RESUMO
Targeted protein degradation is a pharmacological modality that is based on the induced proximity of an E3 ubiquitin ligase and a target protein to promote target ubiquitination and proteasomal degradation. This has been achieved either via proteolysis-targeting chimeras (PROTACs)-bifunctional compounds composed of two separate moieties that individually bind the target and E3 ligase, or via molecular glues that monovalently bind either the ligase or the target1-4. Here, using orthogonal genetic screening, biophysical characterization and structural reconstitution, we investigate the mechanism of action of bifunctional degraders of BRD2 and BRD4, termed intramolecular bivalent glues (IBGs), and find that instead of connecting target and ligase in trans as PROTACs do, they simultaneously engage and connect two adjacent domains of the target protein in cis. This conformational change 'glues' BRD4 to the E3 ligases DCAF11 or DCAF16, leveraging intrinsic target-ligase affinities that do not translate to BRD4 degradation in the absence of compound. Structural insights into the ternary BRD4-IBG1-DCAF16 complex guided the rational design of improved degraders of low picomolar potency. We thus introduce a new modality in targeted protein degradation, which works by bridging protein domains in cis to enhance surface complementarity with E3 ligases for productive ubiquitination and degradation.
Assuntos
Desenho de Fármacos , Proteólise , Especificidade por Substrato , Ubiquitina-Proteína Ligases , Ubiquitinação , Proteínas que Contêm Bromodomínio/metabolismo , Proteínas de Ciclo Celular/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Quimera de Direcionamento de Proteólise , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ligação Proteica , Domínios ProteicosRESUMO
Exposure of cells to heat or oxidative stress causes misfolding of proteins. To avoid toxic protein aggregation, cells have evolved nuclear and cytosolic protein quality control (PQC) systems. In response to proteotoxic stress, cells also limit protein synthesis by triggering transient storage of mRNAs and RNA-binding proteins (RBPs) in cytosolic stress granules (SGs). We demonstrate that the SUMO-targeted ubiquitin ligase (StUbL) pathway, which is part of the nuclear proteostasis network, regulates SG dynamics. We provide evidence that inactivation of SUMO deconjugases under proteotoxic stress initiates SUMO-primed, RNF4-dependent ubiquitylation of RBPs that typically condense into SGs. Impairment of SUMO-primed ubiquitylation drastically delays SG resolution upon stress release. Importantly, the StUbL system regulates compartmentalization of an amyotrophic lateral sclerosis (ALS)-associated FUS mutant in SGs. We propose that the StUbL system functions as surveillance pathway for aggregation-prone RBPs in the nucleus, thereby linking the nuclear and cytosolic axis of proteotoxic stress response.
Assuntos
Esclerose Lateral Amiotrófica/patologia , Núcleo Celular/metabolismo , Grânulos Citoplasmáticos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteína SUMO-1/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitina/metabolismo , Esclerose Lateral Amiotrófica/genética , Núcleo Celular/genética , Células HeLa , Resposta ao Choque Térmico , Humanos , Mutação , Proteínas Nucleares/genética , Proteólise , Proteína FUS de Ligação a RNA/genética , Proteínas de Ligação a RNA/genética , Proteína SUMO-1/genética , Sumoilação , Fatores de Transcrição/genética , UbiquitinaçãoRESUMO
Selective autophagy of mitochondria, mitophagy, is linked to mitochondrial quality control and as such is critical to a healthy organism. We have used a CRISPR/Cas9 approach to screen human E3 ubiquitin ligases for influence on mitophagy under both basal cell culture conditions and upon acute mitochondrial depolarization. We identify two cullin-RING ligase substrate receptors, VHL and FBXL4, as the most profound negative regulators of basal mitophagy. We show that these converge, albeit via different mechanisms, on control of the mitophagy adaptors BNIP3 and BNIP3L/NIX. FBXL4 restricts NIX and BNIP3 levels via direct interaction and protein destabilization, while VHL acts through suppression of HIF1α-mediated transcription of BNIP3 and NIX. Depletion of NIX but not BNIP3 is sufficient to restore mitophagy levels. Our study contributes to an understanding of the aetiology of early-onset mitochondrial encephalomyopathy that is supported by analysis of a disease-associated mutation. We further show that the compound MLN4924, which globally interferes with cullin-RING ligase activity, is a strong inducer of mitophagy, thus providing a research tool in this context and a candidate therapeutic agent for conditions linked to mitochondrial dysfunction.
