Highlights d Complex of cancer-critical MT1-MMP domain with collagen mimic is captured by PRE NMR... more Highlights d Complex of cancer-critical MT1-MMP domain with collagen mimic is captured by PRE NMR d The collagen triple helix is translated 25 Å from MMP-1 complexes d Part of the interface distinguishes MT-MMPs and is attractive for therapeutic development d The domains of MT1-MMP are separated enough for reorientation on collagen
Matrix metalloproteinase 13 (MMP-13) has been shown to be the main collagenase responsible for de... more Matrix metalloproteinase 13 (MMP-13) has been shown to be the main collagenase responsible for degradation of articular cartilage during osteoarthritis and therefore represents a target for drug development. As a result of high-throughput screening and structure−activity relationship studies, we identified a novel, highly selective class of MMP-13 inhibitors (compounds 1 (Q), 2 (Q1), and 3 (Q2)). Mechanistic characterization revealed a noncompetitive nature of these inhibitors with binding constants in the low micromolar range. Crystallographic analyses revealed two binding modes for compound 2 in the MMP-13 S 1 ′ subsite and in an S 1 /S 2 * subsite. Type II collagen-and cartilage-protective effects exhibited by compounds 1, 2, and 3 suggested that these compounds might be efficacious in future in vivo studies. Finally, these compounds were also highly selective when tested against a panel of 30 proteases, which, in combination with a good CYP inhibition profile, suggested low off-target toxicity and drug− drug interactions in humans.
Collagen serves as a structural scaffold and a barrier between tissues, and thus collagen catabol... more 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.
Matrix metalloproteinases (MMPs) are involved in physiological remodeling as well as pathological... more 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.
ADAM10 and ADAM17 have been shown to contribute to the acquired drug resistance of HER2-positive ... more ADAM10 and ADAM17 have been shown to contribute to the acquired drug resistance of HER2-positive breast cancer in response to trastuzumab. The majority of ADAM10 and ADAM17 inhibitor development has been focused on the discovery of compounds that bind the active site zinc, however, in recent years, there has been a shift from active site to secondary substrate binding site (exosite) inhibitor discovery in order to identify non-zinc-binding molecules. In the present work a glycosylated, exosite-binding substrate of ADAM10 and ADAM17 was utilized to screen 370,276 compounds from the MLPCN collection. As a result of this uHTS effort, a selective, time-dependent, non-zinc-binding inhibitor of ADAM10 with Ki = 883 nM was discovered. This compound exhibited low cell toxicity and was able to selectively inhibit shedding of known ADAM10 substrates in several cell-based models. We hypothesize that differential glycosylation of these cognate substrates is the source of selectivity of our nove...
Recently, an unexpected modified residue, γ-hydroxy-D-valine (D-Hyv), was identified within ribos... more Recently, an unexpected modified residue, γ-hydroxy-D-valine (D-Hyv), was identified within ribosomally expressed polypeptide chains of four conopeptides from the venoms of Conus gladiator and Conus mus. In order to assemble Hyv-containing peptides, we have explored several routes for the synthesis of appropriately functionalized Hyv building blocks. D-Hyv was produced from D-Val using a variation of the previously published K 2 PtCl 4 /CuCl 2 oxidative method. Direct synthesis of Boc-or Cbz-D-Hyv lactone proceeded in low yield; additionally, the lactones are too unreative for solid-phase applications. 9-Borabicyclononane or copper-complexed D-Hyv was prepared and treated with tert-butyldimethylsilyl trifluoromethanesulfonate (TBDMSOTf) to produce D-Hyv(O-TBDMS). The most efficient complex disruption was achieved by Chelex 110 resin (Na + form) treatment of copper-complexed D-Hyv(O-TBDMS). Reaction of D-Hyv(O-TBDMS) with Fmoc-OSu produced Fmoc-D-Hyv(O-TBDMS) in 26% yield from D-Val. The Fmoc-D-Hyv(O-TBDMS) diastereomers were separated by preparative RP-HPLC in 13% yield from D-Val. Fmoc-D-Hyv(O-TBDMS) was used for the synthesis of the conopeptide gld-V* from Conus gladiator. The isolated synthetic and natural products had coincidental mass and NMR spectra. The methodology presented herein will greatly facilitate biological studies of Hyv-containing sequences, such as receptor responses to hydroxylated versus non-hydroxylated conopeptides and the relative susceptibility of proteins to modification by oxidative stress.
