RESUMO
Studying the interactions between a protease and its protein substrates at a molecular level is crucial for identifying the factors facilitating selection of particular proteolytic substrates and not others. These selection criteria include both the sequence and the local context of the substrate cleavage site where the active site of the protease initially binds and then performs proteolytic cleavage. Caspase-9, an initiator of the intrinsic apoptotic pathway, mediates activation of executioner procaspase-3 by cleavage of the intersubunit linker (ISL) at site 172IETD↓S. Although procaspase-6, another executioner, possesses two ISL cleavage sites (site 1, 176DVVD↓N; site 2, 190TEVD↓A), neither is directly cut by caspase-9. Thus, caspase-9 directly activates procaspase-3 but not procaspase-6. To elucidate this selectivity of caspase-9, we engineered constructs of procaspase-3 (e.g., swapping the ISL site, 172IETD↓S, with DVVDN and TEVDA) and procaspase-6 (e.g., swapping site 1, 176DVVD↓N, and site 2, 190TEVD↓A, with IETDS). Using the substrate digestion data of these constructs, we show here that the P4-P1' sequence of procaspase-6 ISL site 1 (DVVDN) can be accessed but not cleaved by caspase-9. We also found that caspase-9 can recognize the P4-P1' sequence of procaspase-6 ISL site 2 (TEVDA); however, the local context of this cleavage site is the critical factor that prevents proteolytic cleavage. Overall, our data have demonstrated that both the sequence and the local context of the ISL cleavage sites play a vital role in preventing the activation of procaspase-6 directly by caspase-9.
Assuntos
Caspase 3/química , Caspase 6/química , Caspase 9/metabolismo , Sequência de Aminoácidos/genética , Apoptose/fisiologia , Caspase 3/metabolismo , Caspase 6/metabolismo , Caspase 8/metabolismo , Caspase 9/fisiologia , Caspases/metabolismo , Ativação Enzimática , Humanos , Transdução de Sinais/genéticaRESUMO
Caspase-6 (Casp6) is implicated in Alzheimer disease (AD) cognitive impairment and pathology. Hippocampal atrophy is associated with cognitive impairment in AD. Here, a rare functional exonic missense CASP6 single nucleotide polymorphism (SNP), causing the substitution of asparagine with threonine at amino acid 73 in Casp6 (Casp6N73T), was associated with hippocampal subfield CA1 volume preservation. Compared to wild type Casp6 (Casp6WT), recombinant Casp6N73T altered Casp6 proteolysis of natural substrates Lamin A/C and α-Tubulin, but did not alter cleavage of the Ac-VEID-AFC Casp6 peptide substrate. Casp6N73T-transfected HEK293T cells showed elevated Casp6 mRNA levels similar to Casp6WT-transfected cells, but, in contrast to Casp6WT, did not accumulate active Casp6 subunits nor show increased Casp6 enzymatic activity. Electrophysiological and morphological assessments showed that Casp6N73T recombinant protein caused less neurofunctional damage and neurodegeneration in hippocampal CA1 pyramidal neurons than Casp6WT. Lastly, CASP6 mRNA levels were increased in several AD brain regions confirming the implication of Casp6 in AD. These studies suggest that the rare Casp6N73T variant may protect against hippocampal atrophy due to its altered catalysis of natural protein substrates and intracellular instability thus leading to less Casp6-mediated damage to neuronal structure and function.