Assuntos
Mitofagia , Ubiquitina , Humanos , Mitofagia/fisiologia , Ubiquitina/metabolismo , Proteínas Culina/metabolismo , Mitocôndrias/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Autofagia/fisiologiaRESUMO
Autophagy allows the degradation of cytosolic endogenous and exogenous material in the lysosome. Substrates are engulfed by double-membrane vesicles, coined autophagosomes, which subsequently fuse with lysosomes. Depending on the involvement of specific receptor proteins, autophagy occurs in a selective or nonselective manner. While this process is well understood at the level of bulky cargo such as mitochondria and bacteria, we know very little about individual proteins and protein complexes that are engulfed and degraded by autophagy. In contrast to the critical role of autophagy in balancing proteostasis, our current knowledge of the autophagic degradome is very limited. Here, we combined proximity labeling with quantitative proteomics to systematically map the protein inventory of autophagosomes. Using this strategy, we uncovered a basal, housekeeping mitophagy pathway that involves piecemeal degradation of mitochondrial proteins in a LC3C- and p62-dependent manner and contributes to mitochondrial homeostasis maintenance when cells rely on oxidative phosphorylation.
Assuntos
Proteínas Associadas aos Microtúbulos/metabolismo , Mitofagia/fisiologia , Fosforilação Oxidativa , Fagossomos/metabolismo , Proteólise , Células HeLa , Humanos , Proteínas Associadas aos Microtúbulos/genética , Fagossomos/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
SUMMARY: CRISPR screens are increasingly performed to associate genotypes with genotypes. So far, however, their analysis required specialized computational knowledge to transform high-throughput next-generation sequencing (NGS) data into sequence formats amenable for downstream analysis. We developed ReCo, a stand-alone and user-friendly analytics tool for generating read-count tables of single and combinatorial CRISPR library and screen-based NGS data. Together with cutadapt and bowtie2 for rapid sequence trimming and alignment, ReCo enables the automated generation of read count tables from staggered NGS reads for the downstream identification of gRNA-induced phenotypes. AVAILABILITY AND IMPLEMENTATION: ReCo is published under the MIT license and available at: https://github.com/KaulichLab/ReCo.
Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Software , Sequenciamento de Nucleotídeos em Larga Escala , Biblioteca Gênica , Análise de Sequência de DNARESUMO
Understanding how epigenetic variation in non-coding regions is involved in distal gene-expression regulation is an important problem. Regulatory regions can be associated to genes using large-scale datasets of epigenetic and expression data. However, for regions of complex epigenomic signals and enhancers that regulate many genes, it is difficult to understand these associations. We present StitchIt, an approach to dissect epigenetic variation in a gene-specific manner for the detection of regulatory elements (REMs) without relying on peak calls in individual samples. StitchIt segments epigenetic signal tracks over many samples to generate the location and the target genes of a REM simultaneously. We show that this approach leads to a more accurate and refined REM detection compared to standard methods even on heterogeneous datasets, which are challenging to model. Also, StitchIt REMs are highly enriched in experimentally determined chromatin interactions and expression quantitative trait loci. We validated several newly predicted REMs using CRISPR-Cas9 experiments, thereby demonstrating the reliability of StitchIt. StitchIt is able to dissect regulation in superenhancers and predicts thousands of putative REMs that go unnoticed using peak-based approaches suggesting that a large part of the regulome might be uncharted water.
