RESUMO
The major components of the cartilage extracellular matrix are type II collagen and aggrecan. Matrix metalloproteinase 13 (MMP-13) has been implicated as the protease responsible for collagen degradation in cartilage during osteoarthritis (OA). In the present study, a triple-helical FRET substrate has been utilized for high throughput screening (HTS) of MMP-13 with the MLSCN compound library (n approximately 65,000). Thirty-four compounds from the HTS produced pharmacological dose-response curves. A secondary screen using RP-HPLC validated 25 compounds as MMP-13 inhibitors. Twelve of these compounds were selected for counter-screening with 6 representative MMP family members. Five compounds were found to be broad-spectrum MMP inhibitors, 3 inhibited MMP-13 and one other MMP, and 4 were selective for MMP-13. One of the selective inhibitors was more active against MMP-13 triple-helical peptidase activity compared with single-stranded peptidase activity. Since the THP FRET substrate has distinct conformational features that may interact with MMP secondary binding sites (exosites), novel non-active site-binding inhibitors may be identified via HTS protocols utilizing such assays.
Assuntos
Inibidores de Metaloproteinases de Matriz , Inibidores de Proteases/química , Sítios de Ligação , Avaliação Pré-Clínica de Medicamentos , Transferência Ressonante de Energia de Fluorescência , Humanos , Metaloproteinase 13 da Matriz/metabolismo , Metaloproteinases da Matriz/metabolismo , Peptídeos/química , Inibidores de Proteases/farmacologia , Bibliotecas de Moléculas Pequenas , Especificidade por SubstratoRESUMO
We examined a panel of 26 melanoma and fibroblast samples (tissues and cultured cells) to evaluate the suitability of two commonly used housekeeping genes, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and 18S ribosomal RNA (rRNA), for quantitative real-time PCR. Both genes showed significant variations within the individual cell line and tissue groups. Although no overall trends were observed in the expression of the 18S rRNA, GAPDH was up-regulated in melanoma tissue and cultured cells compared with the corresponding normal samples. In melanoma and fibroblast cell lines and tissues, absolute quantification appears to be more appropriate than normalizing messenger RNA (mRNA) expression via GAPDH or 18S rRNA housekeeping genes.
Assuntos
Perfilação da Expressão Gênica/normas , Regulação Neoplásica da Expressão Gênica , Gliceraldeído-3-Fosfato Desidrogenases/genética , Melanoma/genética , Reação em Cadeia da Polimerase/métodos , RNA Ribossômico 18S/genética , Linhagem Celular Tumoral , Humanos , RNA Mensageiro/genética , Padrões de Referência , Fatores de TempoRESUMO
Protein-protein interactions are governed by a variety of structural features. The sequence specificities of such interactions are usually easier to establish than the "topological specificities," whereby interactions may be classified based on recognition of distinct three-dimensional structural motifs. Approaches to explore topological specificities have been based primarily on assembly of mini-proteins with well defined secondary, tertiary, and/or quarternary structures. The present chapter focuses on three approaches for constructing topologically well defined mini-proteins: template-assembled synthetic proteins (TASPs), disulfide-stabilized structures, and peptide-amphiphiles (PAs). Specific examples are given for applying each approach to explore topologically-dependent protein-protein interactions. TASPs are utilized to identify a metastatic melanoma receptor that binds to the alpha1(IV)1263-1277 region of basement membrane (type IV) collagen. A disulfide-stabilized structure incorporating a sarafotoxin (SRT) 6b model was examined as a matrix metalloproteinase (MMP)-3 inhibitor. PAs were developed as (a) fluorogenic triple-helical or polyPro II substrates for MMPs and aggrecanase members of the a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family and (b) glycosylated and nonglycosylated ligands for metastatic melanoma cells. Topologically constrained mini-proteins have proved to be quite versatile, helping to define critical primary, secondary, and tertiary structural elements that modulate enzyme and receptor functions.