Assuntos
Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Região CA1 Hipocampal/patologia , Caspase 6/genética , Caspase 6/metabolismo , Polimorfismo de Nucleotídeo Único , Transmissão Sináptica , Doença de Alzheimer/enzimologia , Substituição de Aminoácidos , Encéfalo/enzimologia , Encéfalo/patologia , Caspase 1/genética , Caspase 1/metabolismo , Caspase 6/química , Precursores Enzimáticos/metabolismo , Células HEK293 , Hipocampo , Humanos , Lamina Tipo A/metabolismo , Mutação de Sentido Incorreto , Degeneração Neural , Células Piramidais/citologia , Células Piramidais/fisiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/metabolismo , Tubulina (Proteína)/metabolismoRESUMO
The cysteine-containing aspartate specific proteinase (caspase) family plays important roles in apoptosis and the maintenance of homeostasis in lampreys. We conducted genomic and functional comparisons of six distinct lamprey caspase groups with human counterparts to determine how these expanded molecules evolved to adapt to the changing caspase-mediated signaling pathways. Our results showed that lineage-specific duplication and rearrangement were responsible for expanding lamprey caspases 3 and 7, whereas caspases 1, 6, 8, and 9 maintained a relatively stable genome and protein structure. Lamprey caspase family molecules displayed various expression patterns and were involved in the innate immune response. Caspase 1 and 7 functioned as a pattern recognition receptor with a broad-spectrum of microbial recognition and bactericidal effect. Additionally, caspases 1 and 7 may induce cell apoptosis in a time- and dose-dependent manner; however, apoptosis was inhibited by caspase inhibitors. Thus, these molecules may reflect the original state of the vertebrates caspase family. Our phylogenetic and functional data provide insights into the evolutionary history of caspases and illustrate their functional characteristics in primitive vertebrates.
Assuntos
Apoptose/genética , Caspases/genética , Imunidade Inata , Lampreias/genética , Transdução de Sinais/imunologia , Animais , Apoptose/efeitos dos fármacos , Caspase 1/química , Caspase 1/genética , Caspase 1/isolamento & purificação , Caspase 1/metabolismo , Caspase 3/química , Caspase 3/genética , Caspase 3/metabolismo , Caspase 6/química , Caspase 6/genética , Caspase 6/metabolismo , Caspase 7/química , Caspase 7/genética , Caspase 7/isolamento & purificação , Caspase 7/metabolismo , Caspase 8/química , Caspase 8/genética , Caspase 8/metabolismo , Caspase 9/química , Caspase 9/genética , Caspase 9/metabolismo , Inibidores de Caspase/farmacologia , Caspases/química , Caspases/isolamento & purificação , Caspases/metabolismo , Evolução Molecular , Duplicação Gênica , Rearranjo Gênico , Genoma , Genômica , Células HeLa , Humanos , Imunidade Inata/genética , Lampreias/crescimento & desenvolvimento , Lampreias/imunologia , Lampreias/metabolismo , Filogenia , Proteínas Recombinantes , Alinhamento de Sequência , Transdução de Sinais/genética , Staphylococcus aureus/efeitos dos fármacos , Regulação para Cima , Vibrio/efeitos dos fármacosRESUMO
The predominance of Alzheimer's disease (AD) among the aged remains a global challenge. As such, the search for alternative and effective therapeutic options continuous unabated. Among the therapeutic targets explored over the years toward impeding the progression of AD is caspase-6 (Casp6), although selectively targeting Casp6 remains a challenge due to high homology with other members of the caspase family. Methyl 3-[(2,3-dihydro-1-benzofuran-2-yl formamido) methyl]-5-(furan-2-amido) benzoate (C13), a novel allosteric inhibitor, is reportedly shown to exhibit selective inhibition against mutant human Casp6 variants (E35K). However, structural and atomistic insights accounting for the reported inhibitory prowess of C13 remains unresolved. In this study, we seek to unravel the mechanistic selectivity of C13 coupled with the complementary effects of E35K single-nucleotide polymorphism (SNP) relative to Casp6 inhibition. Analyses of binding dynamics revealed that the variant Lysine-35 mediated consistent high-affinity interactions with C13 at the allosteric site, possibly forming the molecular basis of the selectivity of C13 as well as its high binding free energy as estimated. Analysis of residue interaction network around Glu35 and Lys35 revealed prominent residue network distortions in the mutant Casp6 conformation evidenced by a decrease in node degree, reduced number of edges and an increase short in path length relative to a more compact conformation in the wild system. The relatively higher binding free energy of C13 coupled with the stronger intermolecular interactions elicited in the mutant conformation further suggests that the mutation E35K probably favours the inhibitory activity of C13. Further analysis of atomistic changes showed increased C-α atom deviations consistent with structural disorientations in the mutant Casp6. Structural Insights provided could open up a novel paradigm of structure-based design of selective allosteric inhibition of Casp6 towards the treatment of neurodegenerative diseases.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/genética , Caspase 6/genética , Inibidores de Caspase/farmacologia , Mutação , Polimorfismo de Nucleotídeo Único , Sítio Alostérico , Caspase 6/química , Desenho de Fármacos , Humanos , Imageamento Tridimensional , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação ProteicaRESUMO