Assuntos
Cromatina/metabolismo , Análise de Dados , Elementos Facilitadores Genéticos , Epigênese Genética , Regulação da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana , HumanosRESUMO
Combinatorial CRISPR-Cas screens have advanced the mapping of genetic interactions, but their experimental scale limits the number of targetable gene combinations. Here, we describe 3Cs multiplexing, a rapid and scalable method to generate highly diverse and uniformly distributed combinatorial CRISPR libraries. We demonstrate that the library distribution skew is the critical determinant of its required screening coverage. By circumventing iterative cloning of PCR-amplified oligonucleotides, 3Cs multiplexing facilitates the generation of combinatorial CRISPR libraries with low distribution skews. We show that combinatorial 3Cs libraries can be screened with minimal coverages, reducing associated efforts and costs at least 10-fold. We apply a 3Cs multiplexing library targeting 12,736 autophagy gene combinations with 247,032 paired gRNAs in viability and reporter-based enrichment screens. In the viability screen, we identify, among others, the synthetic lethal WDR45B-PIK3R4 and the proliferation-enhancing ATG7-KEAP1 genetic interactions. In the reporter-based screen, we identify over 1,570 essential genetic interactions for autophagy flux, including interactions among paralogous genes, namely ATG2A-ATG2B, GABARAP-MAP1LC3B and GABARAP-GABARAPL2. However, we only observe few genetic interactions within paralogous gene families of more than two members, indicating functional compensation between them. This work establishes 3Cs multiplexing as a platform for genetic interaction screens at scale.
Assuntos
Autofagia/genética , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Técnicas de Inativação de Genes/métodos , Redes Reguladoras de Genes/genética , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Carcinoma de Células Escamosas/mortalidade , Proliferação de Células/genética , Sobrevivência Celular/genética , Bases de Dados Genéticas , Biblioteca Gênica , Genes Essenciais , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Estimativa de Kaplan-Meier , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Neoplasias Pulmonares/mortalidade , Modelos Genéticos , RNA Guia de Cinetoplastídeos , RNA-Seq , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMO
While studies of the autophagy-related (ATG) genes in knockout models have led to an explosion of knowledge about the functions of autophagy components, the exact roles of LC3 and GABARAP family proteins (human ATG8 equivalents) are still poorly understood. A major drawback in understanding their roles is that the available interactome data has largely been acquired using overexpression systems. To overcome these limitations, we employed CRISPR/Cas9-based genome-editing to generate a panel of cells in which human ATG8 genes were tagged at their natural chromosomal locations with an N-terminal affinity epitope. This cellular resource was employed to map endogenous GABARAPL2 protein complexes using interaction proteomics. This approach identified the ER-associated protein and lipid droplet (LD) biogenesis factor ACSL3 as a stabilizing GABARAPL2-binding partner. GABARAPL2 bound ACSL3 in a manner dependent on its LC3-interacting regions, whose binding site in GABARAPL2 was required to recruit the latter to the ER. Through this interaction, the UFM1-activating enzyme UBA5 became anchored at the ER. Furthermore, ACSL3 depletion and LD induction affected the abundance of several ufmylation components and ER-phagy. Together these data allow us to define ACSL3 as a novel regulator of the enigmatic UFM1 conjugation pathway.