Assuntos
Proteínas/síntese química , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Cromatografia de Afinidade/métodos , Cromatografia Líquida de Alta Pressão/métodos , Dissulfetos/química , Estabilidade de Medicamentos , Humanos , Ligantes , Inibidores de Metaloproteinases de Matriz , Modelos Moleculares , Biologia Molecular/métodos , Dados de Sequência Molecular , Domínios e Motivos de Interação entre Proteínas , Proteínas/química , Proteínas/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Inibidores Teciduais de Metaloproteinases/síntese química , Inibidores Teciduais de Metaloproteinases/química , Inibidores Teciduais de Metaloproteinases/farmacologia , Venenos de Víboras/síntese química , Venenos de Víboras/química , Venenos de Víboras/farmacologiaRESUMO
The profiling of protein function is one of the most challenging scientific tasks in the postgenomic age. Traditional protein expression methodologies have focused only on the quantification of proteins under varying conditions or pathologies. Determining the functional differences between protein populations allows for a more accurate view of the outcomes in normal vs diseased proteomes. Because the presence or absence of a protein's function can affect its complex surroundings (consisting of multiple other proteins and substrates), the study of proteome functionality yields information on protein-protein interactions, amplification cascades, signaling pathways, and posttranslational modifications. Of significant interest are proteinases, as proteolysis is responsible for tight regulation of various cellular and tissue processes. Proteinase activities, or lack there of, alter the proteome makeup by regulating other proteins or by generating cleavage products. This chapter describes current proteolytic profiling technologies using activity or target-based formats. In particular, the analysis of collagenolytic matrix metalloproteinase activity using fluorogenic triple-helical substrates is discussed.
Assuntos
Proteínas da Matriz Extracelular/química , Proteínas da Matriz Extracelular/metabolismo , Sequência de Bases , Linhagem Celular Tumoral , Colágeno/química , Colágeno/metabolismo , Primers do DNA/genética , Eletroforese em Gel de Poliacrilamida/métodos , Corantes Fluorescentes , Humanos , Receptores de Hialuronatos/metabolismo , Indicadores e Reagentes , Integrina alfa2beta1/metabolismo , Metaloproteinases da Matriz/química , Metaloproteinases da Matriz/genética , Metaloproteinases da Matriz/metabolismo , Melanoma/enzimologia , Melanoma/genética , Melanoma/secundário , Biologia Molecular/métodos , Neoplasias/genética , Neoplasias/metabolismo , Peptídeo Hidrolases/química , Peptídeo Hidrolases/metabolismo , Análise Serial de Proteínas/métodos , Proteômica/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de SinaisRESUMO
Two matrix metalloproteinases, MMP-2 and MMP-9, contain each three fibronectin type II-like modules, which form their collagen binding domains (CBDs). The contributions of CBD substrate interactions to the catalytic activities of these gelatinases have attracted special interest. Recombinant (r) CBDs retain collagen binding properties and deletions of CBDs in these MMPs reduce activities on collagen and elastin. We have characterized further the requirement of the CBD for MMP-2 cleavage of gelatin. The analyses used intact rMMP-2 and rCBD to eliminate any confounding effects that might result from structural perturbations in rMMP-2 induced by deletion of the approximately 20 kDa internal CBD. In protein-protein binding assays, 2% DMSO disrupted gelatin interactions of both rCBD and rMMP-2. At this concentration, DMSO also reduced the gelatinolytic activity by approximately 70%, pointing to a central role of CBD-substrate interactions during MMP-2 cleavage of gelatin. Subsequently, soluble rCBD was determined to competitively inhibit gelatin binding of unmodified rMMP-2 to gelatin by 73% and to reduce the MMP-2 degradation of gelatin by 70-80%. The residual gelatin cleavage that was not inhibited even by molar excess rCBD could be accounted for by degradation of short substrate molecules. Indeed, rCBD inhibited rMMP-2 cleavage of an 11 amino acid collagen-like peptide substrate (NFF-1) by less than 10%. These observations were confirmed with enzyme extracts from experimental tumors in mice. In the presence of rCBD, approximately 65% of the MMP-derived gelatinolytic activity was eliminated. Together, these results demonstrate that the CBD is absolutely required for MMP-2 cleavage of full-length collagen alpha-chains, but not for short protein fragments such as those generated by hydrolysis of gelatin.
Assuntos
Colágeno Tipo I/metabolismo , Gelatina/metabolismo , Metaloproteinase 2 da Matriz/química , Metaloproteinase 2 da Matriz/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Animais , Neoplasias da Mama/enzimologia , Linhagem Celular Tumoral , Humanos , Hidrólise , Metaloproteinase 2 da Matriz/genética , Camundongos , Mutação , Transplante de Neoplasias , Ligação Proteica , Estrutura Terciária de Proteína , Ratos , Proteínas Recombinantes , Especificidade por SubstratoRESUMO
Proteases play fundamentally important roles in normal physiology and disease pathology. Methods for detection of active proteolysis may greatly aid in the diagnosis of disease progression, and suggest modes of therapeutic intervention. Most assays for proteolytic potential are limited by a lack of specificity and/or quantification. We have developed a solid-phase activity assay for members of the matrix metalloproteinase (MMP) family that is specific and can be used to quantify active enzyme concentration. The assay has two principal components: a capture antibody that immobilizes the MMP without perturbing the enzyme active site, and a fluorescence resonance energy transfer substrate for monitoring proteolysis at low enzyme concentrations. The assay was standardized for MMP-1, MMP-3, MMP-13, and MMP-14. The efficiency of the assay was found to be critically dependent upon the quality of the antibodies, the use of substrates exhibiting high specific activities for the enzymes, and enzyme samples that are fresh. The assay was applied to studies of constitutive and induced MMP activity in human melanoma cells. Analysis of several melanoma cell lines, and comparison with prior studies, correlated higher constitutive MMP-13 activity with higher levels of the cell surface receptor CD44. Ligands to two different melanoma cell surface receptors (the alpha2beta1 integrin or CD44) were found to induce different proteolytic profiles, suggesting that the extracellular matrix can modulate melanoma invasion. Overall, the solid-phase MMP activity assay was found to be valuable for analysis of protease activity in cellular environments. The solid-phase assay is suitably flexible to allow studies of virtually any proteolytic enzyme for which appropriate substrates and antibodies are available.
