RESUMO
A vast ensemble of extracellular proteins influences the development and progression of cancer, shaped and reshaped by a complex network of extracellular proteases. These proteases, belonging to the distinct classes of metalloproteases, serine proteases, cysteine proteases, and aspartic proteases, play a critical role in cancer. They often become dysregulated in cancer, with increases in pathological protease activity frequently driven by the loss of normal latency controls, diminished regulation by endogenous protease inhibitors, and changes in localization. Dysregulated proteases accelerate tumor progression and metastasis by degrading protein barriers within the extracellular matrix (ECM), stimulating tumor growth, reactivating dormant tumor cells, facilitating tumor cell escape from immune surveillance, and shifting stromal cells toward cancer-promoting behaviors through the precise proteolysis of specific substrates to alter their functions. These crucial substrates include ECM proteins and proteoglycans, soluble proteins secreted by tumor and stromal cells, and extracellular domains of cell surface proteins, including membrane receptors and adhesion proteins. The complexity of the extracellular protease web presents a significant challenge to untangle. Nevertheless, technological strides in proteomics, chemical biology, and the development of new probes and reagents are enabling progress and advancing our understanding of the pivotal importance of extracellular proteolysis in cancer.
Assuntos
Metástase Neoplásica , Neoplasias , Peptídeo Hidrolases , Proteólise , Humanos , Neoplasias/metabolismo , Neoplasias/patologia , Neoplasias/enzimologia , Peptídeo Hidrolases/metabolismo , Animais , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Progressão da DoençaRESUMO
Ovarian clear cell carcinoma (OCCC) is an understudied poor prognosis subtype of ovarian cancer lacking in effective targeted therapies. Efforts to define molecular drivers of OCCC malignancy may lead to new therapeutic targets and approaches. Among potential targets are secreted proteases, enzymes which in many cancers serve as key drivers of malignant progression. Here, we found that inhibitors of trypsin-like serine proteases suppressed malignant phenotypes of OCCC cell lines. To identify the proteases responsible for malignancy in OCCC, we employed activity-based protein profiling to directly analyze enzyme activity. We developed an activity-based probe featuring an arginine diphenylphosphonate warhead to detect active serine proteases of trypsin-like specificity and a biotin handle to facilitate affinity purification of labeled proteases. Using this probe, we identified active trypsin-like serine proteases within the complex proteomes secreted by OCCC cell lines, including two proteases in common, tissue plasminogen activator and urokinase-type plasminogen activator. Further interrogation of these proteases showed that both were involved in cancer cell invasion and proliferation of OCCC cells and were also detected in in vivo models of OCCC. We conclude the detection of tissue plasminogen activator and urokinase-type plasminogen activator as catalytically active proteases and significant drivers of the malignant phenotype may point to these enzymes as targets for new therapeutic strategies in OCCC. Our activity-based probe and profiling methodology will also serve as a valuable tool for detection of active trypsin-like serine proteases in models of other cancers and other diseases.
Assuntos
Adenocarcinoma de Células Claras , Neoplasias Ovarianas , Serina Proteases , Adenocarcinoma de Células Claras/enzimologia , Adenocarcinoma de Células Claras/patologia , Feminino , Humanos , Neoplasias Ovarianas/enzimologia , Neoplasias Ovarianas/patologia , Serina Proteases/metabolismo , Ativador de Plasminogênio Tecidual/metabolismo , Tripsina , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
Matrix metalloproteinases (MMPs) have long been known as key drivers in the development and progression of diseases, including cancer and neurodegenerative, cardiovascular, and many other inflammatory and degenerative diseases, making them attractive potential drug targets. Engineering selective inhibitors based upon tissue inhibitors of metalloproteinases (TIMPs), endogenous human proteins that tightly yet nonspecifically bind to the family of MMPs, represents a promising new avenue for therapeutic development. Here, we used a counter-selective screening strategy for directed evolution of yeast-displayed human TIMP-1 to obtain TIMP-1 variants highly selective for the inhibition of MMP-3 in preference over MMP-10. As MMP-3 and MMP-10 are the most similar MMPs in sequence, structure, and function, our results thus clearly demonstrate the capability for engineering full-length TIMP proteins to be highly selective MMP inhibitors. We show using protein crystal structures and models of MMP-3-selective TIMP-1 variants bound to MMP-3 and counter-target MMP-10 how structural alterations within the N-terminal and C-terminal TIMP-1 domains create new favorable and selective interactions with MMP-3 and disrupt unique interactions with MMP-10. While our MMP-3-selective inhibitors may be of interest for future investigation in diseases where this enzyme drives pathology, our platform and screening strategy can be employed for developing selective inhibitors of additional MMPs implicated as therapeutic targets in disease.