Assuntos
Gotículas Lipídicas , Proteínas , Autofagia , Família da Proteína 8 Relacionada à Autofagia , Humanos , Enzimas Ativadoras de UbiquitinaRESUMO
The small GTPases H, K, and NRAS are molecular switches indispensable for proper regulation of cellular proliferation and growth. Several mutations in the genes encoding members of this protein family are associated with cancer and result in aberrant activation of signaling processes caused by a deregulated recruitment of downstream effector proteins. In this study, we engineered variants of the Ras-binding domain (RBD) of the C-Raf proto-oncogene, Ser/Thr kinase (CRAF). These variants bound with high affinity with the effector-binding site of Ras in an active conformation. Structural characterization disclosed how the newly identified RBD mutations cooperate and thereby enhance affinity with the effector-binding site in Ras compared with WT RBD. The engineered RBD variants closely mimicked the interaction mode of naturally occurring Ras effectors and acted as dominant-negative affinity reagents that block Ras signal transduction. Experiments with cancer cells showed that expression of these RBD variants inhibits Ras signaling, reducing cell growth and inducing apoptosis. Using these optimized RBD variants, we stratified patient-derived colorectal cancer organoids with known Ras mutational status according to their response to Ras inhibition. These results revealed that the presence of Ras mutations was insufficient to predict sensitivity to Ras inhibition, suggesting that not all of these tumors required Ras signaling for proliferation. In summary, by engineering the Ras/Raf interface of the CRAF-RBD, we identified potent and selective inhibitors of Ras in its active conformation that outcompete binding of Ras-signaling effectors.
Assuntos
Proteínas Proto-Oncogênicas c-raf/metabolismo , Proteínas ras/metabolismo , Apoptose , Sítios de Ligação , Linhagem Celular Tumoral , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Simulação de Dinâmica Molecular , Mutagênese , Fosfatidilinositol 3-Quinases/metabolismo , Ligação Proteica , Domínios Proteicos , Estrutura Terciária de Proteína , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-raf/química , Proteínas Proto-Oncogênicas c-raf/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Transdução de Sinais , Proteínas ras/antagonistas & inibidores , Proteínas ras/genéticaRESUMO
Genome-wide CRISPR screens are becoming more widespread and allow the simultaneous interrogation of thousands of genomic regions. Although recent progress has been made in the analysis of CRISPR screens, it is still an open problem how to interpret CRISPR mutations in non-coding regions of the genome. Most of the tools concentrate on the interpretation of mutations introduced in gene coding regions. We introduce a computational pipeline that uses epigenomic information about regulatory elements for the interpretation of CRISPR mutations in non-coding regions. We illustrate our analysis protocol on the analysis of a genome-wide CRISPR screen in hTERT-RPE1 cells and reveal novel regulatory elements that mediate chemoresistance against doxorubicin in these cells. We infer links to established and to novel chemoresistance genes. Our analysis protocol is general and can be applied on any cell type and with different CRISPR enzymes.
Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , GenômicaRESUMO
For a long time, gene editing had been a scientific concept, which was limited to a few applications. With recent developments, following the discovery of TALEN zinc-finger endonucleases and in particular the CRISPR/Cas system, gene editing has become a technique applicable in most laboratories. The current gain- and loss-of function models in basic science are revolutionary as they allow unbiased screens of unprecedented depth and complexity and rapid development of transgenic animals. Modifications of CRISPR/Cas have been developed to precisely interrogate epigenetic regulation or to visualize DNA complexes. Moreover, gene editing as a clinical treatment option is rapidly developing with first trials on the way. This article reviews the most recent progress in the field, covering expert opinions gathered during joint conferences on genome editing of the German Cardiac Society (DGK) and the German Center for Cardiovascular Research (DZHK). Particularly focusing on the translational aspect and the combination of cellular and animal applications, the authors aim to provide direction for the development of the field and the most frequent applications with their problems.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes/tendências , Terapia Genética/tendências , Pesquisa Translacional Biomédica/tendências , Animais , Congressos como Assunto , Difusão de Inovações , Modelos Animais de Doenças , Previsões , Predisposição Genética para Doença , Humanos , FenótipoRESUMO
The UDP-glucose ceramide glucosyltransferase (UGCG) is a key enzyme in the synthesis of glycosylated sphingolipids, since this enzyme generates the precursor for all complex glycosphingolipids (GSL), the GlcCer. The UGCG has been associated with several cancer-related processes such as maintaining cancer stem cell properties or multidrug resistance induction. The precise mechanisms underlying these processes are unknown. Here, we investigated the molecular mechanisms occurring after UGCG overexpression in breast cancer cells. We observed alterations of several cellular properties such as morphological changes, which enhanced proliferation and doxorubicin resistance in UGCG overexpressing MCF-7 cells. These cellular effects seem to be mediated by an altered composition of glycosphingolipid-enriched microdomains (GEMs), especially an accumulation of globotriaosylceramide (Gb3) and glucosylceramide (GlcCer), which leads to an activation of Akt and ERK1/2. The induction of the Akt and ERK1/2 signaling pathway results in an increased gene expression of multidrug resistance protein 1 (MDR1) and anti-apoptotic genes and a decrease of pro-apoptotic gene expression. Inhibition of the protein kinase C (PKC) and phosphoinositide 3 kinase (PI3K) reduced MDR1 gene expression. This study discloses how changes in UGCG expression impact several cellular signaling pathways in breast cancer cells resulting in enhanced proliferation and multidrug resistance.