Assuntos
Metaloproteinases da Matriz/análise , Sequência de Aminoácidos , Ensaio de Imunoadsorção Enzimática , Humanos , Metaloproteinases da Matriz/metabolismo , Melanoma/metabolismo , Dados de Sequência Molecular , Especificidade por SubstratoRESUMO
Collagen serves as a structural scaffold and a barrier between tissues, and thus collagen catabolism (collagenolysis) is required to be a tightly regulated process in normal physiology. In turn, the destruction or damage of collagen during pathological states plays a role in tumor growth and invasion, cartilage degradation, or atherosclerotic plaque formation and rupture. Several members of the matrix metalloproteinase (MMP) family catalyze the hydrolysis of collagen triple helical structure. This study has utilized triple helical peptide (THP) substrates and inhibitors to dissect MMP-1 collagenolytic behavior. Analysis of MMP-1/THP interactions by hydrogen/deuterium exchange mass spectrometry followed by evaluation of wild type and mutant MMP-1 kinetics led to the identification of three noncatalytic regions in MMP-1 (residues 285-295, 302-316, and 437-457) and two specific residues (Ile-290 and Arg-291) that participate in collagenolysis. Ile-290 and Arg-291 contribute to recognition of triple helical structure and facilitate both the binding and catalysis of the triple helix. Evidence from this study and prior studies indicates that the MMP-1 catalytic and hemopexin-like domains collaborate in collagen catabolism by properly aligning the triple helix and coupling conformational states to facilitate hydrolysis. This study is the first to document the roles of specific residues within the MMP-1 hemopexin-like domain in substrate binding and turnover. Noncatalytic sites, such as those identified here, can ultimately be utilized to create THP inhibitors that target MMPs implicated in disease progression while sparing proteases with host-beneficial functions.
Assuntos
Colágeno/química , Metaloproteinase 1 da Matriz/química , Animais , Colágeno/metabolismo , Humanos , Metaloproteinase 1 da Matriz/metabolismo , Estrutura Secundária de Proteína/fisiologia , Estrutura Terciária de Proteína/fisiologia , Especificidade por Substrato/fisiologiaRESUMO
Hydroxylysine (Hyl) is a posttranslationally modified amino acid found mainly in collagens, the most abundant protein in mammals. Lysyl hydroxylase (LH) catalyzes the hydroxylation of the C-5 position of a Lys residue, resulting in the production of Hyl. Mechanistically, LH incorporates one oxygen atom into both Lys and 2-oxoglutarate; the latter is decarboxylated to form succinate and CO(2). To develop a convenient, RP-HPLC based LH assay, we used Fmoc solid-phase methodology to synthesize three different peptides designed as LH substrates and one peptide corresponding to an LH product. Peptides were characterized by RP-HPLC, MALDI-TOF mass spectrometry and CD spectroscopy. Separation of peptides was examined under a variety of RP-HPLC conditions. The best results were achieved using peptide derivatization (1-anthroylnitrile for organic phase and dansyl chloride for aqueous phase) prior to RP-HPLC analysis. The products (di- and tetra-substituted Lys- and Hyl-containing peptides) were well resolved by RP-HPLC. The resolution of each peak allows for quantification of peak areas, which in turn, when examined as a function of time, can be utilized for studying the kinetics of LH catalyzed reactions. Most significantly, the RP-HPLC assay directly monitors the Hyl containing product. Prior LH assay methods are multi-step, require radio-labeled substrates, and/or measure depletion of 2-oxoglutarate or formation of CO(2). Since the LH reaction with 2-oxoglutarate is uncoupled from Lys hydroxylation, the most accurate assay of LH activity should monitor the formation of Hyl.