The turnover of collagen triple-helical structure (collagenolysis) is a tightly regulated process... more The turnover of collagen triple-helical structure (collagenolysis) is a tightly regulated process in normal physiology, and has been ascribed to 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 2 triple-helical sequences to compare the cleavage site specificities of 10 MMPs. One substrate featured a continuous Gly-Xxx-Yyy sequence (Pro-Leu-Gly~Met-Arg-Gly) while the other incorporated an interruption in the Gly-Xxx-Yyy repeat (Pro-Val-Asn~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 an 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 membranetype (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, as MT-MMPs could not hydrolyze the Asn-Phe bond, but instead cleaved the triplehelix nearer the C-terminus at a Gly-Gln bond. It is possible that MT-MMPs have a requirement for Gly in the P 1 subsite in order 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, as MMP-13 preferred the interrupted sequence while MMP-8 showed little discrimination between non-interrupted and interrupted triple-helices. Based on the present and prior studies, we can assign unique triple-helical peptidase behaviors to the collagenolytic MMPs. Such † This work was supported by the National Institutes of Health (AR 40994 to K.B., CA 98799 and EB 000289 to G.B.F.) and the FAU Center of Excellence in Biomedical and Marine Biotechnology.
Solvent dynamics can play a major role in enzyme activity, but obtaining an accurate, quantitativ... more Solvent dynamics can play a major role in enzyme activity, but obtaining an accurate, quantitative picture of solvent activity during catalysis is quite challenging. Here, we combine terahertz spectroscopy and X-ray absorption analyses to measure changes in the coupled water-protein motions during peptide hydrolysis by a zinc-dependent human metalloprotease. These changes were tightly correlated with rearrangements at the active site during the formation of productive enzyme-substrate intermediates and were different from those in an enzyme-inhibitor complex. Molecular dynamics simulations showed a steep gradient of fast-to-slow coupled protein-water motions around the protein, active site and substrate. Our results show that water retardation occurs before formation of the functional Michaelis complex. We propose that the observed gradient of coupled protein-water motions may assist enzyme-substrate interactions through water-polarizing mechanisms that are remotely mediated by the catalytic metal ion and the enzyme active site.
Polyphenolic natural products from green tea and red wine have been identified as metalloproteina... more Polyphenolic natural products from green tea and red wine have been identified as metalloproteinase inhibitors. Members from the flavonoid and stilbene families found to possess metalloproteinase inhibitory activities include ())-epigallocatechin gallate, ())-epicatechin gallate and piceatannol, but their minimally active pharmacophores have not been evaluated. The present study has examined compounds that are structural components of or structurally related to ())-epigallocatechin gallate, ())-epicatechin gallate and piceatannol for inhibition of aggrecanases and four representative matrix metalloproteinases. Piceatannol and pyrogallol were found to inhibit all aggrecanases and matrix metalloproteinases studied, indicating a crucial reliance on multiple hydroxyl groups for ())-epigallocatechin gallate, ())-epicatechin gallate and piceatannol activity. Differences in K i values for pyrogallol as determined with two structurally distinct substrates indicated the likelihood that this compound binds in a non-competitive modality. Further analysis showed that pyrogallol acts as an exosite inhibitor, consistent with the action of ())-epigallocatechin gallate. In contrast, piceatannol was shown to be a competitive binding inhibitor and showed no differences in apparent K i values as determined by distinct substrates, illustrating the benefits of using two structurally distinct substrates to assist the analysis of protease inhibitors. The compounds identified here could be utilized to develop novel metalloproteinase probes or as fragment components of more active inhibitors.
Proceedings of the National Academy of Sciences of the United States of America, Jun 23, 1998
Cell surface heparan sulfate proteoglycan (HSPG) interactions with type I collagen may be a ubiqu... more Cell surface heparan sulfate proteoglycan (HSPG) interactions with type I collagen may be a ubiquitous cell adhesion mechanism. However, the HSPG binding sites on type I collagen are unknown. Previously we mapped heparin binding to the vicinity of the type I collagen N terminus by electron microscopy. The present study has identified type I collagen sequences used for heparin binding and endothelial cell-collagen interactions. Using affinity coelectrophoresis, we found heparin to bind as follows: to type I collagen with high affinity (Kd≈ 150 nM); triple-helical peptides (THPs) including the basic N-terminal sequence α 1(I)87-92, KGH-RGF, with intermediate affinities (Kd≈ and THPs including other collagenous sequences, or single-stranded sequences, negligibly (Kd >> 10 μ M). Thus, heparin-type I collagen binding likely relies on an N-terminal basic triple-helical domain represented once within each monomer, and at multiple sites within fibrils. We next defined the features of type I collagen necessary for angiogenesis in a system in which type I collagen and heparin rapidly induce endothelial tube formation in vitro. When peptides, denatured or monomeric type I collagen, or type V collagen was substituted for type I collagen, no tubes formed. However, when peptides and type I collagen were tested together, only the most heparin-avid THPs inhibited tube formation, likely by influencing cell interactions with collagen-heparin complexes. Thus, induction of endothelial tube morphogenesis by type I collagen may depend upon its triple-helical and fibrillar conformations and on the N-terminal heparin-binding site identified here.