Assuntos
Proliferação de Células , Resistencia a Medicamentos Antineoplásicos , Glucosiltransferases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Apoptose/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Colesterol/análise , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Glucosiltransferases/genética , Humanos , Células MCF-7 , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Transdução de Sinais/genética , Esfingolipídeos/análise , Esfingolipídeos/metabolismo , Esfingomielina Fosfodiesterase/genética , Esfingomielina Fosfodiesterase/metabolismo , Esfingosina N-Aciltransferase/genética , Esfingosina N-Aciltransferase/metabolismoRESUMO
PICH is a DNA translocase required for the maintenance of chromosome stability in human cells. Recent data indicate that PICH co-operates with topoisomerase IIα to suppress pathological chromosome missegregation through promoting the resolution of ultra-fine anaphase bridges (UFBs). Here, we identify the BEN domain-containing protein 3 (BEND3) as an interaction partner of PICH in human cells in mitosis. We have purified full length PICH and BEND3 and shown that they exhibit a functional biochemical interaction in vitro. We demonstrate that the PICH-BEND3 interaction occurs via a novel interface between a TPR domain in PICH and a BEN domain in BEND3, and have determined the crystal structure of this TPR-BEN complex at 2.2 Å resolution. Based on the structure, we identified amino acids important for the TPR-BEN domain interaction, and for the functional interaction of the full-length proteins. Our data reveal a proposed new function for BEND3 in association with PICH, and the first example of a specific protein-protein interaction mediated by a BEN domain.
Assuntos
Motivos de Aminoácidos , DNA Helicases/química , Domínios Proteicos , Proteínas Repressoras/química , Sequência de Aminoácidos , Sítios de Ligação/genética , Cristalografia por Raios X , DNA Helicases/genética , DNA Helicases/metabolismo , Células HEK293 , Células HeLa , Humanos , Mitose/genética , Modelos Moleculares , Ligação Proteica , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Atypical protein kinase C (aPKC) isoenzymes are key modulators of insulin signalling, and their dysfunction correlates with insulin-resistant states in both mice and humans. Despite the engaged interest in the importance of aPKCs to type 2 diabetes, much less is known about the molecular mechanisms that govern their cellular functions than for the conventional and novel PKC isoenzymes and the functionally-related protein kinase B (Akt) family of kinases. Here we show that aPKC is constitutively phosphorylated and, using a genetically-encoded reporter for PKC activity, basally active in cells. Specifically, we show that phosphorylation at two key regulatory sites, the activation loop and turn motif, of the aPKC PKCζ in multiple cultured cell types is constitutive and independently regulated by separate kinases: ribosome-associated mammalian target of rapamycin complex 2 (mTORC2) mediates co-translational phosphorylation of the turn motif, followed by phosphorylation at the activation loop by phosphoinositide-dependent kinase-1 (PDK1). Live cell imaging reveals that global aPKC activity is constitutive and insulin unresponsive, in marked contrast to the insulin-dependent activation of Akt monitored by an Akt-specific reporter. Nor does forced recruitment to phosphoinositides by fusing the pleckstrin homology (PH) domain of Akt to the kinase domain of PKCζ alter either the phosphorylation or activity of PKCζ. Thus, insulin stimulation does not activate PKCζ through the canonical phosphatidylinositol-3,4,5-triphosphate-mediated pathway that activates Akt, contrasting with previous literature on PKCζ activation. These studies support a model wherein an alternative mechanism regulates PKCζ-mediated insulin signalling that does not utilize conventional activation via agonist-evoked phosphorylation at the activation loop. Rather, we propose that scaffolding near substrates drives the function of PKCζ.