Assuntos
Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/análise , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , Hidroxilação , Cinética , Espectrometria de Massas , Peptídeos/síntese química , Peptídeos/química , Peptídeos/metabolismo , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/química , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Especificidade por SubstratoRESUMO
Unregulated activities of the matrix metalloproteinase (MMP) family have been implicated in primary and metastatic tumor growth, angiogenesis, and pathological degradation of extracellular matrix components, such as collagen and laminin. However, clinical trials with small molecule MMP inhibitors have been largely unsuccessful, with a lack of selectivity considered particularly problematic. Enhanced selectivity could be achieved by taking advantage of differences in substrate secondary binding sites (exosites) within the MMP family. In this study, triple-helical substrates and triple-helical transition state analog inhibitors have been utilized to dissect the roles of potential exosites in MMP-9 collagenolytic behavior. Substrate and inhibitor sequences were based on either the alpha1(V)436-450 collagen region, which is hydrolyzed at the Gly (downward arrow) Val bond selectively by MMP-2 and MMP-9, or the Gly (downward arrow) Leu cleavage site within the consensus interstitial collagen sequence alpha1(I-III)769-783, which is hydrolyzed by MMP-1, MMP-2, MMP-8, MMP-9, MMP-13, and MT1-MMP. Exosites within the MMP-9 fibronectin II inserts were found to be critical for interactions with type V collagen model substrates and inhibitors and to participate in interactions with an interstitial (types I-III) collagen model inhibitor. A triple-helical peptide incorporating a fibronectin II insert-binding sequence was constructed and found to selectively inhibit MMP-9 type V collagen-based activities compared with interstitial collagen-based activities. This represents the first example of differential inhibition of collagenolytic activities and was achieved via an exosite-binding triple-helical peptide.
Assuntos
Metaloproteinase 9 da Matriz/metabolismo , Sítios de Ligação , Inibidores Enzimáticos/farmacologia , Deleção de Genes , Cinética , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/genética , Inibidores de Metaloproteinases de Matriz , Mutação/genética , Especificidade por Substrato , TemperaturaRESUMO
Matrix metalloproteinase (MMP)-12 (or metalloelastase) efficiently hydrolyzed the gelatinase-selective alpha1(V)436-447 fluorescent triple helical peptide (THP) when the substrate was submicromolar. The sequence of this THP was derived from collagen V, a component of collagen I fibrils. The hemopexin domains of MMP-12 and -9 each increased k(cat)/K(m) toward this substrate by decreasing K(m), just as the hemopexin domain of MMP-1 enhances its triple helical peptidase activity. Non-fluorescent alpha1(V) THP subtly perturbed amide NMR chemical shifts of MMP-12 not only in the active site cleft but also at remote sites of the beta-sheet and adjoining loops. The alpha1(V) THP protected MMP-12 from the NMR line broadening effects of Gd .EDTA in the active site cleft and more dramatically in the V-B loop next to the primed subsites. Mutagenesis of the exosite in the V-B loop at Thr-205 and His-206 that vary among MMP sequences established that this site supports the high specific activity toward alpha1(V) fluorescent THP without affecting general MMP activity. Surprisingly the alpha1(V) THP also protected novel surfaces in the S-shaped metal-binding loop and beta-strands III and V that together form a pocket on the remote side of the zinc binding site. The patterns of protection suggest bending of the triple helical peptide partly around the catalytic domain to reach novel exosites. Partial unwinding or underwinding of the triple helix could accompany this to facilitate its hydrolysis.
Assuntos
Colágeno Tipo V/química , Metaloproteinase 12 da Matriz/química , Peptídeos/química , Sítios de Ligação , Domínio Catalítico , Histidina/química , Humanos , Cinética , Espectroscopia de Ressonância Magnética , Metaloproteinase 12 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/química , Metais , Mutagênese Sítio-Dirigida , Estrutura Secundária de Proteína , Treonina/químicaRESUMO
The major components of the cartilage extracellular matrix are type II collagen and aggrecan. Type II collagen provides cartilage with its tensile strength, whereas the water-binding capacity of aggrecan provides compressibility and elasticity. Aggrecan breakdown leads to an increase in proteolytic susceptibility of articular collagen; hence, aggrecan may also have a protective effect on type II collagen. Given their role in aggrecan degradation and differing substrate specificity profiles, the pursuit of inhibitors for both aggrecanase 1 (a disintegrin and metalloproteinase with thrombospondin motifs-4 [ADAMTS-4]) and aggrecanase 2 (ADAMTS-5) is desirable. We previously described collagen model fluorescence resonance energy transfer (FRET) substrates for aggrecan-degrading members of the ADAMTS family. These FRET substrate assays are also fully compatible with multiwell formats. In the current study, a collagen model FRET substrate was examined for inhibitor screening of ADAMTS-4. ADAMTS-4 was screened against a small compound library (n=960) with known pharmacological activity. Five compounds that inhibited ADAMTS-4>60% at a concentration of 1muM were identified. A secondary screen using reversed-phase high-performance liquid chromatography (RP-HPLC) was developed and performed for verification of the five potential inhibitors. Ultimately, piceatannol was confirmed as a novel inhibitor of ADAMTS-4, with an IC(50) value of 1muM. Because the collagen model FRET substrates have distinct conformational features that may interact with protease secondary substrate sites (exosites), nonactive site-binding inhibitors can be identified via this approach. Selective inhibitors for ADAMTS-4 would allow a more definitive evaluation of this protease in osteoarthritis and also represent a potential next generation in metalloproteinase therapeutics.