Assuntos
Metaloproteinase 3 da Matriz , Inibidor Tecidual de Metaloproteinase-1 , Humanos , Metaloproteinase 10 da Matriz/química , Metaloproteinase 10 da Matriz/genética , Metaloproteinase 10 da Matriz/metabolismo , Metaloproteinase 3 da Matriz/química , Metaloproteinase 3 da Matriz/genética , Metaloproteinase 3 da Matriz/metabolismo , Engenharia de Proteínas , Inibidor Tecidual de Metaloproteinase-1/química , Inibidor Tecidual de Metaloproteinase-1/genética , Inibidor Tecidual de Metaloproteinase-1/metabolismoRESUMO
Since the proposition of the pro-invasive activity of proteolytic enzymes over 70 years ago, several roles for proteases in cancer progression have been established. About half of the 473 active human proteases are expressed in the prostate and many of the most well-characterized members of this enzyme family are regulated by androgens, hormones essential for development of prostate cancer. Most notably, several kallikrein-related peptidases, including KLK3 (prostate-specific antigen, PSA), the most well-known prostate cancer marker, and type II transmembrane serine proteases, such as TMPRSS2 and matriptase, have been extensively studied and found to promote prostate cancer progression. Recent findings also suggest a critical role for proteases in the development of advanced and aggressive castration-resistant prostate cancer (CRPC). Perhaps the most intriguing evidence for this role comes from studies showing that the protease-activated transmembrane proteins, Notch and CDCP1, are associated with the development of CRPC. Here, we review the roles of proteases in prostate cancer, with a special focus on their regulation by androgens.
Assuntos
Peptídeo Hidrolases , Neoplasias da Próstata , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/patologia , Humanos , Animais , Peptídeo Hidrolases/sangue , Peptídeo Hidrolases/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico , Biomarcadores Tumorais/sangueRESUMO
Protein-protein interactions (PPIs) have evolved to display binding affinities that can support their function. As such, cognate and noncognate PPIs could be highly similar structurally but exhibit huge differences in binding affinities. To understand this phenomenon, we study three homologous protease-inhibitor PPIs that span 9 orders of magnitude in binding affinity. Using state-of-the-art methodology that combines protein randomization, affinity sorting, deep sequencing, and data normalization, we report quantitative binding landscapes consisting of ΔΔGbind values for the three PPIs, gleaned from tens of thousands of single and double mutations. We show that binding landscapes of the three complexes are strikingly different and depend on the PPI evolutionary optimality. We observe different patterns of couplings between mutations for the three PPIs with negative and positive epistasis appearing most frequently at hot-spot and cold-spot positions, respectively. The evolutionary trends observed here are likely to be universal to other biological complexes in the cell.
Assuntos
Mapeamento de Interação de ProteínasRESUMO
To facilitate investigations of protein-protein interactions (PPIs), we developed a novel platform for quantitative mapping of protein binding specificity landscapes, which combines the multi-target screening of a mutagenesis library into high- and low-affinity populations with sophisticated next-generation sequencing analysis. Importantly, this method generates accurate models to predict affinity and specificity values for any mutation within a protein complex, and requires only a few experimental binding affinity measurements using purified proteins for calibration. We demonstrated the utility of the approach by mapping quantitative landscapes for interactions between the N-terminal domain of the tissue inhibitor of metalloproteinase 2 (N-TIMP2) and three matrix metalloproteinases (MMPs) having homologous structures but different affinities (MMP-1, MMP-3, and MMP-14). The binding landscapes for N-TIMP2/MMP-1 and N-TIMP2/MMP-3 showed the PPIs to be almost fully optimized, with most single mutations giving a loss of affinity. In contrast, the non-optimized PPI for N-TIMP2/MMP-14 was reflected in a wide range of binding affinities, where single mutations exhibited a far more attenuated effect on the PPI. Our new platform reliably and comprehensively identified not only hot- and cold-spot residues, but also specificity-switch mutations that shape target affinity and specificity. Thus, our approach provides a methodology giving an unprecedentedly rich quantitative analysis of the binding specificity landscape, which will broaden the understanding of the mechanisms and evolutionary origins of specific PPIs and facilitate the rational design of specific inhibitors for structurally similar target proteins.