Assuntos
Fosfatos de Fosfatidilinositol/metabolismo , Proteína Quinase C/metabolismo , Proteínas Quinases Dependentes de 3-Fosfoinositídeo/genética , Proteínas Quinases Dependentes de 3-Fosfoinositídeo/metabolismo , Sequência de Aminoácidos , Animais , Biocatálise , Células Cultivadas , Insulina/farmacologia , Alvo Mecanístico do Complexo 2 de Rapamicina , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Complexos Multiproteicos/metabolismo , Fosforilação , Conformação Proteica , Proteína Quinase C/química , Serina-Treonina Quinases TOR/metabolismoRESUMO
Gene knockout strategies, RNAi and rescue experiments are all employed to study mammalian gene function. However, the disadvantages of these approaches include: loss of function adaptation, reduced viability and gene overexpression that rarely matches endogenous levels. Here, we developed an endogenous gene knockdown/rescue strategy that combines RNAi selectivity with a highly efficient CRISPR directed recombinant Adeno-Associated Virus (rAAV) mediated gene targeting approach to introduce allele-specific mutations plus an allele-selective siRNA Sensitive (siSN) site that allows for studying gene mutations while maintaining endogenous expression and regulation of the gene of interest. CRISPR/Cas9 plus rAAV targeted gene-replacement and introduction of allele-specific RNAi sensitivity mutations in the CDK2 and CDK1 genes resulted in a >85% site-specific recombination of Neo-resistant clones versus â¼8% for rAAV alone. RNAi knockdown of wild type (WT) Cdk2 with siWT in heterozygotic knockin cells resulted in the mutant Cdk2 phenotype cell cycle arrest, whereas allele specific knockdown of mutant CDK2 with siSN resulted in a wild type phenotype. Together, these observations demonstrate the ability of CRISPR plus rAAV to efficiently recombine a genomic locus and tag it with a selective siRNA sequence that allows for allele-selective phenotypic assays of the gene of interest while it remains expressed and regulated under endogenous control mechanisms.
Assuntos
Alelos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Dependovirus/genética , Interferência de RNA , Sequência de Bases , Proteína Quinase CDC2 , Linhagem Celular , Quinase 2 Dependente de Ciclina/genética , Quinases Ciclina-Dependentes/genética , Primers do DNA , Técnicas de Silenciamento de Genes , Humanos , Mutação , Reação em Cadeia da Polimerase , Recombinação GenéticaRESUMO
The spindle assembly checkpoint (SAC) restrains anaphase until all chromosomes become bi-oriented on the mitotic spindle. The SAC protein Mad2 can fold into two distinct conformers, open (O) and closed (C), and can asymmetrically dimerize. Here, we describe a monoclonal antibody that specifically recognizes the dimerization interface of C-Mad2. This antibody revealed several conformation-specific features of Mad2 in human cells. Notably, we show that Mad2 requires association with Mad1 to adopt the closed conformation and that the activity of the Mad1:C-Mad2 complex undergoes regulation by p31comet-dependent 'capping'. Furthermore, C-Mad2 antibody microinjection caused an abrupt termination of the SAC and accelerated mitotic progression. Remarkably, microinjection of a Mad1-neutralizing antibody triggered a comparable mitotic acceleration. Our study provides direct in vivo evidence for the model that a kinetochore complex of Mad1:C-Mad2 acts as a template to sustain the SAC and it challenges the distinction between SAC and mitotic timer.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Anáfase/fisiologia , Proteínas de Ligação ao Cálcio/fisiologia , Proteínas de Ciclo Celular/fisiologia , Proteínas Nucleares/fisiologia , Proteínas Repressoras/fisiologia , Fuso Acromático/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Animais , Anticorpos Monoclonais/imunologia , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/imunologia , Proteínas Cdc20 , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/imunologia , Dimerização , Humanos , Cinetocoros/metabolismo , Substâncias Macromoleculares , Proteínas Mad2 , Camundongos , Camundongos Endogâmicos BALB C , Mitose/efeitos dos fármacos , Mitose/fisiologia , Poro Nuclear/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/imunologia , Conformação Proteica , Dobramento de Proteína , Mapeamento de Interação de Proteínas , RNA Interferente Pequeno/farmacologia , Coelhos , Proteínas Repressoras/química , Proteínas Repressoras/imunologia , Fatores de TempoRESUMO