Acyl phosphates of ATP (ATPAc) and related nucleotides have proven to be useful for the interrogation of known nucleotide binding sites via specific acylation of conserved lysines (K). In addition, occasional K acylations are identified in proteins without such known sites. Here we present a robust and specific acylation of procaspase-6 by ATPAc at K133 in Jurkat cell lysates. The K133 acylation is dependent on π-π stacking interactions between the adenine moiety of ATPAc and a conserved Y198-Y198 site formed at the homodimeric interface of procaspase-6. Significantly, the Y198A mutation in procaspase-6 abolishes K133 acylation but has no effect on the proteolytic activity of the mature, active caspase-6 Y198A variant. Additional in vitro studies show that ATP can inhibit the autoproteolytic activation of procaspase-6. These observations suggest that ATP, and possibly other nucleotides, may serve as the endogenous ligands for the allosteric site at the procaspase-6 dimer interface, a site that has persisted in its "orphan" status for more than a decade.
Assuntos
Trifosfato de Adenosina/metabolismo , Caspase 6/química , Caspase 6/metabolismo , Precursores Enzimáticos/química , Precursores Enzimáticos/metabolismo , Proteômica , Sequência de Aminoácidos , Sítios de Ligação , Humanos , Células Jurkat , Modelos Moleculares , Conformação ProteicaRESUMO
Caspase-6 is a cysteine protease that plays essential roles in programmed cell death, axonal degeneration, and development. The excess neuronal activity of Caspase-6 is associated with Alzheimer disease neuropathology and age-dependent cognitive impairment. Caspase-6 inhibition is a promising strategy to stop early stage neurodegenerative events, yet finding potent and selective Caspase-6 inhibitors has been a challenging task due to the overlapping structural and functional similarities between caspase family members. Here, we investigated how four rare non-synonymous missense single-nucleotide polymorphisms (SNPs), resulting in amino acid substitutions outside human Caspase-6 active site, affect enzyme structure and catalytic efficiency. Three investigated SNPs were found to align with a putative allosteric pocket with low sequence conservation among human caspases. Virtual screening of 57,700 compounds against the putative Caspase-6 allosteric pocket, followed by in vitro testing of the best virtual hits in recombinant human Caspase-6 activity assays identified novel allosteric Caspase-6 inhibitors with IC50 and Ki values ranging from ~2 to 13 µM. This report may pave the way towards the development and optimisation of novel small molecule allosteric Caspase-6 inhibitors and illustrates that functional characterisation of rare natural variants holds promise for the identification of allosteric sites on other therapeutic targets in drug discovery.
Assuntos
Caspase 6/química , Caspase 6/metabolismo , Inibidores de Caspase/farmacologia , Mutação de Sentido Incorreto , Bibliotecas de Moléculas Pequenas/farmacologia , Regulação Alostérica/efeitos dos fármacos , Substituição de Aminoácidos , Caspase 6/genética , Inibidores de Caspase/química , Domínio Catalítico , Simulação por Computador , Cristalografia por Raios X , Humanos , Modelos Moleculares , Polimorfismo de Nucleotídeo Único , Ligação Proteica , Conformação Proteica , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-AtividadeRESUMO
Aims: The coordination of neurons to execute brain functions requires plenty of oxygen. Thus, it is not surprising that the chronic hypoxia resulting from chronic obstructive pulmonary diseases (COPD) can cause neuronal damage. Injury in the cortex can give rise to anxiety and cognitive dysfunction. This study investigated what causes hypoxia-induced neuronal injury and what strategies might be used to protect neurons against such damage. Results: This study found that hypoxia in primary cortical neurons caused neurite retraction, a caspase-6-dependent process. The hypoxic stress activated caspase-6 within the neurite, leading to microtubule disassembly and neurite retraction. The effect of hypoxia on caspase-6 activation, microtubule disassembly, and neurite retraction was alleviated by nitrite treatment. The protective role of nitrite was further supported by the observation that the active-site Cys146 of caspase-6 was S-nitrosylated in hypoxic neuro-2a cells treated with nitrite. We further validated the beneficial effect of nitrite on neuronal function against hypoxic stress in vivo. Using the wild-type or Apo E-/- mice exposed to chronic hypoxia as a model, we demonstrated that supplementing drinking water with nitrite suppressed active caspase-6 in the cortex of the brain, concomitant with the prevention of hypoxia-induced anxiety in the animals. Innovation: These results are the first evidence of a new pathway for the activation of caspase-6 and the first to indicate that nitrite can protect neurons against chronic hypoxic insult. Conclusion: Our findings suggest that nitrite holds great potential for the treatment of diseases such as COPD associated with hypoxia-induced neuronal injury.
