RESUMO
The SARS-CoV-2 main protease (Mpro ) holds significant importance as a biological target in combating coronaviruses due to its importance in virus replication. Considering the emergence of novel SARS-CoV-2 variants and the mutations observed in the Mpro sequence, we hypothesized that these mutations may have a potential impact on the protease's specificity. To test this, we expressed Mpro corresponding to the original strain and variants Beta1, Beta2, and Omicron and analyzed their activity on protein-based and peptide substrates. Although we observed differential activity on the protein-based substrate, there was very little difference when analyzed on the peptide substrate. We conclude that mutations on the Mpro sequence, despite having a minor effect on a peptide substrate cleavage, did not change the catalytic site environment enough to build resistance to inhibition. Therefore, we propose that inhibitors initially designed for the Mpro of the original strain will be effective in all the variants. Thus, Mpro is likely to continue to be a target of therapeutic interest as mutations in its sequence are rare and, as we show here, have a minor effect on the protease's recognition of peptide-based molecules.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , COVID-19/genética , Proteínas não Estruturais Virais/genética , Peptídeos/genética , Proteases 3C de Coronavírus/genética , Inibidores de Proteases/química , Antivirais/farmacologia , Peptídeo HidrolasesRESUMO
Activated effector caspases 3, 6 and 7 are responsible for cleaving a number of target substrates, leading to the ultimate destruction of cells via apoptosis. The functions of caspases 3 and 7 in apoptosis execution have been widely studied over the years with multiple chemical probes for both of these enzymes. In contrast, caspase 6 seems to be largely neglected when compared to the heavily studied caspases 3 and 7. Therefore, the development of new small-molecule reagents for the selective detection and visualization of caspase 6 activity can improve our understanding of molecular circuits of apoptosis and shed new light on how they intertwine with other types of programmed cell death. In this study, we profiled caspase 6 substrate specificity at the P5 position and discovered that, similar to caspase 2, caspase 6 prefers pentapeptide substrates over tetrapeptides. Based on these data, we developed a set of chemical reagents for caspase 6 investigation, including coumarin-based fluorescent substrates, irreversible inhibitors and selective aggregation-induced emission luminogens (AIEgens). We showed that AIEgens are able to distinguish between caspase 3 and caspase 6 in vitro. Finally, we validated the efficiency and selectivity of the synthesized reagents by monitoring lamin A and PARP cleavage via mass cytometry and western blot analysis. We propose that our reagents may provide new research prospects for single-cell monitoring of caspase 6 activity to reveal its function in programmed cell death pathways.
RESUMO
A patient diagnosed with multiple myeloma, bicuspid aortic valve, and Von Hippel-Lindau syndrome underwent whole-exome sequencing seeking a unified genetic cause for these three pathologies. The patient possessed a single-point mutation of arginine to cysteine (R24C) in the N-terminal region(pro-domain) of matrix metalloproteinase 9 (MMP-9). The pro-domain interacts with the catalytic site of this enzyme rendering it inactive. MMP-9 has previously been associated with all three pathologies suffered by the patient. We hypothesized that the observed mutation in the pro-domain would influence the activity of this enzyme. We expressed recombinant versions of MMP-9 and an investigation of their biochemical properties revealed that MMP-9 R24C is a constitutively active zymogen. To our knowledge, this is the first example of a mutation that discloses catalytic activity in the pro-form in any of the 24 human MMPs.
Assuntos
Doença da Válvula Aórtica Bicúspide , Mieloma Múltiplo , Doença de von Hippel-Lindau , Mutação com Ganho de Função , Humanos , Metaloproteinase 9 da Matriz/genética , Mieloma Múltiplo/complicações , Mieloma Múltiplo/genética , Doença de von Hippel-Lindau/complicações , Doença de von Hippel-Lindau/genéticaRESUMO
Many proteases recognize their substrates with high specificities, with this in mind, it should theoretically be possible to utilize the substrate binding cleft of a protease as a scaffold to engineer an affinity reagent. In this study, we sought to develop reagents that would differentiate between substrates and products of proteolysis, based on a caspase 7 scaffold. Firstly, we engineered a form of caspase 7 that can undergo conversion to a substrate binding conformation without catalysis. Seeking to generate a product-only trap, we further engineered this construct by incorporating mutations that compensate for the generation of a negative charge in the neo C terminus of a newly generated product. This was accomplished with only three substitutions within the substrate binding cleft. Moreover, the affinity of the product trap for peptides was comparable to the affinity of caspase 7 to parental substrates. Finally, generation of a hybrid fluorescent protein with the product trap provided a reagent that specifically recognized apoptotic cells and highlights the versatility of such an approach in developing affinity and imaging agents for a variety of cysteine and serine proteases.