Assuntos
Metaloproteinase 14 da Matriz/metabolismo , Metaloproteinase 1 da Matriz/metabolismo , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas/métodos , Inibidor Tecidual de Metaloproteinase-2/genética , Biologia Computacional/métodos , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Metaloproteinase 3 da Matriz/metabolismo , Mutagênese , Mutação , Engenharia de Proteínas/métodos , Inibidor Tecidual de Metaloproteinase-2/química , Inibidor Tecidual de Metaloproteinase-2/metabolismoRESUMO
Serine protease inhibitors of the Kunitz-bovine pancreatic trypsin inhibitor (BPTI) family are ubiquitous biological regulators of proteolysis. These small proteins are resistant to proteolysis, but can be slowly cleaved within the protease-binding loop by target proteases, thereby compromising their activity. For the human protease mesotrypsin, this cleavage is especially rapid. Here, we aimed to stabilize the Kunitz domain structure against proteolysis through disulfide engineering. Substitution within the Kunitz inhibitor domain of the amyloid precursor protein (APPI) that incorporated a new disulfide bond between residues 17 and 34 reduced proteolysis by mesotrypsin 74-fold. Similar disulfide engineering of tissue factor pathway inhibitor-1 Kunitz domain 1 (KD1TFPI1) and bikunin Kunitz domain 2 (KD2bikunin) likewise stabilized these inhibitors against mesotrypsin proteolysis 17- and 6.6-fold, respectively. Crystal structures of disulfide-engineered APPI and KD1TFPI1 variants in a complex with mesotrypsin at 1.5 and 2.0 Å resolution, respectively, confirmed the formation of well-ordered disulfide bonds positioned to stabilize the binding loop. Long all-atom molecular dynamics simulations of disulfide-engineered Kunitz domains and their complexes with mesotrypsin revealed conformational stabilization of the primed side of the inhibitor-binding loop by the engineered disulfide, along with global suppression of conformational dynamics in the Kunitz domain. Our findings suggest that the Cys-17-Cys-34 disulfide slows proteolysis by dampening conformational fluctuations in the binding loop and minimizing motion at the enzyme-inhibitor interface. The generalizable approach developed here for the stabilization against proteolysis of Kunitz domains, which can serve as important scaffolds for therapeutics, may thus find applications in drug development.
Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Aprotinina/metabolismo , Tripsina/metabolismo , Precursor de Proteína beta-Amiloide/química , Precursor de Proteína beta-Amiloide/genética , Animais , Aprotinina/química , Aprotinina/genética , Cristalografia por Raios X , Dissulfetos/química , Dissulfetos/metabolismo , Humanos , Modelos Moleculares , Conformação Proteica , Domínios Proteicos , Engenharia de Proteínas , Proteólise , Tripsina/químicaRESUMO
Tissue inhibitors of metalloproteinases (TIMPs) are natural inhibitors of matrix metalloproteinases (MMPs), enzymes that contribute to cancer and many inflammatory and degenerative diseases. The TIMP N-terminal domain binds and inhibits an MMP catalytic domain, but the role of the TIMP C-terminal domain in MMP inhibition is poorly understood. Here, we employed yeast surface display for directed evolution of full-length human TIMP-1 to develop MMP-3-targeting ultrabinders. By simultaneously incorporating diversity into both domains, we identified TIMP-1 variants that were up to 10-fold improved in binding MMP-3 compared with WT TIMP-1, with inhibition constants (Ki ) in the low picomolar range. Analysis of individual and paired mutations from the selected TIMP-1 variants revealed cooperative effects between distant residues located on the N- and C-terminal TIMP domains, positioned on opposite sides of the interaction interface with MMP-3. Crystal structures of MMP-3 complexes with TIMP-1 variants revealed conformational changes in TIMP-1 near the cooperative mutation sites. Affinity was strengthened by cinching of a reciprocal "tyrosine clasp" formed between the N-terminal domain of TIMP-1 and proximal MMP-3 interface and by changes in secondary structure within the TIMP-1 C-terminal domain that stabilize interdomain interactions and improve complementarity to MMP-3. Our protein engineering and structural studies provide critical insight into the cooperative function of TIMP domains and the significance of peripheral TIMP epitopes in MMP recognition. Our findings suggest new strategies to engineer TIMP proteins for therapeutic applications, and our directed evolution approach may also enable exploration of functional domain interactions in other protein systems.