Assuntos
Proteínas ADAM/antagonistas & inibidores , Colágeno/química , Pró-Colágeno N-Endopeptidase/antagonistas & inibidores , Proteína ADAMTS4 , Transferência Ressonante de Energia de Fluorescência , Inibidores de Proteases/farmacologiaRESUMO
Identification of the biochemical pathways involved in the transformation from primary to metastatic melanoma is an area under intense investigation. A 2DE proteomics approach has been applied herein to the matched patient primary and metastatic melanoma cell lines WM-115 and WM-266-4, respectively, to better understand the processes that underlie tumor progression. Image analysis between samples aligned 470 common gel spots. Quantitative gel analysis indicated 115 gel spots of greater intensity in the metastatic line compared with the primary one, leading to the identification of 131 proteins via database searching of nano-LC-ESI-Q-TOF-MS/MS data. This more than tripled the number of proteins previously shown to be of higher abundance during melanoma progression. Also observed were 22 gel spots to be of lesser intensity in the metastatic line with respect to the primary one. Of these gel spots 15 proteins could be identified. Numerous proteins from both groups had not been reported previously to participate in melanoma progression. Further analysis of one protein, cyclophilin A, confirmed that this protein is expressed at higher levels in metastatic melanoma compared with primary melanoma and normal fibroblasts. Overall, this study expands our knowledge of protein modulation during melanoma stages, and suggests new targets for inhibitor development.
Assuntos
Melanoma/metabolismo , Metástase Neoplásica , Proteínas de Neoplasias/metabolismo , Proteoma , Linhagem Celular Tumoral , Cromatografia Líquida , Eletroforese em Gel Bidimensional , Feminino , Humanos , Melanoma/patologia , Pessoa de Meia-Idade , Mapeamento de Peptídeos , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em TandemRESUMO
The sarafotoxins and endothelins are approximately 25-residue peptides that spontaneously fold into a defined tertiary structure with specific pairing of four cysteines into two disulfide bonds. Their structures show an interesting topological similarity to the core of the metalloproteinase interaction sites of the tissue inhibitors of metalloproteinases. Previous work indicates that sarafotoxins and endothelins can be engineered to eliminate or greatly reduce their vasopressive action and that their structural framework can withstand multiple sequence changes. When sarafotoxin 6b, which possesses modest matrix metalloproteinase inhibitory activity, was C-terminally truncated to remove its toxic vasopressive activity, the metalloproteinase inhibitory activity was essentially abolished. However, further changes, based on the sequences of peptides selected from libraries of sarafotoxin variants or suggested by analogy with tissue inhibitors of metalloproteinases, progressively enhanced the matrix metalloproteinase inhibitory activity. Peptide variants with multiple substitutions folded correctly and formed native disulfide bonds. Improvements in matrix metalloproteinase affinity have generated a peptide with micromolar K(i) values for matrix metalloproteinase-1 and -9 that are selective inhibitors of different metalloproteinases. Characterization of its solution structure indicates a close similarity to sarafotoxin but with a more extended C-terminal helix. The effects of N-acetylation and other changes, as well as docking studies, support the hypothesis that the engineered sarafotoxins bind to matrix metalloproteinases in a manner analogous to the tissue inhibitors of metalloproteinases.