The putative chromatin remodeling enzyme Plk1-interacting checkpoint helicase (PICH) was discovered as an interaction partner and substrate of the mitotic kinase Plk1. During mitosis PICH associates with centromeres and kinetochores and, most interestingly, constitutes a robust marker for ultrafine DNA bridges (UFBs) that connect separating chromatids in anaphase cells. The precise roles of PICH remain to be clarified. Here, we have used antibody microinjection and siRNA-rescue experiments to study PICH function and localization during M phase progression, with particular emphasis on the role of the predicted ATPase domain and the regulation of PICH localization by Plk1. We show that interference with PICH function results in chromatin bridge formation and micronucleation and that ATPase activity is critical for PICH function. Interestingly, an intact ATPase domain of PICH is required for prevention of chromatin bridge formation but not for UFB resolution, and quantitative analyses of UFB and chromatin bridge frequencies suggest that these structures are of different etiologies. We also show that the ATPase activity of PICH is required for temporal and spatial control of PICH localization to chromatin and that Plk1 likely controls PICH localization through phosphorylation of proteins distinct from PICH itself. This work strengthens the view that PICH is an important, Plk1-regulated enzyme, whose ATPase activity is essential for maintenance of genome integrity. Although not required for the spindle assembly checkpoint, PICH is clearly important for faithful chromosome segregation.
Assuntos
Adenosina Trifosfatases/genética , Segregação de Cromossomos , DNA Helicases/genética , Adenosina Trifosfatases/metabolismo , Anáfase/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Divisão Celular , Cromatina/genética , Cromatina/metabolismo , DNA Helicases/metabolismo , Regulação da Expressão Gênica , Células HeLa , Humanos , Cinetocoros/metabolismo , Mitose , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Fuso Acromático/metabolismo , Quinase 1 Polo-LikeRESUMO
The selective autophagic degradation of mitochondria via mitophagy is essential for preserving mitochondrial homeostasis and, thereby, disease maintenance and progression in acute myeloid leukemia (AML). Mitophagy is orchestrated by a variety of mitophagy receptors whose interplay is not well understood. Here, we established a pairwise multiplexed CRISPR screen targeting mitophagy receptors to elucidate redundancies and gain a deeper understanding of the functional interactome governing mitophagy in AML. We identified OPTN (optineurin) as sole non-redundant mitophagy receptor and characterized its unique role in AML. Knockdown and overexpression experiments demonstrated that OPTN expression is rate-limiting for AML cell proliferation. In a MN1-driven murine transplantation model, loss of OPTN prolonged overall median survival by 7 days (+21%). Mechanistically, we found broadly impaired mitochondrial respiration and function with increased mitochondrial ROS, that most likely caused the proliferation defect. Our results decipher the intertwined network of mitophagy receptors in AML for both ubiquitin-dependent and receptor-mediated mitophagy, identify OPTN as a non-redundant tool to study mitophagy in the context of leukemia and suggest OPTN inhibition as an attractive therapeutic strategy.Abbreviations: AML: acute myeloid leukemia; CRISPR: Clustered Regularly Interspaced Short Palindromic Repeats; CTRL: control; DFP: deferiprone; GI: genetic interaction; KD: knockdown; KO: knockout; ldMBM, lineage-depleted murine bone marrow; LFC: log2 fold change; LIR: LC3-interacting region; LSC: leukemic stem cell; MAGeCK: Model-based Analysis of Genome-wide CRISPR-Cas9 Knockout; MDIVI-1: mitochondrial division inhibitor 1; MOI: multiplicity of infection; MOM: mitochondrial outer membrane; NAC: N-acetyl-L-cysteine; OA: oligomycin-antimycin A; OCR: oxygen consumption rate; OE: overexpression; OPTN: optineurin; PINK1: PTEN induced putative kinase 1; ROS: reactive oxygen species; SEM: standard error of the mean; TCGA: The Cancer Genome Atlas; TEM: transmission electron microscopy; UBD: ubiquitin-binding domain; WT: wild type.