Assuntos
Evolução Molecular Direcionada , Metaloproteinase 3 da Matriz/metabolismo , Inibidores de Metaloproteinases de Matriz/metabolismo , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Humanos , Metaloproteinase 3 da Matriz/química , Metaloproteinase 3 da Matriz/genética , Inibidores de Metaloproteinases de Matriz/química , Mutação , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Inibidor Tecidual de Metaloproteinase-1/química , Inibidor Tecidual de Metaloproteinase-1/genética , Técnicas do Sistema de Duplo-HíbridoRESUMO
Human tissue kallikrein (KLK) proteases are hormone-like signaling molecules with important functions in cancer pathophysiology. KLK-related peptidase 6 (KLK6), specifically, is highly up-regulated in several types of cancer, where its increased activity promotes cancer invasion and metastasis. This characteristic suggests KLK6 as an attractive target for therapeutic interventions. However, inhibitors that specifically target KLK6 have not yet been reported, possibly because KLK6 shares a high sequence homology and structural similarity with other serine proteases and resists inhibition by many polypeptide inhibitors. Here, we present an innovative combinatorial approach to engineering KLK6 inhibitors via flow cytometry-based screening of a yeast-displayed mutant library of the human amyloid precursor protein Kunitz protease inhibitor domain (APPI), an inhibitor of other serine proteases, such as anionic and cationic trypsins. On the basis of this screening, we generated APPIM17L,I18F,S19F,F34V (APPI-4M), an APPI variant with a KLK6 inhibition constant (Ki ) of 160 pm and a turnover time of 10 days. To the best of our knowledge, APPI-4M is the most potent KLK6 inhibitor reported to date, displaying 146-fold improved affinity and 13-fold improved proteolytic stability compared with WT APPI (APPIWT). We further demonstrate that APPI-4M acts as a functional inhibitor in a cell-based model of KLK6-dependent breast cancer invasion. Finally, the crystal structures of the APPIWT/KLK6 and APPI-4M/KLK6 complexes revealed the structural and mechanistic bases for the improved KLK6 binding and proteolytic resistance of APPI-4M. We anticipate that APPI-4M will have substantial translational potential as both imaging agent and therapeutic.
Assuntos
Precursor de Proteína beta-Amiloide/farmacologia , Neoplasias da Mama/tratamento farmacológico , Engenharia Genética , Calicreínas/antagonistas & inibidores , Inibidores de Proteases/farmacologia , Proteólise , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Movimento Celular , Proliferação de Células , Feminino , Ensaios de Triagem em Larga Escala , Humanos , Calicreínas/química , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Células Tumorais CultivadasRESUMO
High structural and sequence similarity within protein families can pose significant challenges to the development of selective inhibitors, especially toward proteolytic enzymes. Such enzymes usually belong to large families of closely similar proteases and may also hydrolyze, with different rates, protein- or peptide-based inhibitors. To address this challenge, we employed a combinatorial yeast surface display library approach complemented with a novel pre-equilibrium, competitive screening strategy for facile assessment of the effects of multiple mutations on inhibitor association rates and binding specificity. As a proof of principle for this combined approach, we utilized this strategy to alter inhibitor/protease association rates and to tailor the selectivity of the amyloid ß-protein precursor Kunitz protease inhibitor domain (APPI) for inhibition of the oncogenic protease mesotrypsin, in the presence of three competing serine proteases, anionic trypsin, cationic trypsin and kallikrein-6. We generated a variant, designated APPIP13W/M17G/I18F/F34V, with up to 30-fold greater specificity relative to the parental APPIM17G/I18F/F34V protein, and 6500- to 230 000-fold improved specificity relative to the wild-type APPI protein in the presence of the other proteases tested. A series of molecular docking simulations suggested a mechanism of interaction that supported the biochemical results. These simulations predicted that the selectivity and specificity are affected by the interaction of the mutated APPI residues with nonconserved enzyme residues located in or near the binding site. Our strategy will facilitate a better understanding of the binding landscape of multispecific proteins and will pave the way for design of new drugs and diagnostic tools targeting proteases and other proteins.
Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Biblioteca de Peptídeos , Inibidores de Proteases/química , Inibidores de Proteases/metabolismo , Tripsina/metabolismo , Precursor de Proteína beta-Amiloide/química , Precursor de Proteína beta-Amiloide/genética , Ligação Competitiva , Humanos , Modelos Moleculares , Simulação de Acoplamento Molecular , Especificidade por Substrato , Tripsina/genéticaRESUMO
Degradation of the extracellular matrices in the human body is controlled by matrix metalloproteinases (MMPs), a family of more than 20 homologous enzymes. Imbalance in MMP activity can result in many diseases, such as arthritis, cardiovascular diseases, neurological disorders, fibrosis, and cancers. Thus, MMPs present attractive targets for drug design and have been a focus for inhibitor design for as long as 3 decades. Yet, to date, all MMP inhibitors have failed in clinical trials because of their broad activity against numerous MMP family members and the serious side effects of the proposed treatment. In this study, we integrated a computational method and a yeast surface display technique to obtain highly specific inhibitors of MMP-14 by modifying the natural non-specific broad MMP inhibitor protein N-TIMP2 to interact optimally with MMP-14. We identified an N-TIMP2 mutant, with five mutations in its interface, that has an MMP-14 inhibition constant (Ki ) of 0.9 pm, the strongest MMP-14 inhibitor reported so far. Compared with wild-type N-TIMP2, this variant displays â¼900-fold improved affinity toward MMP-14 and up to 16,000-fold greater specificity toward MMP-14 relative to other MMPs. In an in vitro and cell-based model of MMP-dependent breast cancer cellular invasiveness, this N-TIMP2 mutant acted as a functional inhibitor. Thus, our study demonstrates the enormous potential of a combined computational/directed evolution approach to protein engineering. Furthermore, it offers fundamental clues into the molecular basis of MMP regulation by N-TIMP2 and identifies a promising MMP-14 inhibitor as a starting point for the development of protein-based anticancer therapeutics.
Assuntos
Desenho de Fármacos , Metaloproteinase 14 da Matriz/metabolismo , Inibidores de Metaloproteinases de Matriz/química , Inibidores de Metaloproteinases de Matriz/farmacologia , Inibidor Tecidual de Metaloproteinase-2/química , Inibidor Tecidual de Metaloproteinase-2/farmacologia , Sequência de Aminoácidos , Animais , Bovinos , Cristalografia por Raios X , Evolução Molecular Direcionada , Humanos , Metaloproteinase 14 da Matriz/química , Inibidores de Metaloproteinases de Matriz/metabolismo , Simulação de Acoplamento Molecular , Mutação , Inibidor Tecidual de Metaloproteinase-2/genéticaRESUMO
The molecular basis of enzyme catalytic power and specificity derives from dynamic interactions between enzyme and substrate during catalysis. Although considerable effort has been devoted to understanding how conformational dynamics within enzymes affect catalysis, the role of conformational dynamics within protein substrates has not been addressed. Here, we examine the importance of substrate dynamics in the cleavage of Kunitz-bovine pancreatic trypsin inhibitor protease inhibitors by mesotrypsin, finding that the varied conformational dynamics of structurally similar substrates can profoundly impact the rate of catalysis. A 1.4-Å crystal structure of a mesotrypsin-product complex formed with a rapidly cleaved substrate reveals a dramatic conformational change in the substrate upon proteolysis. By using long all-atom molecular dynamics simulations of acyl-enzyme intermediates with proteolysis rates spanning 3 orders of magnitude, we identify global and local dynamic features of substrates on the nanosecond-microsecond time scale that correlate with enzymatic rates and explain differential susceptibility to proteolysis. By integrating multiple enhanced sampling methods for molecular dynamics, we model a viable conformational pathway between substrate-like and product-like states, linking substrate dynamics on the nanosecond-microsecond time scale with large collective substrate motions on the much slower time scale of catalysis. Our findings implicate substrate flexibility as a critical determinant of catalysis.
Assuntos
Aprotinina/química , Simulação de Dinâmica Molecular , Proteólise , Tripsina/química , Animais , Catálise , Bovinos , Cristalografia por Raios X , Domínios ProteicosRESUMO
Matrix metalloproteinases (MMPs) are a family of zinc endopeptidases that cleave nearly all components of the extracellular matrix as well as many other soluble and cell-associated proteins. MMPs have been implicated in normal physiological processes, including development, and in the acquisition and progression of the malignant phenotype. Disappointing results from a series of clinical trials testing small molecule, broad spectrum MMP inhibitors as cancer therapeutics led to a re-evaluation of how MMPs function in the tumor microenvironment, and ongoing research continues to reveal that these proteins play complex roles in cancer development and progression. It is now clear that effective targeting of MMPs for therapeutic benefit will require selective inhibition of specific MMPs. Here, we provide an overview of the MMP family and its biological regulators, the tissue inhibitors of metalloproteinases (TIMPs). We then summarize recent research from model systems that elucidate how specific MMPs drive the malignant phenotype of breast cancer cells, including acquisition of cancer stem cell features and induction of the epithelial-mesenchymal transition, and we also outline clinical studies that implicate specific MMPs in breast cancer outcomes. We conclude by discussing ongoing strategies for development of inhibitors with therapeutic potential that are capable of selectively targeting the MMPs most responsible for tumor promotion, with special consideration of the potential of biologics including antibodies and engineered proteins based on the TIMP scaffold. J. Cell. Biochem. 