Assuntos
Caspase 6/metabolismo , Hipóxia Encefálica/prevenção & controle , Fármacos Neuroprotetores/administração & dosagem , Nitritos/administração & dosagem , Animais , Caspase 6/química , Domínio Catalítico , Linhagem Celular , Modelos Animais de Doenças , Hipóxia Encefálica/metabolismo , Camundongos , Camundongos Knockout para ApoE , Neuritos/efeitos dos fármacos , Neuritos/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fármacos Neuroprotetores/farmacologia , Nitritos/farmacologiaRESUMO
Caspases are cysteine-aspartic proteases involved in the regulation of programmed cell death (apoptosis) and a number of other biological processes. Despite overall similarities in structure and active-site composition, caspases show striking selectivity for particular protein substrates. Exosites are emerging as one of the mechanisms by which caspases can recruit, engage, and orient these substrates for proper hydrolysis. Following computational analyses and database searches for candidate exosites, we utilized site-directed mutagenesis to identify a new exosite in caspase-6 at the hinge between the disordered N-terminal domain (NTD), residues 23-45, and core of the caspase-6 structure. We observed that substitutions of the tri-arginine patch Arg-42-Arg-44 or the R44K cancer-associated mutation in caspase-6 markedly alter its rates of protein substrate hydrolysis. Notably, turnover of protein substrates but not of short peptide substrates was affected by these exosite alterations, underscoring the importance of this region for protein substrate recruitment. Hydrogen-deuterium exchange MS-mediated interrogation of the intrinsic dynamics of these enzymes suggested the presence of a substrate-binding platform encompassed by the NTD and the 240's region (containing residues 236-246), which serves as a general exosite for caspase-6-specific substrate recruitment. In summary, we have identified an exosite on caspase-6 that is critical for protein substrate recognition and turnover and therefore highly relevant for diseases such as cancer in which caspase-6-mediated apoptosis is often disrupted, and in neurodegeneration in which caspase-6 plays a central role.
Assuntos
Caspase 6/química , Mutação de Sentido Incorreto , Proteínas de Neoplasias/química , Neoplasias/enzimologia , Doenças Neurodegenerativas/enzimologia , Substituição de Aminoácidos , Arginina/química , Arginina/genética , Arginina/metabolismo , Caspase 6/genética , Caspase 6/metabolismo , Humanos , Hidrólise , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/patologia , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/patologia , Domínios ProteicosRESUMO
The cysteine protease Caspase-6 (Casp6) is a potential therapeutic target of Alzheimer Disease (AD) and age-dependent cognitive impairment. To assess if Casp6 is essential to human health, we investigated the effect of CASP6 variants sequenced from healthy humans on Casp6 activity. Here, we report the effects of two rare Casp6 amino acid polymorphisms, R65W and G66R, on the catalytic function and structure of Casp6. The G66R substitution eliminated and R65W substitution significantly reduced Casp6 catalytic activity through impaired substrate binding. In contrast to wild-type Casp6, both Casp6 variants were unstable and inactive in transfected mammalian cells. In addition, Casp6-G66R acted as a dominant negative inhibitor of wild-type Casp6. The R65W and G66R substitutions caused perturbations in substrate recognition and active site organization as revealed by molecular dynamics simulations. Our results suggest that full Casp6 activity may not be essential for healthy humans and support the use of Casp6 inhibitors against Casp6-dependent neurodegeneration in age-dependent cognitive impairment and AD. Furthermore, this work illustrates that studying natural single amino acid polymorphisms of enzyme drug targets is a promising approach to uncover previously uncharacterized regulatory sites important for enzyme activity.