Assuntos
Caspase 7/genética , Proteínas Mutantes/metabolismo , Mutação , Engenharia de Proteínas/métodos , Apoptose/efeitos dos fármacos , Sítios de Ligação/genética , Caspase 7/química , Caspase 7/metabolismo , Linhagem Celular Tumoral , Endopeptidases/metabolismo , Humanos , Proteínas Mutantes/química , Proteínas Mutantes/genética , Ligação Proteica , Domínios Proteicos , Proteólise , Especificidade por Substrato , Ligante Indutor de Apoptose Relacionado a TNF/farmacologiaRESUMO
Viral papain-like cysteine protease (PLpro, NSP3) is essential for SARS-CoV-2 replication and represents a promising target for the development of antiviral drugs. Here, we used a combinatorial substrate library and performed comprehensive activity profiling of SARS-CoV-2 PLpro. On the scaffold of the best hits from positional scanning, we designed optimal fluorogenic substrates and irreversible inhibitors with a high degree of selectivity for SARS PLpro. We determined crystal structures of two of these inhibitors in complex with SARS-CoV-2 PLpro that reveals their inhibitory mechanisms and provides a molecular basis for the observed substrate specificity profiles. Last, we demonstrate that SARS-CoV-2 PLpro harbors deISGylating activity similar to SARSCoV-1 PLpro but its ability to hydrolyze K48-linked Ub chains is diminished, which our sequence and structure analysis provides a basis for. Together, this work has revealed the molecular rules governing PLpro substrate specificity and provides a framework for development of inhibitors with potential therapeutic value or drug repurposing.
Assuntos
Betacoronavirus/enzimologia , Desenho de Fármacos , Inibidores de Proteases/química , Proteínas não Estruturais Virais/antagonistas & inibidores , Sequência de Aminoácidos , Betacoronavirus/isolamento & purificação , Sítios de Ligação , COVID-19 , Domínio Catalítico , Proteases 3C de Coronavírus , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Cristalografia por Raios X , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Humanos , Cinética , Simulação de Dinâmica Molecular , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Pandemias , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Inibidores de Proteases/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , SARS-CoV-2 , Especificidade por Substrato , Ubiquitinas/metabolismo , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismoRESUMO
Deubiquitinating enzymes (DUBs) are responsible for removing ubiquitin (Ub) from its protein conjugates. DUBs have been implicated as attractive therapeutic targets in the treatment of viral diseases, neurodegenerative disorders and cancer. The lack of selective chemical tools for the exploration of these enzymes significantly impairs the determination of their roles in both normal and pathological states. Commercially available fluorogenic substrates are based on the C-terminal Ub motif or contain Ub coupled to a fluorophore (Z-LRGG-AMC, Ub-AMC); therefore, these substrates suffer from lack of selectivity. By using a hybrid combinatorial substrate library (HyCoSuL) and a defined P2 library containing a wide variety of nonproteinogenic amino acids, we established a full substrate specificity profile for two DUBs-MERS PLpro and human UCH-L3. Based on these results, we designed and synthesized Ub-based substrates and activity-based probes (ABPs) containing selected unnatural amino acids located in the C-terminal Ub motif. Biochemical analysis and cell lysate experiments confirmed the activity and selectivity of engineered Ub-based substrates and probes. Using this approach, we propose that for any protease that recognizes Ub and Ub-like substrates, a highly active and selective unnatural substrate or probe can be engineered.