Biomimetic membrane surfaces functionalized with fragments of the extracellular matrix protein, f... more Biomimetic membrane surfaces functionalized with fragments of the extracellular matrix protein, fibronectin, are constructed from mixtures of peptide and polyethylene glycol (PEG) amphiphiles. Peptides from the primary binding loop, GRGDSP, were used in conjunction with the synergy site peptide, PHSRN, in the III9–10 sites of human fibronectin. These peptides were attached to dialkyl lipid tails to form peptide amphiphiles. PEG amphiphiles were mixed in the layer to minimize non-specific adhesion in the background. GRGDSP and PEG amphiphiles or GRGDSP, PHSRN, and PEG amphiphiles were mixed in various ratios and deposited on solid substrates from the air–water interface using Langmuir–Blodgett techniques. In this method, peptide composition, density, and presentation could be controlled accurately. The effectiveness of these substrates to mimic native fibronectin is evaluated by their ability to generate adhesive forces when they are in contact with purified activated α5β1 integrin receptors that are immobilized on an opposing surface. Adhesion is measured using a contact mechanical approach (JKR experiment). The effects of membrane composition, density, temperature, and peptide conformation on adhesion to activated integrins in this simulated cell adhesion setup were determined. Addition of the synergy site, PHSRN, was found to increase adhesion of α5β1 to biomimetic substrates markedly. Increased peptide mobility (due to increased experimental temperature) increased integrin adhesion markedly at low peptide concentrations. A balance between peptide density and steric accessibility of the receptor binding face to α5β1 integrin was required for highest adhesion.
Journal of the American College of Cardiology, Jan 22, 2015
Proteolytically released extracellular matrix (ECM) fragments, matricryptins, are biologically ac... more Proteolytically released extracellular matrix (ECM) fragments, matricryptins, are biologically active and play important roles in wound healing. Following myocardial infarction (MI), collagen I, a major component of cardiac ECM, is cleaved by matrix metalloproteinases (MMPs). This study identified novel collagen-derived matricryptins generated post-MI that mediate remodeling of the left ventricle (LV). Recombinant collagen Ia1 was used in MMPs cleavage assays, the products were analyzed by mass spectrometry for identification of cleavage sites. C57BL6/J mice were given MI and animals were treated either with vehicle control or p1158/59 matricryptin. Seven days post-MI, LV function and parameters of LV remodeling were measured. Levels of p1158/59 were also measured in plasma of MI patients and healthy controls. In situ, MMP-2 and -9 generate a collagen Iα1 C-1158/59 fragment, and MMP-9 can further degrade it. The C-1158/59 fragment was identified post-MI, both in human plasma and mou...
A great variety of cells, such as melanoma cells, fibroblasts, platelets, keratinocytes, and epit... more A great variety of cells, such as melanoma cells, fibroblasts, platelets, keratinocytes, and epithelial cells, adhere to and migrate on specific regions within the triple-helical domains of types I, III, and IV collagen. The relative importance of collagen primary, secondary, and tertiary structures on these cellular activities has not been ascertained, as no general synthetic methodology exists to allow for the study of peptides incorporating biologically active sequences in triple-helical conformation. We have thus developed a novel, generally applicable solid-phase branching methodology for the synthesis of aligned, triple-helical collagen-model polypeptides (i.e. "mini-collagens"). Three nascent peptide chains are carboxyl-terminally linked through one N alpha-amino and two N epsilon-amino groups of Lys, while repeating Gly-Pro-Hyp triplets induce triple helicity. A homotrimeric triple-helical polypeptide (THP) of 124 amino acids, incorporating residues 1263-1277 of al...