Assuntos
Doença de Alzheimer/enzimologia , Substituição de Aminoácidos , Caspase 6/genética , Caspase 6/metabolismo , Disfunção Cognitiva/enzimologia , Doença de Alzheimer/tratamento farmacológico , Caspase 6/química , Domínio Catalítico , Disfunção Cognitiva/tratamento farmacológico , Desenho de Fármacos , Humanos , Modelos Moleculares , Mutação de Sentido Incorreto , Polimorfismo de Nucleotídeo Único , Ligação Proteica , Conformação Proteica , Sequências Reguladoras de Ácido NucleicoRESUMO
Caspase-6 is critical to the neurodegenerative pathways of Alzheimer's, Huntington's, and Parkinson's diseases and has been identified as a potential molecular target for treatment of neurodegeneration. Thus, understanding the global and regional changes in dynamics and conformation provides insights into the unique properties of caspase-6 that may contribute to achieving control of its function. In this work, hydrogen/deuterium exchange MS (H/DX-MS) was used to map the local changes in the conformational flexibility of procaspase-6 at the discrete states that reflect the series of cleavage events that ultimately lead to the fully active, substrate-bound state. Intramolecular self-cleavage at Asp-193 evoked higher solvent exposure in the regions of the substrate-binding loops L1, L3, and L4 and in the 130s region, the intersubunit linker region, the 26-32 region as well as in the stabilized loop 2. Additional removal of the linker allowed caspase-6 to gain more flexibility in the 130s region and in the L2 region converting caspase-6 to a competent substrate-binding state. The prodomain region was found to be intrinsically disordered independent of the activation state of caspase-6; however, its complete removal resulted in the protection of the adjacent 26-32 region, suggesting that this region may play a regulatory role. The molecular details of caspase-6 dynamics in solution provide a comprehensive scaffold for strategic design of therapeutic approaches for neurodegenerative disorders.
Assuntos
Caspase 6/química , Simulação de Dinâmica Molecular , Proteólise , Caspase 6/metabolismo , Medição da Troca de Deutério , Humanos , Domínios Proteicos , Estrutura Secundária de ProteínaRESUMO
Unregulated, particularly suppressed programmed cell death is one of the distinguishing features of many cancer cells. The cysteine protease caspase-6, one of the executioners of apoptotic cell death, plays a crucial role in regulation of apoptosis. Several somatic mutations in the CASP6 gene in tumor tissues have been reported. This work explores the effect of CASP6 tumor-associated mutations on the catalytic efficiency and structure of caspase-6. In general, these mutations showed decreased overall rates of catalytic turnover. Mutations within 8 Å of the substrate-binding pocket of caspase-6 were found to be the most catalytically deactivating. Notably, the R259H substitution decreased activity by 457-fold. This substitution disrupts the cation-π stacking interaction between Arg-259 and Trp-227, which is indispensable for proper assembly of the substrate-binding loops in caspase-6. Sequence conservation analysis at the homologous position across the caspase family suggests a role for this cation-π stacking in the catalytic function of caspases generally. These data suggest that caspase-6 deactivating mutations may contribute to multifactorial carcinogenic transformations.