RESUMO
Pyroptosis is the caspase-dependent inflammatory cell death mechanism that underpins the innate immune response against pathogens and is dysregulated in inflammatory disorders. Pyroptosis occurs via two pathways: the canonical pathway, signaled by caspase-1, and the noncanonical pathway, regulated by mouse caspase-11 and human caspase-4/5. All inflammatory caspases activate the pyroptosis effector protein gasdermin D, but caspase-1 mostly activates the inflammatory cytokine precursors prointerleukin-18 and prointerleukin-1ß (pro-IL18/pro-IL1ß). Here, in vitro cleavage assays with recombinant proteins confirmed that caspase-11 prefers cleaving gasdermin D over the pro-ILs. However, we found that caspase-11 recognizes protein substrates through a mechanism that is different from that of most caspases. Results of kinetics analysis with synthetic fluorogenic peptides indicated that P1'-P4', the C-terminal gasdermin D region adjacent to the cleavage site, influences gasdermin D recognition by caspase-11. Furthermore, introducing the gasdermin D P1'-P4' region into pro-IL18 enhanced catalysis by caspase-11 to levels comparable with that of gasdermin D cleavage. Pro-IL1ß cleavage was only moderately enhanced by similar substitutions. We conclude that caspase-11 specificity is mediated by the P1'-P4' region in its substrate gasdermin D, and similar experiments confirmed that the substrate specificities of the human orthologs of caspase-11, i.e. caspase-4 and caspase-5, are ruled by the same mechanism. We propose that P1'-P4'-based inhibitors could be exploited to specifically target inflammatory caspases.
Assuntos
Caspases/metabolismo , Inflamação/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Proteínas de Ligação a Fosfato/fisiologia , Piroptose , Animais , Catálise , Humanos , Imunidade Inata , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Proteínas de Ligação a Fosfato/química , Proteínas de Ligação a Fosfato/metabolismo , Proteólise , Especificidade por SubstratoRESUMO
In December 2019, the first cases of a novel coronavirus infection causing COVID-19 were diagnosed in Wuhan, China. Viral Papain-Like cysteine protease (PLpro, NSP3) is essential for SARS-CoV-2 replication and represents a promising target for the development of antiviral drugs. Here, we used a combinatorial substrate library containing natural and a wide variety of nonproteinogenic amino acids and performed comprehensive activity profiling of SARS-CoV-2-PLpro. On the scaffold of best hits from positional scanning we designed optimal fluorogenic substrates and irreversible inhibitors with a high degree of selectivity for SARS PLpro variants versus other proteases. We determined crystal structures of two of these inhibitors (VIR250 and VIR251) in complex with SARS-CoV-2-PLpro which reveals their inhibitory mechanisms and provides a structural basis for the observed substrate specificity profiles. Lastly, we demonstrate that SARS-CoV-2-PLpro harbors deISGylating activities similar to SARS-CoV-1-PLpro but its ability to hydrolyze K48-linked Ub chains is diminished, which our sequence and structure analysis provides a basis for. Altogether this work has revealed the molecular rules governing PLpro substrate specificity and provides a framework for development of inhibitors with potential therapeutic value or drug repositioning.
RESUMO
Natural killer (NK) cells are key innate immunity effectors that combat viral infections and control several cancer types. For their immune function, human NK cells rely largely on five different cytotoxic proteases, called granzymes (A/B/H/K/M). Granzyme B (GrB) initiates at least three distinct cell death pathways, but key aspects of its function remain unexplored because selective probes that detect its activity are currently lacking. In this study, we used a set of unnatural amino acids to fully map the substrate preferences of GrB, demonstrating previously unknown GrB substrate preferences. We then used these preferences to design substrate-based inhibitors and a GrB-activatable activity-based fluorogenic probe. We show that our GrB probes do not significantly react with caspases, making them ideal for in-depth analyses of GrB localization and function in cells. Using our quenched fluorescence substrate, we observed GrB within the cytotoxic granules of human YT cells. When used as cytotoxic effectors, YT cells loaded with GrB attacked MDA-MB-231 target cells, and active GrB influenced its target cell-killing efficiency. In summary, we have developed a set of molecular tools for investigating GrB function in NK cells and demonstrate noninvasive visual detection of GrB with an enzyme-activated fluorescent substrate.