Assuntos
Leucemia Mieloide Aguda , Mitofagia , Animais , Camundongos , Autofagia , Mitofagia/genética , Espécies Reativas de Oxigênio/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinas , HumanosRESUMO
CRISPR-based gene perturbation enables unbiased investigations of single and combinatorial genotype-to-phenotype associations. In light of efforts to map combinatorial gene dependencies at scale, choosing an efficient and robust CRISPR-associated (Cas) nuclease is of utmost importance. Even though SpCas9 and AsCas12a are widely used for single, combinatorial, and orthogonal screenings, side-by-side comparisons remain sparse. Here, we systematically compared combinatorial SpCas9, AsCas12a, and CHyMErA in hTERT-immortalized retinal pigment epithelial cells and extracted performance-critical parameters for combinatorial and orthogonal CRISPR screens. Our analyses identified SpCas9 to be superior to enhanced and optimized AsCas12a, with CHyMErA being largely inactive in the tested conditions. Since AsCas12a contains RNA processing activity, we used arrayed dual-gRNAs to improve AsCas12a and CHyMErA applications. While this negatively influenced the effect size range of combinatorial AsCas12a applications, it enhanced the performance of CHyMErA. This improved performance, however, was limited to AsCas12a dual-gRNAs, as SpCas9 gRNAs remained largely inactive. To avoid the use of hybrid gRNAs for orthogonal applications, we engineered the multiplex SpCas9-enAsCas12a approach (multiSPAS) that avoids RNA processing for efficient orthogonal gene editing.
Assuntos
Benchmarking , Sistemas CRISPR-Cas , Edição de Genes , Endonucleases/genéticaRESUMO
Ribosome biogenesis is a multi-step process, in which a network of trans-acting factors ensures the coordinated assembly of pre-ribosomal particles in order to generate functional ribosomes. Ribosome biogenesis is tightly coordinated with cell proliferation and its perturbation activates a p53-dependent cell-cycle checkpoint. How p53-independent signalling networks connect impaired ribosome biogenesis to the cell-cycle machinery has remained largely enigmatic. We demonstrate that inactivation of the nucleolar SUMO isopeptidases SENP3 and SENP5 disturbs distinct steps of 40S and 60S ribosomal subunit assembly pathways, thereby triggering the canonical p53-dependent impaired ribosome biogenesis checkpoint. However, inactivation of SENP3 or SENP5 also induces a p53-independent checkpoint that converges on the specific downregulation of the key cell-cycle regulator CDK6. We further reveal that impaired ribosome biogenesis generally triggers the downregulation of CDK6, independent of the cellular p53 status. Altogether, these data define the role of SUMO signalling in ribosome biogenesis and unveil a p53-independent checkpoint of impaired ribosome biogenesis.