Assuntos
Caspase 6/química , Mutação de Sentido Incorreto , Proteínas de Neoplasias/química , Neoplasias/enzimologia , Substituição de Aminoácidos , Caspase 6/genética , Caspase 6/metabolismo , Domínio Catalítico , Humanos , Proteínas de Neoplasias/metabolismo , Neoplasias/genéticaRESUMO
Caspases are cysteine aspartate proteases that are major players in key cellular processes, including apoptosis and inflammation. Specifically, caspase-6 has also been implicated in playing a unique and critical role in neurodegeneration; however, structural similarities between caspase-6 and other caspase active sites have hampered precise targeting of caspase-6. All caspases can exist in a canonical conformation, in which the substrate binds atop a ß-strand platform in the 130's region. This caspase-6 region can also adopt a helical conformation that has not been seen in any other caspases. Understanding the dynamics and interconversion between the helical and strand conformations in caspase-6 is critical to fully assess its unique function and regulation. Here, hydrogen/deuterium exchange mass spectrometry indicated that caspase-6 is inherently and dramatically more conformationally dynamic than closely related caspase-7. In contrast to caspase-7, which rests constitutively in the strand conformation before and after substrate binding, the hydrogen/deuterium exchange data in the L2' and 130's regions suggested that before substrate binding, caspase-6 exists in a dynamic equilibrium between the helix and strand conformations. Caspase-6 transitions exclusively to the canonical strand conformation only upon substrate binding. Glu-135, which showed noticeably different calculated pK a values in the helix and strand conformations, appears to play a key role in the interconversion between the helix and strand conformations. Because caspase-6 has roles in several neurodegenerative diseases, exploiting the unique structural features and conformational changes identified here may provide new avenues for regulating specific caspase-6 functions for therapeutic purposes.
Assuntos
Caspase 6/metabolismo , Caspase 6/química , Caspase 7/química , Caspase 7/metabolismo , Estabilidade Enzimática , Humanos , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica , Conformação Proteica em alfa-Hélice , PrótonsRESUMO
The ability to routinely engineer protease specificity can allow us to better understand and modulate their biology for expanded therapeutic and industrial applications. Here, we report a new approach based on a caged green fluorescent protein (CA-GFP) reporter that allows for flow-cytometry-based selection in bacteria or other cell types enabling selection of intracellular protease specificity, regardless of the compositional complexity of the protease. Here, we apply this approach to introduce the specificity of caspase-6 into caspase-7, an intracellular cysteine protease important in cellular remodeling and cell death. We found that substitution of substrate-contacting residues from caspase-6 into caspase-7 was ineffective, yielding an inactive enzyme, whereas saturation mutagenesis at these positions and selection by directed evolution produced active caspases. The process produced a number of nonobvious mutations that enabled conversion of the caspase-7 specificity to match caspase-6. The structures of the evolved-specificity caspase-7 (esCasp-7) revealed alternate binding modes for the substrate, including reorganization of an active site loop. Profiling the entire human proteome of esCasp-7 by N-terminomics demonstrated that the global specificity toward natural protein substrates is remarkably similar to that of caspase-6. Because the esCasp-7 maintained the core of caspase-7, we were able to identify a caspase-6 substrate, lamin C, that we predict relies on an exosite for substrate recognition. These reprogrammed proteases may be the first tool built with the express intent of distinguishing exosite dependent or independent substrates. This approach to specificity reprogramming should also be generalizable across a wide range of proteases.
Assuntos
Caspase 7/metabolismo , Evolução Molecular Direcionada , Caspase 6/química , Caspase 7/genética , Citometria de Fluxo , Proteínas de Fluorescência Verde/genética , Humanos , Células Jurkat , Laminas/metabolismo , Modelos Moleculares , Mutação , Engenharia de Proteínas , Proteoma/metabolismo , Especificidade por SubstratoRESUMO
Caspases, a family of cysteine proteases, are major mediators of apoptosis and inflammation. Caspase-6 is classified as an apoptotic effector, and it mediates nuclear shrinkage during apoptosis, but it possesses unique activation and regulation mechanisms that differ from those of other effector caspases. Furthermore, increasing evidence has shown that caspase-6 is highly involved in axon degeneration and neurodegenerative diseases, such as Huntington's disease and Alzheimer's disease. Cleavage at the caspase-6 site in mutated huntingtin protein is a prerequisite for the development of the characteristic behavioral and neuropathological features of Huntington's disease. Active caspase-6 is present in early stages of Alzheimer's disease, and caspase-6 activity is associated with the disease's pathological lesions. In this review, we discuss the evidence relevant to the role of caspase-6 in neurodegenerative diseases and summarize its activation and regulation mechanisms. In doing so, we provide new insight about potential therapeutic approaches that incorporate the modulation of caspase-6 function for the treatment of neurodegenerative diseases.
