RESUMO
Caspases are a highly conserved family of cysteine-aspartyl proteases known for their essential roles in regulating apoptosis, inflammation, cell differentiation, and proliferation. Complementary to genetic approaches, small-molecule probes have emerged as useful tools for modulating caspase activity. However, due to the high sequence and structure homology of all 12 human caspases, achieving selectivity remains a central challenge for caspase-directed small-molecule inhibitor development efforts. Here, using mass spectrometry-based chemoproteomics, we first identify a highly reactive noncatalytic cysteine that is unique to caspase-2. By combining both gel-based activity-based protein profiling (ABPP) and a tobacco etch virus (TEV) protease activation assay, we then identify covalent lead compounds that react preferentially with this cysteine and afford a complete blockade of caspase-2 activity. Inhibitory activity is restricted to the zymogen or precursor form of monomeric caspase-2. Focused analogue synthesis combined with chemoproteomic target engagement analysis in cellular lysates and in cells yielded both pan-caspase-reactive molecules and caspase-2 selective lead compounds together with a structurally matched inactive control. Application of this focused set of tool compounds to stratify the functions of the zymogen and partially processed (p32) forms of caspase-2 provide evidence to support that caspase-2-mediated response to DNA damage is largely driven by the partially processed p32 form of the enzyme. More broadly, our study highlights future opportunities for the development of proteoform-selective caspase inhibitors that target nonconserved and noncatalytic cysteine residues.
Assuntos
Caspase 2 , Inibidores de Caspase , Proteômica , Humanos , Caspase 2/metabolismo , Caspase 2/química , Proteômica/métodos , Inibidores de Caspase/farmacologia , Inibidores de Caspase/química , Inibidores de Caspase/metabolismo , Estrutura Molecular , Cisteína EndopeptidasesRESUMO
Kinetochores control eukaryotic chromosome segregation by connecting chromosomal centromeres to spindle microtubules. Duplication of centromeric DNA necessitates kinetochore disassembly and subsequent reassembly on nascent sisters. To search for a regulatory mechanism that controls the earliest steps of this process, we studied Mif2/CENP-C, an essential basal component of the kinetochore. We found that phosphorylation of a central region of Mif2 (Mif2-PEST) enhances inner kinetochore assembly. Eliminating Mif2-PEST phosphorylation sites progressively impairs cellular fitness. The most severe Mif2-PEST mutations are lethal in cells lacking otherwise non-essential inner kinetochore factors. These data show that multi-site phosphorylation of Mif2/CENP-C controls inner kinetochore assembly.
Assuntos
Cinetocoros , Proteínas de Saccharomyces cerevisiae , Cinetocoros/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Fosforilação , Proteínas Cromossômicas não Histona/metabolismo , Centrômero/metabolismo , Mitose , Proteína Centromérica A/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Eukaryotic DNA mismatch repair (MMR) depends on recruitment of the Mlh1-Pms1 endonuclease (human MLH1-PMS2) to mispaired DNA. Both Mlh1 and Pms1 contain a long unstructured linker that connects the N- and carboxyl-terminal domains. Here, we demonstrated the Mlh1 linker contains a conserved motif (Saccharomyces cerevisiae residues 391-415) required for MMR. The Mlh1-R401A,D403A-Pms1 linker motif mutant protein was defective for MMR and endonuclease activity in vitro, even though the conserved motif could be >750 Å from the carboxyl-terminal endonuclease active site or the N-terminal adenosine triphosphate (ATP)-binding site. Peptides encoding this motif inhibited wild-type Mlh1-Pms1 endonuclease activity. The motif functioned in vivo at different sites within the Mlh1 linker and within the Pms1 linker. Motif mutations in human cancers caused a loss-of-function phenotype when modeled in S. cerevisiae. These results suggest that the Mlh1 motif promotes the PCNA-activated endonuclease activity of Mlh1-Pms1 via interactions with DNA, PCNA, RFC, or other domains of the Mlh1-Pms1 complex.
Assuntos
Neoplasias , Proteínas de Saccharomyces cerevisiae , Trifosfato de Adenosina/metabolismo , DNA/metabolismo , Reparo de Erro de Pareamento de DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endonucleases/genética , Endonucleases/metabolismo , Humanos , Endonuclease PMS2 de Reparo de Erro de Pareamento/genética , Proteína 1 Homóloga a MutL/genética , Proteína 1 Homóloga a MutL/metabolismo , Proteínas MutL , Proteína 2 Homóloga a MutS/metabolismo , Proteínas Mutantes/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Timely completion of eukaryotic genome duplication requires coordinated DNA replication initiation at multiple origins. Replication begins with the loading of the Mini-Chromosome Maintenance (MCM) complex, proceeds by the activation of the Cdc45-MCM-GINS (CMG) helicase, and ends with CMG removal after chromosomes are fully replicated. Post-translational modifications on the MCM and associated factors ensure an orderly transit of these steps. Although the mechanisms of CMG activation and removal are partially understood, regulated MCM loading is not, leaving an incomplete understanding of how DNA replication begins. Here we describe a site-specific modification of Mcm3 by the Small Ubiquitin-like MOdifier (SUMO). Mutations that prevent this modification reduce the MCM loaded at replication origins and lower CMG levels, resulting in impaired cell growth, delayed chromosomal replication, and the accumulation of gross chromosomal rearrangements (GCRs). These findings demonstrate the existence of a SUMO-dependent regulation of origin-bound MCM and show that this pathway is needed to prevent genome rearrangements.