Assuntos
Corantes Fluorescentes/química , Granzimas , Imagem Óptica , Peptídeos/química , Linfócitos T/enzimologia , Linhagem Celular Tumoral , Granzimas/química , Granzimas/metabolismo , HumanosRESUMO
Cytotoxic T-lymphocytes (CTLs) and natural killer cells (NKs) kill compromised cells to defend against tumor and viral infections. Both effector cell types use multiple strategies to induce target cell death including Fas/CD95 activation and the release of perforin and a group of lymphocyte granule serine proteases called granzymes. Granzymes have relatively broad and overlapping substrate specificities and may hydrolyze a wide range of peptidic epitopes; it is therefore challenging to identify their natural and synthetic substrates and to distinguish their localization and functions. Here, we present a specific and potent substrate, an inhibitor, and an activity-based probe of Granzyme A (GrA) that can be used to follow functional GrA in cells.
Assuntos
Cumarínicos/farmacologia , Corantes Fluorescentes/farmacologia , Granzimas/análise , Oligopeptídeos/farmacologia , Inibidores de Serina Proteinase/farmacologia , Linhagem Celular Tumoral , Cumarínicos/síntese química , Cumarínicos/toxicidade , Desenho de Fármacos , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/toxicidade , Granzimas/química , Humanos , Oligopeptídeos/síntese química , Oligopeptídeos/toxicidade , Inibidores de Serina Proteinase/síntese química , Inibidores de Serina Proteinase/toxicidade , Especificidade por SubstratoRESUMO
Caspases participate in regulated cell death mechanisms and are divided into apoptotic and proinflammatory caspases. The main problem in identifying the unique role of a particular caspase in the mechanisms of regulated cell death is their overlapping substrate specificity; caspases recognize and hydrolyze similar peptide substrates. Most studies focus on examining the non-prime sites of the caspases, yet there is a need for novel and more precise chemical tools to identify the molecular participants and mechanisms of programmed cell death pathways. Therefore, we developed an innovative chemical approach that examines the prime area of the caspase active sites. This method permits the agile parallel solid-phase synthesis of caspase inhibitors with a high yield and purity. Using synthesized compounds we have shown the similarities and differences in the prime area of the caspase active site and, as a proof of concept, we demonstrated the exclusive role of caspase-8 in necroptosis.
Assuntos
Caspases/metabolismo , Neoplasias do Colo/metabolismo , Sítios de Ligação/efeitos dos fármacos , Inibidores de Caspase/síntese química , Inibidores de Caspase/química , Inibidores de Caspase/farmacologia , Caspases/química , Morte Celular/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/patologia , Células HT29 , HumanosRESUMO
Neutrophils are primary host innate immune cells defending against pathogens. One proposed mechanism by which neutrophils prevent the spread of pathogens is NETosis, the extrusion of cellular DNA resulting in neutrophil extracellular traps (NETs). The protease neutrophil elastase (NE) has been implicated in the formation of NETs through proteolysis of nuclear proteins leading to chromatin decondensation. In addition to NE, neutrophils contain three other serine proteases that could compensate if the activity of NE was neutralized. However, whether they do play such a role is unknown. Thus, we deployed recently described specific inhibitors against all four of the neutrophil serine proteases (NSPs). Using specific antibodies to the NSPs along with our labeled inhibitors, we show that catalytic activity of these enzymes is not required for the formation of NETs. Moreover, the NSPs that decorate NETs are in an inactive conformation and thus cannot participate in further catalytic events. These results indicate that NSPs play no role in either NETosis or arming NETs with proteolytic activity.