Assuntos
Doença de Alzheimer/enzimologia , Encéfalo/enzimologia , Caspase 6/metabolismo , Doença de Huntington/enzimologia , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/patologia , Sequência de Aminoácidos , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Caspase 6/química , Inibidores de Caspase/uso terapêutico , Desenho de Fármacos , Ativação Enzimática , Humanos , Proteína Huntingtina , Doença de Huntington/tratamento farmacológico , Doença de Huntington/patologia , Modelos Moleculares , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/metabolismo , Conformação Proteica , Transdução de Sinais , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Caspases are a family of cysteine proteases that play an important role in the apoptotic pathway. Caspase-6 is an apoptosis effector that cleaves a variety of cellular substrates. The active form of the enzyme is required for use in research. However, it has been difficult to obtain sufficient quantities of active caspase-6 from Escherichia coli. In the present study, we constructed a caspase-6 with a 23-amino-acid deletion in the pro-domain. This engineered enzyme was expressed as a soluble protein in E. coli and was purified using affinity resin. In vitro enzyme assay and cleavage analysis revealed that the engineered active caspase-6 protein had characteristics similar to those of wild-type caspase-6. This novel method can be a valuable tool for obtaining active caspase-6 that can be used for screening caspase-6-specific substrates, which in turn can be used to elucidate the function of caspase-6 in apoptosis.
Assuntos
Caspase 6/genética , Caspase 6/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Domínios e Motivos de Interação entre Proteínas/genética , Deleção de Sequência , Caspase 6/química , Caspase 6/isolamento & purificação , Ativação Enzimática , Proteínas RecombinantesRESUMO
Caspase 6 (CASP6) is a neuron degeneration-related protease and is widely considered to be a potential drug-design target against neurodegenerative diseases such as Huntington's disease and Alzheimer's disease. The N-terminal pro-peptide of CASP6, also referred to as the pro-domain, contains 23 residues and its functional role remains elusive. In this study, the crystal structure of a full-length CASP6 zymogen mutant, proCASP6H121A, was solved. Although the pro-domain was flexible in the crystal, without visible electron density, structural analyses combined with biochemical assays revealed that the pro-domain inhibited CASP6 auto-activation by inhibiting intramolecular cleavage at the intersubunit cleavage site TEVD(193) and also by preventing this site from intermolecular cleavage at low protein concentration through a so-called `suicide-protection' mechanism. Further experiments showed that the length of the pro-domain and the side chain of Asn18 played critical roles in suicide protection. These results disclosed a new inhibitory mechanism of CASP6 and shed light on the pathogenesis and therapeutically relevant study of CASP6-related neurodegenerative diseases.
Assuntos
Caspase 6/química , Caspase 6/genética , Caspase 6/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Ativação Enzimática , Humanos , Modelos Moleculares , Mutação , Doenças Neurodegenerativas/enzimologia , Estrutura Terciária de ProteínaRESUMO
This study was designed to investigate the effects of desmin oxidation on its degradation by proteolytic enzymes. Desmin was isolated from bovine muscle and exposed to varying oxidative conditions, and then incubated with µ-calpain, caspase-3 or -6, respectively. The extent of protein degradation was subsequently determined using SDS-PAGE and Western-blotting. Furthermore, the oxidative modification of the secondary structure of desmin was measured by circular dichroism (CD). Our results revealed that, compared with the native desmin, degradation of oxidised desmin was enhanced by caspases, but suppressed by µ-calpain. The CD spectra of desmin showed that the content of α-helix decreased from 76.2% to 52% while random coil increased from 8% to 22.4% after oxidation. These findings demonstrated that oxidative modifications of desmin changed their susceptibility to µ-calpain, caspase-3 and -6 as well as their secondary structure.
