF153 as an important regulator of BAK apoptotic function. Deleting MARCHF5 in diverse cell lines dependent on BAK conferred profound resistance to BH3-mimetic drugs. The loss of MARCHF5 or its E3 ubiquitin ligase activity surprisingly drove BAK to adopt an activated conformation, with resistance to BH3-mimetics afforded by the formation of inhibitory complexes with pro-survival proteins MCL-1 and BCL-XL. Importantly, these changes to BAK conformation and pro-survival association occurred independently of BH3-only proteins and influence on pro-survival proteins. This study identifies a new mechanism by which MARCHF5 regulates apoptotic cell death by restraining BAK activating conformation change and provides new insight into how cancer cells respond to BH3-mimetic drugs. These data also highlight the emerging role of ubiquitin signalling in apoptosis that may be exploited therapeutically.


Assuntos
Ubiquitina-Proteína Ligases , Proteína Killer-Antagonista Homóloga a bcl-2 , Proteína bcl-X/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Apoptose/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo
4.
J Immunol ; 184(2): 859-68, 2010 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-20007538

RESUMO

Autoimmune lymphocytic infiltration of the salivary glands, termed sialadenitis, is a pathologic feature of Sjögren's syndrome (SjS) that is also prominent in nonobese diabetic (NOD) mice. Genetic factors regulate sialadenitis, and a previous (NOD x NZW)F2 study detected linkage to murine chromosome (Chr) 7. The locus, subsequently annotated as Ssial3, maps to the distal end of Chr7 and overlaps a region associated with type 1 diabetes susceptibility in NOD mice. To examine whether Ssial3 could contribute to both diseases, or was specific for SjS, we generated a congenic mouse strain that harbored an NZW-derived Chr7 interval on the NOD genetic background. This congenic strain exhibited reduced sialadenitis compared with NOD mice and confirmed Ssial3. This reduction, however, did not ameliorate saliva abnormalities associated with SjS-like disease in NOD mice, nor were congenic mice protected against insulitis (lymphocytic infiltration of the pancreatic islets) or diabetes onset. Thus, the Ssial3 locus appears to have a tissue-specific effect for which the NZW allele is unable to prevent other autoimmune traits in the NOD mouse. Anomalous increases for antinuclear Ab production and frequency of marginal-zone B cells were also identified in congenic mice, indicating that the NZW-derived Chr7 interval has a complex effect on the NOD immune system.


Assuntos
Diabetes Mellitus Tipo 1/genética , Ligação Genética , Sialadenite/genética , Animais , Anticorpos Antinucleares/biossíntese , Linfócitos B/patologia , Mapeamento Cromossômico , Cromossomos , Predisposição Genética para Doença , Camundongos , Camundongos Congênicos , Camundongos Endogâmicos NOD
5.
Nat Commun ; 9(1): 4976, 2018 11 26.
Artigo em Inglês | MEDLINE | ID: mdl-30478310

RESUMO

Intrinsic apoptosis is critical to prevent tumor formation and is engaged by many anti-cancer agents to eliminate tumor cells. BAX and BAK, the two essential mediators of apoptosis, are thought to be regulated through similar mechanisms and act redundantly to drive apoptotic cell death. From an unbiased genome-wide CRISPR/Cas9 screen, we identified VDAC2 (voltage-dependent anion channel 2) as important for BAX, but not BAK, to function. Genetic deletion of VDAC2 abrogated the association of BAX and BAK with mitochondrial complexes containing VDAC1, VDAC2, and VDAC3, but only inhibited BAX apoptotic function. Deleting VDAC2 phenocopied the loss of BAX in impairing both the killing of tumor cells by anti-cancer agents and the ability to suppress tumor formation. Together, our studies show that efficient BAX-mediated apoptosis depends on VDAC2, and reveal a striking difference in how BAX and BAK are functionally impacted by their interactions with VDAC2.


Assuntos
Apoptose , Carcinogênese/metabolismo , Carcinogênese/patologia , Canal de Ânion 2 Dependente de Voltagem/metabolismo , Proteína X Associada a bcl-2/metabolismo , Animais , Sistemas CRISPR-Cas/genética , Desenvolvimento Embrionário , Células HCT116 , Células HeLa , Humanos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Regiões Promotoras Genéticas/genética , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo
6.
Nat Commun ; 6: 6841, 2015 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-25880232

RESUMO

During apoptosis, Bak permeabilizes mitochondria after undergoing major conformational changes, including poorly defined N-terminal changes. Here, we characterize those changes using 11 antibodies that were epitope mapped using peptide arrays and mutagenesis. After Bak activation by Bid, epitopes throughout the α1 helix are exposed indicating complete dissociation of α1 from α2 in the core and from α6-α8 in the latch. Moreover, disulfide tethering of α1 to α2 or α6 blocks cytochrome c release, suggesting that α1 dissociation is required for further conformational changes during apoptosis. Assaying epitope exposure when α1 is tethered shows that Bid triggers α2 movement, followed by α1 dissociation. However, α2 reaches its final position only after α1 dissociates from the latch. Thus, α1 dissociation is a key step in unfolding Bak into three major components, the N terminus, the core (α2-α5) and the latch (α6-α8).


Assuntos
Apoptose , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Animais , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Linhagem Celular , Linhagem Celular Transformada , Mapeamento de Epitopos , Humanos , Camundongos , Mutagênese Sítio-Dirigida , Análise Serial de Proteínas
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