118: 3531-3548, 2017. © 2017 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals, Inc.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Colagenases/metabolismo , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Inibidores de Metaloproteinases de Matriz/uso terapêutico , Proteínas de Neoplasias , Microambiente Tumoral/efeitos dos fármacos , Animais , Neoplasias da Mama/enzimologia , Feminino , Humanos , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/metabolismo , Inibidores Teciduais de Metaloproteinases/metabolismoRESUMO
Engineered protein therapeutics offer advantages, including strong target affinity, selectivity and low toxicity, but like natural proteins can be susceptible to proteolytic degradation, thereby limiting their effectiveness. A compelling therapeutic target is mesotrypsin, a protease up-regulated with tumour progression, associated with poor prognosis, and implicated in tumour growth and progression of many cancers. However, with its unique capability for cleavage and inactivation of proteinaceous inhibitors, mesotrypsin presents a formidable challenge to the development of biological inhibitors. We used a powerful yeast display platform for directed evolution, employing a novel multi-modal library screening strategy, to engineer the human amyloid precursor protein Kunitz protease inhibitor domain (APPI) simultaneously for increased proteolytic stability, stronger binding affinity and improved selectivity for mesotrypsin inhibition. We identified a triple mutant APPIM17G/I18F/F34V, with a mesotrypsin inhibition constant (Ki) of 89 pM, as the strongest mesotrypsin inhibitor yet reported; this variant displays 1459-fold improved affinity, up to 350 000-fold greater specificity and 83-fold improved proteolytic stability compared with wild-type APPI. We demonstrated that APPIM17G/I18F/F34V acts as a functional inhibitor in cell-based models of mesotrypsin-dependent prostate cancer cellular invasiveness. Additionally, by solving the crystal structure of the APPIM17G/I18F/F34V-mesotrypsin complex, we obtained new insights into the structural and mechanistic basis for improved binding and proteolytic resistance. Our study identifies a promising mesotrypsin inhibitor as a starting point for development of anticancer protein therapeutics and establishes proof-of-principle for a novel library screening approach that will be widely applicable for simultaneously evolving proteolytic stability in tandem with desired functionality for diverse protein scaffolds.
Assuntos
Precursor de Proteína beta-Amiloide/química , Inibidores de Proteases/metabolismo , Engenharia de Proteínas/métodos , Tripsina/metabolismo , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Evolução Molecular Direcionada/métodos , Citometria de Fluxo , Humanos , Cinética , Masculino , Neoplasias/tratamento farmacológico , Especificidade por SubstratoRESUMO
Human mesotrypsin is highly homologous to other mammalian trypsins, and yet it is functionally unique in possessing resistance to inhibition by canonical serine protease inhibitors and in cleaving these inhibitors as preferred substrates. Arg-193 and Ser-39 have been identified as contributors to the inhibitor resistance and cleavage capability of mesotrypsin, but it is not known whether these residues fully account for the unusual properties of mesotrypsin. Here, we use human cationic trypsin as a template for engineering a gain of catalytic function, assessing mutants containing mesotrypsin-like mutations for resistance to inhibition by bovine pancreatic trypsin inhibitor (BPTI) and amyloid precursor protein Kunitz protease inhibitor (APPI), and for the ability to hydrolyze these inhibitors as substrates. We find that Arg-193 and Ser-39 are sufficient to confer mesotrypsin-like resistance to inhibition; however, compared with mesotrypsin, the trypsin-Y39S/G193R double mutant remains 10-fold slower at hydrolyzing BPTI and 2.5-fold slower at hydrolyzing APPI. We identify two additional residues in mesotrypsin, Lys-74 and Asp-97, which in concert with Arg-193 and Ser-39 confer the full catalytic capability of mesotrypsin for proteolysis of BPTI and APPI. Novel crystal structures of trypsin mutants in complex with BPTI suggest that these four residues function cooperatively to favor conformational dynamics that assist in dissociation of cleaved inhibitors. Our results reveal that efficient inhibitor cleavage is a complex capability to which at least four spatially separated residues of mesotrypsin contribute. These findings suggest that inhibitor cleavage represents a functional adaptation of mesotrypsin that may have evolved in response to positive selection pressure.