Highlights d Complex of cancer-critical MT1-MMP domain with collagen mimic is captured by PRE NMR... more Highlights d Complex of cancer-critical MT1-MMP domain with collagen mimic is captured by PRE NMR d The collagen triple helix is translated 25 Å from MMP-1 complexes d Part of the interface distinguishes MT-MMPs and is attractive for therapeutic development d The domains of MT1-MMP are separated enough for reorientation on collagen
Matrix metalloproteinase 13 (MMP-13) has been shown to be the main collagenase responsible for de... more Matrix metalloproteinase 13 (MMP-13) has been shown to be the main collagenase responsible for degradation of articular cartilage during osteoarthritis and therefore represents a target for drug development. As a result of high-throughput screening and structure−activity relationship studies, we identified a novel, highly selective class of MMP-13 inhibitors (compounds 1 (Q), 2 (Q1), and 3 (Q2)). Mechanistic characterization revealed a noncompetitive nature of these inhibitors with binding constants in the low micromolar range. Crystallographic analyses revealed two binding modes for compound 2 in the MMP-13 S 1 ′ subsite and in an S 1 /S 2 * subsite. Type II collagen-and cartilage-protective effects exhibited by compounds 1, 2, and 3 suggested that these compounds might be efficacious in future in vivo studies. Finally, these compounds were also highly selective when tested against a panel of 30 proteases, which, in combination with a good CYP inhibition profile, suggested low off-target toxicity and drug− drug interactions in humans.
Collagen serves as a structural scaffold and a barrier between tissues, and thus collagen catabol... more 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.
Matrix metalloproteinases (MMPs) are involved in physiological remodeling as well as pathological... more 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.
ADAM10 and ADAM17 have been shown to contribute to the acquired drug resistance of HER2-positive ... more ADAM10 and ADAM17 have been shown to contribute to the acquired drug resistance of HER2-positive breast cancer in response to trastuzumab. The majority of ADAM10 and ADAM17 inhibitor development has been focused on the discovery of compounds that bind the active site zinc, however, in recent years, there has been a shift from active site to secondary substrate binding site (exosite) inhibitor discovery in order to identify non-zinc-binding molecules. In the present work a glycosylated, exosite-binding substrate of ADAM10 and ADAM17 was utilized to screen 370,276 compounds from the MLPCN collection. As a result of this uHTS effort, a selective, time-dependent, non-zinc-binding inhibitor of ADAM10 with Ki = 883 nM was discovered. This compound exhibited low cell toxicity and was able to selectively inhibit shedding of known ADAM10 substrates in several cell-based models. We hypothesize that differential glycosylation of these cognate substrates is the source of selectivity of our nove...
Recently, an unexpected modified residue, γ-hydroxy-D-valine (D-Hyv), was identified within ribos... more Recently, an unexpected modified residue, γ-hydroxy-D-valine (D-Hyv), was identified within ribosomally expressed polypeptide chains of four conopeptides from the venoms of Conus gladiator and Conus mus. In order to assemble Hyv-containing peptides, we have explored several routes for the synthesis of appropriately functionalized Hyv building blocks. D-Hyv was produced from D-Val using a variation of the previously published K 2 PtCl 4 /CuCl 2 oxidative method. Direct synthesis of Boc-or Cbz-D-Hyv lactone proceeded in low yield; additionally, the lactones are too unreative for solid-phase applications. 9-Borabicyclononane or copper-complexed D-Hyv was prepared and treated with tert-butyldimethylsilyl trifluoromethanesulfonate (TBDMSOTf) to produce D-Hyv(O-TBDMS). The most efficient complex disruption was achieved by Chelex 110 resin (Na + form) treatment of copper-complexed D-Hyv(O-TBDMS). Reaction of D-Hyv(O-TBDMS) with Fmoc-OSu produced Fmoc-D-Hyv(O-TBDMS) in 26% yield from D-Val. The Fmoc-D-Hyv(O-TBDMS) diastereomers were separated by preparative RP-HPLC in 13% yield from D-Val. Fmoc-D-Hyv(O-TBDMS) was used for the synthesis of the conopeptide gld-V* from Conus gladiator. The isolated synthetic and natural products had coincidental mass and NMR spectra. The methodology presented herein will greatly facilitate biological studies of Hyv-containing sequences, such as receptor responses to hydroxylated versus non-hydroxylated conopeptides and the relative susceptibility of proteins to modification by oxidative stress.