Assuntos
Armadilhas Extracelulares/metabolismo , Neutrófilos/enzimologia , Serina Proteases/metabolismo , Animais , Anticorpos/química , Anticorpos/imunologia , Candida albicans/fisiologia , DNA/metabolismo , Escherichia coli/fisiologia , Armadilhas Extracelulares/efeitos dos fármacos , Humanos , Elastase de Leucócito/antagonistas & inibidores , Elastase de Leucócito/imunologia , Elastase de Leucócito/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Microscopia Confocal , Neutrófilos/efeitos dos fármacos , Piroptose/efeitos dos fármacos , Células RAW 264.7 , Serina Proteases/química , Serina Proteases/imunologia , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/metabolismo , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Neutrophil granule serine proteases contribute to immune responses through cleavage of microbial toxins and structural proteins. They induce tissue damage and modulate inflammation if levels exceed their inhibitors. Here, we show that the intracellular protease inhibitors Serpinb1a and Serpinb6a contribute to monocyte and neutrophil survival in steady-state and inflammatory settings by inhibiting cathepsin G (CatG). Importantly, we found that CatG efficiently cleaved gasdermin D (GSDMD) to generate the signature N-terminal domain GSDMD-p30 known to induce pyroptosis. Yet GSDMD deletion did not rescue neutrophil survival in Sb1a.Sb6a-/- mice. Furthermore, Sb1a.Sb6a-/- mice released high levels of pro-inflammatory cytokines upon endotoxin challenge in vivo in a CatG-dependent manner. Canonical inflammasome activation in Sb1a.Sb6a-/- macrophages showed increased IL-1ß release that was dependent on CatG and GSDMD. Together, our findings demonstrate that cytosolic serpins expressed in myeloid cells prevent cell death and regulate inflammatory responses by inhibiting CatG and alternative activation of GSDMD.
Assuntos
Catepsina G/antagonistas & inibidores , Inflamação/prevenção & controle , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Monócitos/patologia , Neutrófilos/patologia , Proteínas de Ligação a Fosfato/metabolismo , Serpinas/fisiologia , Animais , Apoptose , Endotoxinas/toxicidade , Feminino , Inflamassomos , Inflamação/induzido quimicamente , Inflamação/metabolismo , Inflamação/patologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Macrófagos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/metabolismo , Necrose , Neutrófilos/metabolismo , Proteínas de Ligação a Fosfato/genética , PiroptoseRESUMO
Most caspases can be positioned unambiguously within the regulated cell death networks of apoptosis and pyroptosis, but the role of caspase-2, a highly conserved protease within the family, remains enigmatic. This is mainly due to lack of selective chemical and biochemical tools for the investigation of this protease. In this study, we used our hybrid combinatorial substrate library (HyCoSuL) approach to broadly profile caspase-2 substrate specificity using peptide scanning libraries. This screen uncovered previously unknown caspase-2 peptidyl substrate preferences, which were further used to develop caspase-2 selective fluorogenic substrates and covalent, irreversible AOMK inhibitors. Finally, we used the champion inhibitor (NH-23-C2) in reversine-treated HCT-116 colon cancer cells to selectively block caspase-2 activity and caspase-2-mediated MDM-2 cleavage. In addition, we showed that NH-23-C2 does not block caspase-3 or caspase-8, which makes it a powerful chemical tool to dissect the true role of caspase-2 in various biological setups.
Assuntos
Caspase 2/metabolismo , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/metabolismo , Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Morfolinas/uso terapêutico , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Purinas/uso terapêutico , Apoptose/efeitos dos fármacos , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Células HCT116 , Humanos , Morfolinas/farmacologia , Purinas/farmacologia , Especificidade por SubstratoRESUMO
Apical caspases initiate and effector caspases execute apoptosis. Reagents that can distinguish between caspases, particularly apical caspases-8, 9, and 10 are scarce and generally nonspecific. Based upon a previously described large-scale screen of peptide-based caspase substrates termed HyCoSuL, we sought to develop reagents to distinguish between apical caspases in order to reveal their function in apoptotic cell death paradigms. To this end, we selected tetrapeptide-based sequences that deliver optimal substrate selectivity and converted them to inhibitors equipped with a detectable tag (activity-based probes-ABPs). We demonstrate a strong relationship between substrate kinetics and ABP kinetics. To evaluate the utility of selective substrates and ABPs, we examined distinct apoptosis pathways in Jurkat T lymphocyte and MDA-MB-231 breast cancer lines triggered to undergo cell death via extrinsic or intrinsic apoptosis. We report the first highly selective substrate appropriate for quantitation of caspase-8 activity during apoptosis. Converting substrates to ABPs promoted loss-of-activity and selectivity, thus we could not define a single ABP capable of detecting individual apical caspases in complex mixtures. To overcome this, we developed a panel strategy utilizing several caspase-selective ABPs to interrogate apoptosis, revealing the first chemistry-based approach to uncover the participation of caspase-8, but not caspase-9 or -10 in TRAIL-induced extrinsic apoptosis. We propose that using select panels of ABPs can provide information regarding caspase-8 apoptotic signaling more faithfully than can single, generally nonspecific reagents.