Assuntos
Aminoácidos/genética , Evolução Molecular , Inibidores da Tripsina/metabolismo , Tripsina/química , Tripsina/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Aprotinina/química , Aprotinina/metabolismo , Biocatálise , Bovinos , Sequência Conservada , Cristalografia por Raios X , Humanos , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Proteólise , Ratos , Especificidade por SubstratoRESUMO
Human cationic trypsinogen, precursor of the digestive enzyme trypsin, can be rapidly degraded to protect the pancreas when pathological conditions threaten, while trypsin itself is impressively resistant to degradation. For either form, degradation is controlled by two necessary initial proteolytic events: cleavage of the Leu81-Glu82 peptide bond by chymotrypsin C (CTRC) and cleavage of the Arg122-Val123 peptide bond by trypsin. Here we demonstrate that the Leu81-Glu82 peptide bond of human cationic trypsin, but not trypsinogen, is thermodynamically stable, such that cleavage by CTRC leads to an equilibrium mixture containing 10% cleaved and 90% uncleaved trypsin. When cleaved trypsin was incubated with CTRC, the Leu81-Glu82 peptide bond was re-synthesized to establish the same equilibrium. The thermodynamic stability of the scissile peptide bond was not dependent on CTRC or Leu-81, as re-synthesis was also accomplished by other proteases acting on mutated cationic trypsin. The Leu81-Glu82 peptide bond is located within a calcium binding loop, and thermodynamic stability of the bond was strictly dependent on calcium and on the calcium-coordinated residue Glu-85. Trypsinolytic cleavage of the Arg122-Val123 site was also delayed in trypsin relative to trypsinogen in a calcium-dependent manner, but for this bond cleavage was modulated by kinetic rather than thermodynamic control. Our results reveal that the trypsinogen to trypsin conformational switch modulates cleavage susceptibility of nick sites by altering both the thermodynamics and kinetics of cleavage to protect human cationic trypsin from premature degradation.
Assuntos
Precursores Enzimáticos/metabolismo , Peptídeos/metabolismo , Proteólise , Tripsina/metabolismo , Tripsinogênio/metabolismo , Aminoácidos/metabolismo , Cálcio/metabolismo , Cátions , Quimotripsina/farmacologia , Estabilidade Enzimática/efeitos dos fármacos , Humanos , Ligação de Hidrogênio/efeitos dos fármacos , Hidrólise/efeitos dos fármacos , Cinética , Modelos Moleculares , Mutação/genética , Elastase Pancreática/metabolismo , Inibidores de Proteases/farmacologia , Proteólise/efeitos dos fármacos , Termodinâmica , Tripsina/químicaRESUMO
Mesotrypsin is an isoform of trypsin that is uniquely resistant to polypeptide trypsin inhibitors and can cleave some inhibitors rapidly. Previous studies have shown that the amyloid precursor protein Kunitz protease inhibitor domain (APPI) is a specific substrate of mesotrypsin and that stabilization of the APPI cleavage site in a canonical conformation contributes to recognition by mesotrypsin. We hypothesized that other proteins possessing potential cleavage sites stabilized in a similar conformation might also be mesotrypsin substrates. Here we evaluated a series of candidate substrates, including human Kunitz protease inhibitor domains from amyloid precursor-like protein 2 (APLP2), bikunin, hepatocyte growth factor activator inhibitor type 2 (HAI2), tissue factor pathway inhibitor-1 (TFPI1), and tissue factor pathway inhibitor-2 (TFPI2), as well as E-selectin, an unrelated protein possessing a potential cleavage site displaying canonical conformation. We find that Kunitz domains within APLP2, bikunin, and HAI2 are cleaved by mesotrypsin with kinetic profiles of specific substrates. TFPI1 and TFPI2 Kunitz domains are cleaved less efficiently by mesotrypsin, and E-selectin is not cleaved at the anticipated site. Cocrystal structures of mesotrypsin with HAI2 and bikunin Kunitz domains reveal the mode of mesotrypsin interaction with its canonical substrates. Our data suggest that major determinants of mesotrypsin substrate specificity include sequence preferences at the P1 and P'2 positions along with conformational stabilization of the cleavage site in the canonical conformation. Mesotrypsin up-regulation has been implicated previously in cancer progression, and proteolytic clearance of Kunitz protease inhibitors offers potential mechanisms by which mesotrypsin may mediate pathological effects in cancer.