Assuntos
Inibidores Teciduais de Metaloproteinases/farmacologia , Venenos de Víboras/farmacologia , Sequência de Aminoácidos , Cristalografia , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Engenharia de Proteínas , Relação Estrutura-Atividade , Inibidores Teciduais de Metaloproteinases/química , Venenos de Víboras/químicaRESUMO
Protease-substrate interactions are governed by a variety of structural features. Although the substrate sequence specificities of numerous proteases have been established, "topological specificities," whereby proteases may be classified based on recognition of distinct three-dimensional structural motifs, have not. The aggrecanase members of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family cleave a variety of proteins but do not seem to possess distinct sequence specificities. In the present study, the topological substrate specificity of ADAMTS-4 (aggrecanase-1) was examined using triple-helical or single-stranded poly(Pro) II helical peptides. Substrate topology modulated the affinity and sequence specificity of ADAMTS-4 with K(m) values indicating a preference for triple-helical structure. In turn, non-catalytic ADAMTS-4 domains were critical for hydrolysis of triple-helical and poly(Pro) II helical substrates. Comparison of ADAMTS-4 with MMP-1 (collagenase 1), MMP-13 (collagenase 3), trypsin, and thermolysin using triple-helical peptide (THP) and single-stranded peptide (SSP) substrates demonstrated that all five proteases possessed efficient "triple-helical peptidase" activity and fell into one of two categories: (k(cat)/K(m))(SSP) > (k(cat)/K(m))(THP) (thermolysin, trypsin, and MMP-13) or (k(cat)/K(m))(THP) > or = (k(cat)/K(m))(SSP) and (K(m))(SSP) > (K(m))(THP) (MMP-1 and ADAMTS-4). Overall these results suggest that topological specificity may be a guiding principle for protease behavior and can be utilized to design specific substrates and inhibitors. The triple-helical and single-stranded poly(Pro) II helical peptides represent the first synthetic substrates successfully designed for aggrecanases.
Assuntos
Proteínas ADAM/química , Pró-Colágeno N-Endopeptidase/química , Proteínas ADAM/metabolismo , Proteína ADAMTS4 , Sequência de Aminoácidos , Dicroísmo Circular , Escherichia coli/metabolismo , Humanos , Cinética , Metaloproteinase 1 da Matriz/química , Metaloproteinase 13 da Matriz/química , Dados de Sequência Molecular , Peptídeos/química , Pró-Colágeno N-Endopeptidase/metabolismo , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína , Especificidade por SubstratoRESUMO
The turnover of the collagen triple-helical structure (collagenolysis) is a tightly regulated process in normal physiology and has been ascribed to a small number of proteases. Several members of the matrix metalloproteinase (MMPs) family possess collagenolytic activity, and the mechanisms by which these enzymes process triple helices are beginning to be unraveled. The present study has utilized two triple-helical sequences to compare the cleavage-site specificities of 10 MMPs. One substrate featured a continuous Gly-Xxx-Yyy sequence (Pro-Leu-Gly approximately Met-Arg-Gly), while the other incorporated an interruption in the Gly-Xxx-Yyy repeat (Pro-Val-Asn approximately Phe-Arg-Gly). Both sequences were selectively cleaved by MMP-13 while in linear form, but neither proved to be selective within a triple helix. This suggests that the conformational presentation of substrate sequences to a MMP active site is critical for enzyme specificity, in that activities differ when sequences are presented from an unwound triple helix versus an independent single strand. Differences in specificity between secreted and membrane-type (MT) MMPs were also observed for both sequences, where MMP-2 and MT-MMPs showed an ability to hydrolyze a triple helix at an additional site (Gly-Gln bond). Interruption of the triple helix had different effects on secreted MMPs and MT-MMPs, because MT-MMPs could not hydrolyze the Asn-Phe bond but instead cleaved the triple helix closer to the C terminus at a Gly-Gln bond. It is possible that MT-MMPs have a requirement for Gly in the P1 subsite to be able to efficiently process a triple-helical molecule. Analysis of individual kinetic parameters and activation energies indicated different substrate preferences within secreted MMPs, because MMP-13 preferred the interrupted sequence, while MMP-8 showed little discrimination between non-interrupted and interrupted triple helices. On the basis of the present and prior studies, we can assign unique triple-helical peptidase behaviors to the collagenolytic MMPs. Such differences may be significant for understanding MMP mechanisms of action and aid in the development of selective MMP inhibitors.