Assuntos
Caspase 10/isolamento & purificação , Caspase 8/isolamento & purificação , Caspase 9/isolamento & purificação , Peptídeos/química , Apoptose/genética , Caspase 10/química , Caspase 10/genética , Caspase 3/química , Caspase 3/genética , Caspase 3/isolamento & purificação , Caspase 8/química , Caspase 8/genética , Caspase 9/química , Caspase 9/genética , Inibidores de Caspase/química , Inibidores de Caspase/farmacologia , Humanos , Células Jurkat , Cinética , Transdução de Sinais , Especificidade por SubstratoRESUMO
Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) belongs to the CD clan of cysteine proteases. MALT1 is a unique enzyme among this clan because it recognizes the basic amino acid arginine in the P1 pocket. Previous studies carried out with natural amino acids revealed the substrate specificity of the P4-P1 pockets of MALT1 but have provided only limited information about the catalytic preferences of this enzyme. In this study, we exploited Hybrid Combinatorial Substrate Library and Internally Quenched Fluorescence substrate technologies to interrogate the extended substrate specificity profile of the S5-S2' active site pockets using unnatural amino acids. This strategy resulted in the design of a peptide-based fluorogenic substrate, which exhibited significant activity toward MALT1. Subsequently, the substrate sequence was further utilized to develop potent, irreversible activity-based probes.
Assuntos
Sequência de Aminoácidos/genética , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/química , Peptídeos/química , Aminoácidos/química , Aminoácidos/genética , Animais , Arginina/química , Cinética , Camundongos , Modelos Moleculares , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/genética , Peptídeos/genética , Especificidade por SubstratoRESUMO
The proteasome is an enzyme complex critical for maintaining protein homeostasis. Perturbed proteasome function leads to pathologies including cancer and autoimmune and neurodegenerative disease. Therefore, the proteasome constitutes an excellent molecular target for pharmaceutical development. Here, using the HyCoSuL approach, we designed and synthesized novel and selective fluorogenic substrates for each of these three constitutive 20S proteasome activities and applied them to assess inhibition of proteasome subunits by MG-132 and a clinically used inhibitor bortezomib. Our results confirm the utility of designed substrates in biochemical assays. Furthermore, selective peptide sequences obtained in this manner were used to construct fluorophore-labeled activity-based probes and then utilized to detect each constitutive 20S proteasome subunit simultaneously in lysates of HEK-293F cells and red blood cells. Overall, we describe a simple and rapid method useful to measure constitutive 20S proteasome activity in whole human blood samples that could enable early diagnosis of pathological states associated with aberrantly upregulated proteasome activity.
Assuntos
Imagem Molecular/métodos , Sondas Moleculares/química , Complexo de Endopeptidases do Proteassoma/análise , Corantes Fluorescentes/química , Células HEK293 , Humanos , Leupeptinas/farmacologia , Complexo de Endopeptidases do Proteassoma/sangue , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Especificidade por Substrato , Espectrometria de Massas em TandemRESUMO
Inflammatory cell death, or pyroptosis, is triggered by pathogenic infections or events. It is executed by caspase-1 (in the canonical pyroptosis pathway) or caspase-11 (noncanonical pathway), each via production of a cell-lytic domain from the pyroptosis effector protein gasdermin D through specific and limited proteolysis. Pyroptosis is accompanied by the release of inflammatory mediators, including the proteolytically processed forms of interleukin-1ß (IL-1ß) and IL-18. Given the similar inflammatory outcomes of the canonical and noncanonical pyroptosis pathways, we hypothesized that caspase-1 and -11 should have very similar activities and substrate specificities. To test this hypothesis, we purified recombinant murine caspases and analyzed their primary specificities by massive hybrid combinatorial substrate library (HyCoSuL) screens. We correlated the substrate preferences of each caspase with their activities on the recombinant natural substrates IL-1ß, IL-18, and gasdermin D. Although we identified highly selective and robust peptidyl substrates for caspase-1, we were unable to do so for caspase-11, because caspase-1 cleaved even the best caspase-11 substrates equally well. Caspase-1 rapidly processed pro-IL-1ß and -18, but caspase-11 processed these two pro-ILs extremely poorly. However, both caspase-1 and -11 efficiently produced the cell-lytic domain from the gasdermin D precursor. We hypothesize that caspase-11 may have evolved a specific exosite to selectively engage pyroptosis without directly activating pro-IL-1ß or -18. In summary, comparing the activities of caspase-1 and -11 in HyCoSuL screens and with three endogenous protein substrates, we conclude that caspase-11 has highly restricted substrate specificity, preferring gasdermin D over all other substrates examined.