Assuntos
Replicação do DNA , Sumoilação , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , DNA Helicases/genética , Replicação do DNA/genética , Proteínas de Manutenção de Minicromossomo/genética , Proteínas de Manutenção de Minicromossomo/metabolismo , Origem de Replicação/genética , Sumoilação/genéticaRESUMO
Protein sumoylation, especially when catalyzed by the Mms21 SUMO E3 ligase, plays a major role in suppressing duplication-mediated gross chromosomal rearrangements (dGCRs). How Mms21 targets its substrates in the cell is insufficiently understood. Here, we demonstrate that Esc2, a protein with SUMO-like domains (SLDs), recruits the Ubc9 SUMO conjugating enzyme to specifically facilitate Mms21-dependent sumoylation and suppress dGCRs. The D430R mutation in Esc2 impairs its binding to Ubc9 and causes a synergistic growth defect and accumulation of dGCRs with mutations that delete the Siz1 and Siz2 E3 ligases. By contrast, esc2-D430R does not appreciably affect sensitivity to DNA damage or the dGCRs caused by the catalytically inactive mms21-CH. Moreover, proteome-wide analysis of intracellular sumoylation demonstrates that esc2-D430R specifically down-regulates sumoylation levels of Mms21-preferred targets, including the nucleolar proteins, components of the SMC complexes and the MCM complex that acts as the catalytic core of the replicative DNA helicase. These effects closely resemble those caused by mms21-CH, and are relatively unaffected by deleting Siz1 and Siz2. Thus, by recruiting Ubc9, Esc2 facilitates Mms21-dependent sumoylation to suppress the accumulation of dGCRs independent of Siz1 and Siz2.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteína SUMO-1/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Dano ao DNA , Replicação do DNA , Regulação para Baixo , Mutagênese , Ligação Proteica , Domínios Proteicos , Estabilidade Proteica , Proteômica , Proteína SUMO-1/química , Proteína SUMO-1/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Alinhamento de Sequência , Sumoilação , Enzimas de Conjugação de Ubiquitina/química , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Adipose tissue is an ideal mesenchymal stem cell (MSC) source, as it is dispensable and accessible with minimal morbidity. However, the stromal vascular fraction (SVF) of adipose tissue is a heterogeneous cell population, which has disadvantages for tissue regeneration. In the present study, we prospectively purified human perivascular stem cells (PSCs) from n = 60 samples of human lipoaspirate and documented their frequency, viability, and variation with patient demographics. PSCs are a fluorescence-activated cell sorting-sorted population composed of pericytes (CD45-, CD146+, CD34-) and adventitial cells (CD45-, CD146-, CD34+), each of which we have previously reported to have properties of MSCs. Here, we found that PSCs make up, on average, 43.2% of SVF from human lipoaspirate (19.5% pericytes and 23.8% adventitial cells). These numbers were minimally changed by age, gender, or body mass index of the patient or by length of refrigerated storage time between liposuction and processing. In a previous publication, we observed that human PSCs (hPSCs) formed significantly more bone in vivo in comparison with unsorted human SVF (hSVF) in an intramuscular implantation model. We now extend this finding to a bone injury model, observing that purified hPSCs led to significantly greater healing of mouse critical-size calvarial defects than hSVF (60.9% healing as opposed to 15.4% healing at 2 weeks postoperative by microcomputed tomography analysis). These studies suggest that adipose-derived hPSCs are a new cell source for future efforts in skeletal regenerative medicine. Moreover, hPSCs are a stem cell-based therapeutic that is readily approvable by the U.S. Food and Drug Administration, with potentially increased safety, purity, identity, potency, and efficacy.
Assuntos
Regeneração Óssea , Osso e Ossos , Células-Tronco Mesenquimais/metabolismo , Engenharia Tecidual , Tecido Adiposo/citologia , Túnica Adventícia , Animais , Antígenos CD34/análise , Antígeno CD146/análise , Separação Celular , Humanos , Antígenos Comuns de Leucócito/análise , Camundongos , Pericitos , Alicerces Teciduais , Cicatrização , Ferimentos e Lesões/terapiaRESUMO
Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a major role in cholesterol homeostasis through enhanced degradation of the LDL receptor (LDLR) in liver. As novel inhibitors/silencers of PCSK9 are now being tested in clinical trials to treat hypercholesterolemia, it is crucial to define the physiological consequences of the lack of PCSK9 in various organs. LDLR regulation by PCSK9 has not been extensively described during mouse brain development and injury. Herein, we show that PCSK9 and LDLR are co-expressed in mouse brain during development and at adulthood. Although the protein levels of LDLR and apolipoprotein E (apoE) in the adult brain of Pcsk9(-/-) mice are similar to those of wild-type (WT) mice, LDLR levels increased and were accompanied by a reduction of apoE levels during development. This suggests that the upregulation of LDLR protein levels in Pcsk9(-/-) mice enhances apoE degradation. Upon ischemic stroke, PCSK9 was expressed in the dentate gyrus between 24 h and 72 h following brain reperfusion. Although mouse behavior and lesion volume were similar, LDLR protein levels dropped â¼2-fold less in the Pcsk9(-/-)-lesioned hippocampus, without affecting apoE levels and neurogenesis. Thus, PCSK9 downregulates LDLR levels during brain development and following transient ischemic stroke in adult mice.