Assuntos
Calpaína/química , Caspase 3/química , Caspase 6/química , Desmina/química , Carne/análise , Músculo Esquelético/metabolismo , Animais , Calpaína/metabolismo , Caspase 3/metabolismo , Caspase 6/metabolismo , Bovinos , Desmina/isolamento & purificação , Desmina/metabolismo , Músculo Esquelético/química , Músculo Esquelético/enzimologia , Oxirredução , Conformação ProteicaRESUMO
Although they represent attractive therapeutic targets, caspases have so far proven recalcitrant to the development of drugs targeting the active site. Allosteric modulation of caspase activity is an alternate strategy that potentially avoids the need for anionic and electrophilic functionality present in most active-site inhibitors. Caspase-6 has been implicated in neurodegenerative disease, including Huntington's and Alzheimer's diseases. Herein we describe a fragment-based lead discovery effort focused on caspase-6 in its active and zymogen forms. Fragments were identified for procaspase-6 using surface plasmon resonance methods and subsequently shown by X-ray crystallography to bind a putative allosteric site at the dimer interface. A fragment-merging strategy was employed to produce nanomolar-affinity ligands that contact residues in the L2 loop at the dimer interface, significantly stabilizing procaspase-6. Because rearrangement of the L2 loop is required for caspase-6 activation, our results suggest a strategy for the allosteric control of caspase activation with drug-like small molecules.
Assuntos
Caspase 6/metabolismo , Bibliotecas de Moléculas Pequenas/química , Sítio Alostérico , Sítios de Ligação , Caspase 6/química , Cristalografia por Raios X , Dimerização , Desenho de Fármacos , Precursores Enzimáticos/química , Precursores Enzimáticos/metabolismo , Concentração de Íons de Hidrogênio , Simulação de Acoplamento Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Bibliotecas de Moléculas Pequenas/metabolismo , Temperatura de TransiçãoRESUMO
Apoptosis, programmed cell death, is a process involved in the development and maintenance of cell homeostasis in multicellular organisms. It is typically accompanied by the activation of a class of cysteine proteases called caspases. Apoptotic caspases are classified into the initiator caspases and the executioner caspases, according to the stage of their action in apoptotic processes. Although caspase-3, a typical executioner caspase, has been studied for its mechanism and substrates, little is known of caspase-6, one of the executioner caspases. To understand the biological functions of caspase-6, we performed proteomics analyses, to seek for novel caspase-6 substrates, using recombinant caspase-6 and HepG2 extract. Consequently, 34 different candidate proteins were identified, through 2-dimensional electrophoresis/MALDI-TOF analyses. Of these identified proteins, 8 proteins were validated with in vitro and in vivo cleavage assay. Herein, we report that HAUSP, Kinesin5B, GEP100, SDCCAG3 and PARD3 are novel substrates for caspase-6 during apoptosis.
Assuntos
Apoptose , Caspase 6/metabolismo , Apoptose/efeitos dos fármacos , Caspase 6/química , Caspase 6/genética , Inibidores de Caspase/metabolismo , Inibidores de Caspase/farmacologia , Eletroforese em Gel Bidimensional , Células HeLa , Células Hep G2 , Humanos , Proteômica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Especificidade por SubstratoRESUMO
Dysregulation of apoptosis is associated with several human diseases. The main apoptotic mediators are caspases, which propagate death signals to downstream targets. Executioner caspase-3 is responsible for the majority of cleavage events and its therapeutic potential is of high interest with to date several available active site peptide inhibitors. These molecules inhibit caspase-3, but also homologous caspases. Here, we describe caspase-3 specific inhibitors D3.4 and D3.8, which have been selected from a library of designed ankyrin repeat proteins (DARPins). The crystal structures of D3.4 and mutants thereof show how high specificity and inhibition is achieved. They also show similarities in the binding mode with that of the natural caspase inhibitor XIAP (X-linked inhibitor of apoptosis). The kinetic data reveal a competitive inhibition mechanism. D3.4 is specific for caspase-3 and does not bind the highly homologous caspase-7. D3.4 therefore is an excellent tool to define the precise role of caspase-3 in the various apoptotic pathways.