Assuntos
Inibidores de Proteases/química , Conformação Proteica , Estrutura Terciária de Proteína , Tripsina/química , alfa-Globulinas/química , alfa-Globulinas/genética , alfa-Globulinas/metabolismo , Sequência de Aminoácidos , Precursor de Proteína beta-Amiloide/química , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Aprotinina/química , Aprotinina/genética , Aprotinina/metabolismo , Sítios de Ligação/genética , Cristalografia por Raios X , Selectina E/química , Selectina E/genética , Selectina E/metabolismo , Glicoproteínas/química , Glicoproteínas/genética , Glicoproteínas/metabolismo , Humanos , Cinética , Lipoproteínas/química , Lipoproteínas/genética , Lipoproteínas/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Inibidores de Proteases/metabolismo , Ligação Proteica , Especificidade por Substrato , Tripsina/genética , Tripsina/metabolismoRESUMO
Human chymotrypsin C (CTRC) is a pancreatic serine protease that regulates activation and degradation of trypsinogens and procarboxypeptidases by targeting specific cleavage sites within their zymogen precursors. In cleaving these regulatory sites, which are characterized by multiple flanking acidic residues, CTRC shows substrate specificity that is distinct from that of other isoforms of chymotrypsin and elastase. Here, we report the first crystal structure of active CTRC, determined at 1.9-Å resolution, revealing the structural basis for binding specificity. The structure shows human CTRC bound to the small protein protease inhibitor eglin c, which binds in a substrate-like manner filling the S6-S5' subsites of the substrate binding cleft. Significant binding affinity derives from burial of preferred hydrophobic residues at the P1, P4, and P2' positions of CTRC, although acidic P2' residues can also be accommodated by formation of an interfacial salt bridge. Acidic residues may also be specifically accommodated in the P6 position. The most unique structural feature of CTRC is a ring of intense positive electrostatic surface potential surrounding the primarily hydrophobic substrate binding site. Our results indicate that long-range electrostatic attraction toward substrates of concentrated negative charge governs substrate discrimination, which explains CTRC selectivity in regulating active digestive enzyme levels.
Assuntos
Quimotripsina/química , Regulação Enzimológica da Expressão Gênica , Sítios de Ligação , Biofísica/métodos , Cálcio/química , Carboxipeptidases/química , Cristalografia por Raios X/métodos , Ativação Enzimática , Células HEK293 , Humanos , Cinética , Modelos Moleculares , Conformação Molecular , Mutação , Elastase Pancreática/química , Isoformas de Proteínas , Proteínas/química , Eletricidade Estática , Especificidade por Substrato , Propriedades de Superfície , Tripsinogênio/químicaRESUMO
The yeast surface display platform provides a powerful approach for screening protein diversity libraries to identify binders with an enhanced affinity toward a binding partner. Here, we describe an adaptation of the approach to identify binders with enhanced specificity toward one among multiple closely related binding partners. Specifically, we describe methods for engineering selective matrix metalloproteinase (MMP) inhibitors via yeast surface display of a tissue inhibitor of metalloproteinase (TIMP) diversity library coupled with a counter-selective screening strategy. This protocol may also be employed for developing selective protein binders or inhibitors toward other targets.
Assuntos
Inibidores de Metaloproteinases de Matriz , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Inibidores de Metaloproteinases de Matriz/metabolismo , Proteínas , Metaloproteases , Inibidor Tecidual de Metaloproteinase-1/metabolismoRESUMO
Matrix Metalloproteinases (MMPs) are drivers of many diseases including cancer and are established targets for drug development. Tissue inhibitors of metalloproteinases (TIMPs) are human proteins that inhibit MMPs and are being pursued for the development of anti-MMP therapeutics. TIMPs possess many attractive properties of a drug candidate, such as complete MMP inhibition, low toxicity and immunogenicity, high tissue permeability and others. A major challenge with TIMPs, however, is their formulation and delivery, as these proteins are quickly cleared from the bloodstream due to their small size. In this study, we explore a new method for plasma half-life extension for the N-terminal domain of TIMP2 (N-TIMP2) through appending it with a long intrinsically unfolded tail containing a random combination of Pro, Ala, and Thr (PATylation). We design, produce and explore two PATylated N-TIMP2 constructs with a tail length of 100- and 200-amino acids (N-TIMP2-PAT100 and N-TIMP2-PAT200, respectively). We demonstrate that both PATylated N-TIMP2 constructs possess apparent higher molecular weights compared to the wild-type protein and retain high inhibitory activity against MMP-9. Furthermore, when injected into mice, N-TIMP2-PAT200 exhibited a significant increase in plasma half-life compared to the non-PATylated variant, enhancing the therapeutic potential of the protein. Thus, we establish that PATylation could be successfully applied to TIMP-based therapeutics and offers distinct advantages as an approach for half-life extension, such as fully genetic encoding of the gene construct, mono-dispersion, and biodegradability. Furthermore, PATylation could be easily applied to N-TIMP2 variants engineered to possess high affinity and selectivity toward individual MMP family members, thus creating attractive candidates for drug development against MMP-related diseases.