The turnover of collagen triple-helical structure (collagenolysis) is a tightly regulated process... more The turnover of collagen triple-helical structure (collagenolysis) is a tightly regulated process in normal physiology, and has been ascribed to 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 2 triple-helical sequences to compare the cleavage site specificities of 10 MMPs. One substrate featured a continuous Gly-Xxx-Yyy sequence (Pro-Leu-Gly~Met-Arg-Gly) while the other incorporated an interruption in the Gly-Xxx-Yyy repeat (Pro-Val-Asn~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 an 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 membranetype (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, as MT-MMPs could not hydrolyze the Asn-Phe bond, but instead cleaved the triplehelix nearer the C-terminus at a Gly-Gln bond. It is possible that MT-MMPs have a requirement for Gly in the P 1 subsite in order 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, as MMP-13 preferred the interrupted sequence while MMP-8 showed little discrimination between non-interrupted and interrupted triple-helices. Based on the present and prior studies, we can assign unique triple-helical peptidase behaviors to the collagenolytic MMPs. Such † This work was supported by the National Institutes of Health (AR 40994 to K.B., CA 98799 and EB 000289 to G.B.F.) and the FAU Center of Excellence in Biomedical and Marine Biotechnology.
Solvent dynamics can play a major role in enzyme activity, but obtaining an accurate, quantitativ... more Solvent dynamics can play a major role in enzyme activity, but obtaining an accurate, quantitative picture of solvent activity during catalysis is quite challenging. Here, we combine terahertz spectroscopy and X-ray absorption analyses to measure changes in the coupled water-protein motions during peptide hydrolysis by a zinc-dependent human metalloprotease. These changes were tightly correlated with rearrangements at the active site during the formation of productive enzyme-substrate intermediates and were different from those in an enzyme-inhibitor complex. Molecular dynamics simulations showed a steep gradient of fast-to-slow coupled protein-water motions around the protein, active site and substrate. Our results show that water retardation occurs before formation of the functional Michaelis complex. We propose that the observed gradient of coupled protein-water motions may assist enzyme-substrate interactions through water-polarizing mechanisms that are remotely mediated by the catalytic metal ion and the enzyme active site.
Polyphenolic natural products from green tea and red wine have been identified as metalloproteina... more Polyphenolic natural products from green tea and red wine have been identified as metalloproteinase inhibitors. Members from the flavonoid and stilbene families found to possess metalloproteinase inhibitory activities include ())-epigallocatechin gallate, ())-epicatechin gallate and piceatannol, but their minimally active pharmacophores have not been evaluated. The present study has examined compounds that are structural components of or structurally related to ())-epigallocatechin gallate, ())-epicatechin gallate and piceatannol for inhibition of aggrecanases and four representative matrix metalloproteinases. Piceatannol and pyrogallol were found to inhibit all aggrecanases and matrix metalloproteinases studied, indicating a crucial reliance on multiple hydroxyl groups for ())-epigallocatechin gallate, ())-epicatechin gallate and piceatannol activity. Differences in K i values for pyrogallol as determined with two structurally distinct substrates indicated the likelihood that this compound binds in a non-competitive modality. Further analysis showed that pyrogallol acts as an exosite inhibitor, consistent with the action of ())-epigallocatechin gallate. In contrast, piceatannol was shown to be a competitive binding inhibitor and showed no differences in apparent K i values as determined by distinct substrates, illustrating the benefits of using two structurally distinct substrates to assist the analysis of protease inhibitors. The compounds identified here could be utilized to develop novel metalloproteinase probes or as fragment components of more active inhibitors.
Proceedings of the National Academy of Sciences of the United States of America, Jun 23, 1998
Cell surface heparan sulfate proteoglycan (HSPG) interactions with type I collagen may be a ubiqu... more Cell surface heparan sulfate proteoglycan (HSPG) interactions with type I collagen may be a ubiquitous cell adhesion mechanism. However, the HSPG binding sites on type I collagen are unknown. Previously we mapped heparin binding to the vicinity of the type I collagen N terminus by electron microscopy. The present study has identified type I collagen sequences used for heparin binding and endothelial cell-collagen interactions. Using affinity coelectrophoresis, we found heparin to bind as follows: to type I collagen with high affinity (Kd≈ 150 nM); triple-helical peptides (THPs) including the basic N-terminal sequence α 1(I)87-92, KGH-RGF, with intermediate affinities (Kd≈ and THPs including other collagenous sequences, or single-stranded sequences, negligibly (Kd >> 10 μ M). Thus, heparin-type I collagen binding likely relies on an N-terminal basic triple-helical domain represented once within each monomer, and at multiple sites within fibrils. We next defined the features of type I collagen necessary for angiogenesis in a system in which type I collagen and heparin rapidly induce endothelial tube formation in vitro. When peptides, denatured or monomeric type I collagen, or type V collagen was substituted for type I collagen, no tubes formed. However, when peptides and type I collagen were tested together, only the most heparin-avid THPs inhibited tube formation, likely by influencing cell interactions with collagen-heparin complexes. Thus, induction of endothelial tube morphogenesis by type I collagen may depend upon its triple-helical and fibrillar conformations and on the N-terminal heparin-binding site identified here.