Assuntos
Colágeno/química , Metaloproteinases da Matriz Associadas à Membrana/química , Sequência de Aminoácidos , Animais , Colágeno/genética , Colágeno/metabolismo , Humanos , Metaloproteinases da Matriz Associadas à Membrana/genética , Metaloproteinases da Matriz Associadas à Membrana/metabolismo , Estrutura Secundária de Proteína , Especificidade por Substrato/genéticaRESUMO
The hydrolysis of collagen (collagenolysis) is one of the committed steps in extracellular matrix turnover. Within the matrix metalloproteinase (MMP) family distinct preferences for collagen types are seen. The substrate determinants that may guide these specificities are unknown. In this study, we have utilized 12 triple-helical substrates in combination with 10 MMPs to better define the contributions of substrate sequence and thermal stability toward triple helicase activity and collagen specificity. In general, MMP-13 was found to be distinct from MMP-8 and MT1-MMP(Delta279-523), in that enhanced substrate thermal stability has only a modest effect on activity, regardless of sequence. This result correlates to the unique collagen specificity of MMP-13 compared with MMP-8 and MT1-MMP, in that MMP-13 hydrolyzes type II collagen efficiently, whereas MMP-8 and MT1-MMP are similar in their preference for type I collagen. In turn, MMP-1 was the least efficient of the collagenolytic MMPs at processing increasingly thermal stable triple helices and thus favors type III collagen, which has a relatively flexible cleavage site. Gelatinases (MMP-2 and MMP-9(Delta444-707)) appear incapable of processing more stable helices and are thus mechanistically distinct from collagenolytic MMPs. The collagen specificity of MMPs appears to be based on a combination of substrate sequence and thermal stability. Analysis of the hydrolysis of triple-helical peptides by an MMP mutant indicated that Tyr(210) functions in triple helix binding and hydrolysis, but not in processing triple helices of increasing thermal stabilities. Further exploration of MMP active sites and exosites, in combination with substrate conformation, may prove valuable for additional dissection of collagenolysis and yield information useful in the design of more selective MMP inhibitors.
Assuntos
Colágeno/metabolismo , Metaloproteinases da Matriz/metabolismo , Peptídeo Hidrolases/metabolismo , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , Estabilidade Enzimática , Hidrólise , Cinética , Dados de Sequência Molecular , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Especificidade por SubstratoRESUMO
Matrix metalloproteinase (MMP) family members are involved in the physiological remodeling of tissues and embryonic development as well as pathological destruction of extracellular matrix components. To study the mechanisms of MMP action on collagenous substrates, non-fluorogenic and fluorogenic triple-helical peptide models of MMP-1 cleavage sites in interstitial collagens have been constructed. Triple-helical peptides were assembled by either (a) covalent branching or (b) self-association driven by hydrophobic interactions. Fluorogenic triple-helical peptide (fTHP) substrates contained the fluorophore/quencher pair of (7-methoxycoumarin-4-yl)acetyl (Mca) and N-2,4-dinitrophenyl (Dnp) in the P5 and P5' positions, respectively. Investigation of MMP family hydrolysis of THPs showed kcat/Km values in the order of MMP-13 > MMP-1 approximately MMP-1(delta243-450) approximately MMP-2 >> MMP-3. Studies on the effect of temperature on fTHP and an analogous fluorogenic single-stranded peptide (fSSP) hydrolysis by MMP-1 showed that the activation energies between these two substrates differed by 3.4-fold, similar to the difference in activation energies for MMP-1 hydrolysis of type I collagen and gelatin. The general proteases trypsin and thermolysin were also studied for triple-helical peptidase activity. Both of these enzymes exhibited similar activation energies to MMP-1 for hydrolysis of fTHP versus fSSP. These results suggest that 'triple-helical peptidase' activity can be distinguished from 'collagenolytic' activity, and that mechanistically distinct enzymes convergently evolved to develop collagenolytic activity.
Assuntos
Metaloproteinases da Matriz/metabolismo , Fragmentos de Peptídeos/química , Sítios de Ligação , Colágeno/química , Colágeno/metabolismo , Corantes Fluorescentes , Humanos , Cinética , Metaloproteinases da Matriz/química , Fragmentos de Peptídeos/metabolismo , Conformação ProteicaRESUMO
The matrix metalloproteinase (MMP)/matrixin family has been implicated in both normal tissue remodeling and a variety of diseases associated with abnormal turnover of extracellular matrix components. The mechanism by which MMPs catabolize collagen (collagenolysis) is still largely unknown. Substrate flexibility, MMP active sites, and MMP exosites all contribute to collagen degradation. It has recently been demonstrated that the ability to cleave a triple helix (triple-helical peptidase activity) can be distinguished from the ability to cleave collagen (collagenolytic activity). This suggests that the ability to cleave a triple helix is not the limiting factor for collagenolytic activity-the ability to properly orient and potentially destabilize collagen is. For the MMP family, the catalytic domain can unwind and cleave a triple-helical structure, while the C-terminal hemopexin-like domain appears to be responsible for properly orienting collagen and destabilizing it to some degree. It is also possible that exosites within the catalytic and/or C-terminal hemopexin-like domain may exclude some MMPs from cleaving collagen. Overall, it appears that many proteases of distinct mechanisms possess triple-helical peptidase activity, and that convergent evolution led to a few proteases possessing collagenolytic activity. Proper orientation and distortion of the triple helix may be the key factor for collagenolysis.