Assuntos
Caspase 1/metabolismo , Caspases/metabolismo , Peptídeos/metabolismo , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Caspases Iniciadoras , Morte Celular , Citocinas/metabolismo , Mediadores da Inflamação/metabolismo , Interleucina-10/metabolismo , Interleucina-1beta/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Proteínas de Ligação a Fosfato , Proteólise , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
SHARPIN, an adaptor for the linear ubiquitin chain assembly complex (LUBAC), plays important roles in NF-κB signaling and inflammation. Here, we have demonstrated a LUBAC-independent role for SHARPIN in regulating melanoma growth. We observed that SHARPIN interacted with PRMT5, a type II protein arginine methyltransferase, and increased its multiprotein complex and methyltransferase activity. Activated PRMT5 controlled the expression of the transcription factors SOX10 and MITF by SHARPIN-dependent arginine dimethylation and inhibition of the transcriptional corepressor SKI. Activation of PRMT5 by SHARPIN counteracted PRMT5 inhibition by methylthioadenosine, a substrate of methylthioadenosine phosphorylase, which is codeleted with cyclin-dependent kinase inhibitor 2A (CDKN2A) in approximately 15% of human cancers. Collectively, we identified a LUBAC-independent role for SHARPIN in enhancing PRMT5 activity that contributes to melanomagenesis through the SKI/SOX10 regulatory axis.
Assuntos
Melanoma/metabolismo , Proteínas de Neoplasias/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Ubiquitinas/metabolismo , Linhagem Celular Tumoral , Células HEK293 , Humanos , Melanoma/genética , Melanoma/patologia , Proteínas de Neoplasias/genética , Proteína-Arginina N-Metiltransferases/genética , Fatores de Transcrição SOXE/genética , Fatores de Transcrição SOXE/metabolismo , Ubiquitinas/genéticaRESUMO
Internally quenched fluorescent (IQF) peptide substrates originating from FRET (Förster Resonance Energy Transfer) are powerful tool for examining the activity and specificity of proteases, and a variety of donor/acceptor pairs are extensively used to design individual substrates and combinatorial libraries. We developed a highly sensitive and adaptable donor/acceptor pair that can be used to investigate the substrate specificity of cysteine proteases, serine proteases and metalloproteinases. This novel pair comprises 7-amino-4-carbamoylmethylcoumarin (ACC) as the fluorophore and 2,4-dinitrophenyl-lysine (Lys(DNP)) as the quencher. Using caspase-3, caspase-7, caspase-8, neutrophil elastase, legumain, and two matrix metalloproteinases (MMP2 and MMP9), we demonstrated that substrates containing ACC/Lys(DNP) exhibit 7 to 10 times higher sensitivity than conventional 7-methoxy-coumarin-4-yl acetic acid (MCA)/Lys(DNP) substrates; thus, substantially lower amounts of substrate and enzyme can be used for each assay. We therefore propose that the ACC/Lys(DNP) pair can be considered a novel and sensitive scaffold for designing substrates for any group of endopeptidases. We further demonstrate that IQF substrates containing unnatural amino acids can be used to investigate protease activities/specificities for peptides containing post-translationally modified amino acids. Finally, we used IQF substrates to re-investigate the P1-Asp characteristic of caspases, thus demonstrating that some human caspases can also hydrolyze substrates after glutamic acid.