Biomimetic membrane surfaces functionalized with fragments of the extracellular matrix protein, f... more Biomimetic membrane surfaces functionalized with fragments of the extracellular matrix protein, fibronectin, are constructed from mixtures of peptide and polyethylene glycol (PEG) amphiphiles. Peptides from the primary binding loop, GRGDSP, were used in conjunction with the synergy site peptide, PHSRN, in the III9–10 sites of human fibronectin. These peptides were attached to dialkyl lipid tails to form peptide amphiphiles. PEG amphiphiles were mixed in the layer to minimize non-specific adhesion in the background. GRGDSP and PEG amphiphiles or GRGDSP, PHSRN, and PEG amphiphiles were mixed in various ratios and deposited on solid substrates from the air–water interface using Langmuir–Blodgett techniques. In this method, peptide composition, density, and presentation could be controlled accurately. The effectiveness of these substrates to mimic native fibronectin is evaluated by their ability to generate adhesive forces when they are in contact with purified activated α5β1 integrin receptors that are immobilized on an opposing surface. Adhesion is measured using a contact mechanical approach (JKR experiment). The effects of membrane composition, density, temperature, and peptide conformation on adhesion to activated integrins in this simulated cell adhesion setup were determined. Addition of the synergy site, PHSRN, was found to increase adhesion of α5β1 to biomimetic substrates markedly. Increased peptide mobility (due to increased experimental temperature) increased integrin adhesion markedly at low peptide concentrations. A balance between peptide density and steric accessibility of the receptor binding face to α5β1 integrin was required for highest adhesion.
Journal of the American College of Cardiology, Jan 22, 2015
Proteolytically released extracellular matrix (ECM) fragments, matricryptins, are biologically ac... more Proteolytically released extracellular matrix (ECM) fragments, matricryptins, are biologically active and play important roles in wound healing. Following myocardial infarction (MI), collagen I, a major component of cardiac ECM, is cleaved by matrix metalloproteinases (MMPs). This study identified novel collagen-derived matricryptins generated post-MI that mediate remodeling of the left ventricle (LV). Recombinant collagen Ia1 was used in MMPs cleavage assays, the products were analyzed by mass spectrometry for identification of cleavage sites. C57BL6/J mice were given MI and animals were treated either with vehicle control or p1158/59 matricryptin. Seven days post-MI, LV function and parameters of LV remodeling were measured. Levels of p1158/59 were also measured in plasma of MI patients and healthy controls. In situ, MMP-2 and -9 generate a collagen Iα1 C-1158/59 fragment, and MMP-9 can further degrade it. The C-1158/59 fragment was identified post-MI, both in human plasma and mou...
A great variety of cells, such as melanoma cells, fibroblasts, platelets, keratinocytes, and epit... more A great variety of cells, such as melanoma cells, fibroblasts, platelets, keratinocytes, and epithelial cells, adhere to and migrate on specific regions within the triple-helical domains of types I, III, and IV collagen. The relative importance of collagen primary, secondary, and tertiary structures on these cellular activities has not been ascertained, as no general synthetic methodology exists to allow for the study of peptides incorporating biologically active sequences in triple-helical conformation. We have thus developed a novel, generally applicable solid-phase branching methodology for the synthesis of aligned, triple-helical collagen-model polypeptides (i.e. "mini-collagens"). Three nascent peptide chains are carboxyl-terminally linked through one N alpha-amino and two N epsilon-amino groups of Lys, while repeating Gly-Pro-Hyp triplets induce triple helicity. A homotrimeric triple-helical polypeptide (THP) of 124 amino acids, incorporating residues 1263-1277 of al...
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