Assuntos
Colágeno/metabolismo , Metaloproteinases da Matriz/metabolismo , Sequência de Carboidratos , Colagenases/metabolismo , Endopeptidases/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos/metabolismo , Ligação Proteica/fisiologia , Estrutura Terciária de ProteínaRESUMO
Matrix metalloproteinases (MMPs) are involved in physiological remodeling as well as pathological destruction of tissues. The turnover of the collagen triple-helical structure has been ascribed to several members of the MMP family, but the determinants for collagenolytic specificity have not been identified. The present study has compared the triple-helical peptidase activities of MMP-1 and MMP-14 (membrane-type 1 MMP; MT1-MMP). The ability of each enzyme to efficiently hydrolyze the triple helix was quantified using chemically synthesized fluorogenic triple-helical substrates that, via addition of N-terminal alkyl chains, differ in their thermal stabilities. One series of substrates was modeled after a collagenolytic MMP consensus cleavage site from types I-III collagen, while the other series had a single substitution in the P(1)' subsite of the consensus sequence. The substitution of Cys(4-methoxybenzyl) for Leu in the P(1)' subsite was greatly favored by MMP-14 but disfavored by MMP-1. An increase in substrate triple-helical thermal stability led to the decreased ability of the enzyme to cleave such substrates, but with a much more pronounced effect for MMP-1. Increased thermal stability was detrimental to enzyme turnover of substrate (k(cat)), but not binding (K(M)). Activation energies were considerably lower for MMP-14 hydrolysis of triple-helical substrates compared with MMP-1. Overall, MMP-1 was found to be less efficient at processing triple-helical structures than MMP-14. These results demonstrate that collagenolytic MMPs have subtle differences in their abilities to hydrolyze triple helices and may explain the relative collagen specificity of MMP-1.
Assuntos
Exopeptidases/química , Metaloendopeptidases/química , Sequência de Aminoácidos , Substituição de Aminoácidos , Colágeno Tipo I/metabolismo , Colágeno Tipo II/metabolismo , Colágeno Tipo III/metabolismo , Ativação Enzimática , Estabilidade Enzimática , Exopeptidases/metabolismo , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/metabolismo , Humanos , Hidrólise , Metaloproteinases da Matriz Associadas à Membrana , Metaloendopeptidases/metabolismo , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Especificidade por Substrato , TemperaturaRESUMO
Collagen is a multifunctional protein, serving as a structural scaffold and a modulator of cellular responses. Prior work has identified distinct regions from several collagen types that promote cell adhesion, spreading, migration, and signal transduction. One of these regions, alpha1(IV)1263-1277 from type IV collagen, mediates these responses via melanoma cell CD44-chondrotin sulfate proteoglycan receptors. In the study presented here, we have used a triple-helical model of alpha1(IV)1263-1277 to evaluate (a) conformational stability and (b) cellular responses based on single-site incorporation of trans-4-fluoro-L-proline (trans-Flp) or cis-4-fluoro-L-proline (cis-Flp) for trans-4-hydroxy-L-proline (trans-Hyp). The structural effects of cis-Flp and trans-Flp substitution were studied by circular dichroism and NMR spectroscopies. The peptide containing a single trans-Flp instead of trans-Hyp was slightly more thermally stable than the parent peptide (T(m) = 37 vs 34 degrees C), while the peptide containing cis-Flp was considerably less stable than the parent peptide (T(m) = 30 degrees C). Melanoma cell adhesion and spreading were examined under conditions where the trans-Hyp-, trans-Flp-, and cis-Flp-containing ligands were approximately 15, <10, and approximately 65% denatured, respectively. Adhesion to each of the three ligands was remarkably sensitive to the respective ligand conformation, with EC(50) values of approximately 2.5, approximately 0.35, and >5.0 microM for the trans-Hyp-, trans-Flp-, and cis-Flp-containing ligands, respectively. Melanoma cell spreading was quantitated over a ligand concentration range of 0.01-50 microM and, in a fashion similar to adhesion, was more extensive on the trans-Flp ligand than on the trans-Hyp ligand. Very low levels of spreading were observed with the cis-Flp-containing ligand at all concentrations tested. Melanoma cell adhesion to and spreading on the three ligands suggested the dramatic biological consequence of even subtle changes in relative triple-helical content. Such subtle changes may model those occurring in the basement membrane during the tumor cell invasion process, and thus provide mechanistic insight into this stage of metastasis.