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Papers by Philippe Cuniasse

Nucleic Acids Research, May 5, 2009

culaire des Proté ines (SIMOPRO) and 2 Groupe informatique pour les scientifiques d'

Journal of Biological Chemistry, 1998

The influence of the substrate P 1 position on the specificity of two zinc matrix metalloprotease... more The influence of the substrate P 1 position on the specificity of two zinc matrix metalloproteases, membrane type-1 matrix metalloprotease (MT1-MMP) and stromelysin-3 (ST3), was evaluated by synthesizing a series of fluorogenic substrates of general formula dansyl-Pro-Leu-Ala-Xaa-Trp-Ala-Arg-NH 2 , where Xaa in the P 1 position represents unusual amino acids containing either long arylalkyl or alkyl side chains. Our data demonstrate that both MT1-MMP and ST3 cleave substrates containing in their P 1 position unusual amino acids with extremely long side chains more efficiently than the corresponding substrates with natural phenylalanine or leucine amino acids. In this series of substrates, the replacement of leucine by S-para-methoxybenzyl cysteine increased the k cat /K m ratio by a factor of 37 for MT1-MMP and 9 for ST3. The substrate with a S-paramethoxybenzyl cysteine residue in the P 1 position displayed a k cat /K m value of 1.59 10 6 M ؊1 s ؊1 and 1.67 10 4 M ؊1 s ؊1 , when assayed with MT1-MMP and ST3, respectively. This substrate is thus one of the most rapidly hydrolyzed substrates so far reported for matrixins, and is the first synthetic peptide efficiently cleaved by ST3. These unexpected results for these two matrixins suggest that extracellular proteins may be cleaved by matrixins at sites containing amino acids with unusual long side chains, like those generated in vivo by some post-translational modifications.

Journal of Medicinal Chemistry, Dec 17, 2003

Phosphinic peptides were previously reported to be potent inhibitors of several matrixins (MMPs).... more Phosphinic peptides were previously reported to be potent inhibitors of several matrixins (MMPs). To identify more selective inhibitors of MMP-11, a matrixin overexpressed in breast cancer, a series of phosphinic pseudopeptides bearing a variety of P(1)'-side chains has been synthesized, by parallel diversification of a phosphinic template. The potencies of these compounds were evaluated against a set of seven MMPs (MMP-2, MMP-7, MMP-8, MMP-9, MMP-11, MMP-13, and MMP-14). The chemical strategy applied led to the identification of several phosphinic inhibitors displaying high selectivity toward MMP-11. One of the most selective inhibitors of MMP-11 in this series, compound 22, exhibits a K(i) value of 0.23 microM toward MMP-11, while its potency toward the other MMPs tested is 2 orders of magnitude lower. This remarkable selectivity may rely on interactions of the P(1)'-side chain atoms of these inhibitors with residues located at the entrance of the S(1)'-cavity of MMP-11. The design of inhibitors able to interact with residues located at the entrance of MMPs' S(1)'-cavity might represent an alternative strategy to identify selective inhibitors that will fully differentiate one MMP among the others.

bioRxiv (Cold Spring Harbor Laboratory), Dec 9, 2021

Microbial Cell Factories, 2013

Background: Disulfide-rich proteins or DRPs are versatile bioactive compounds that encompass a wi... more Background: Disulfide-rich proteins or DRPs are versatile bioactive compounds that encompass a wide variety of pharmacological, therapeutic, and/or biotechnological applications. Still, the production of DRPs in sufficient quantities is a major bottleneck for their complete structural or functional characterization. Recombinant expression of such small proteins containing multiple disulfide bonds in the bacteria E. coli is considered difficult and general methods and protocols, particularly on a high throughput scale, are limited. Results: Here we report a high throughput screening approach that allowed the systematic investigation of the solubilizing and folding influence of twelve cytoplasmic partners on 28 DRPs in the strains BL21 (DE3) pLysS, Origami B (DE3) pLysS and SHuffle® T7 Express lysY (1008 conditions). The screening identified the conditions leading to the successful soluble expression of the 28 DRPs selected for the study. Amongst 336 conditions tested per bacterial strain, soluble expression was detected in 196 conditions using the strain BL21 (DE3) pLysS, whereas only 44 and 50 conditions for soluble expression were identified for the strains Origami B (DE3) pLysS and SHuffle® T7 Express lysY respectively. To assess the redox states of the DRPs, the solubility screen was coupled with mass spectrometry (MS) to determine the exact masses of the produced DRPs or fusion proteins. To validate the results obtained at analytical scale, several examples of proteins expressed and purified to a larger scale are presented along with their MS and functional characterization. Conclusions: Our results show that the production of soluble and functional DRPs with cytoplasmic partners is possible in E. coli. In spite of its reducing cytoplasm, BL21 (DE3) pLysS is more efficient than the Origami B (DE3) pLysS and SHuffle® T7 Express lysY trxB-/gorstrains for the production of DRPs in fusion with solubilizing partners. However, our data suggest that oxidation of the proteins occurs ex vivo. Our protocols allow the production of a large diversity of DRPs using DsbC as a fusion partner, leading to pure active DRPs at milligram scale in many cases. These results open up new possibilities for the study and development of DRPs with therapeutic or biotechnological interest whose production was previously a limitation.

F1000Research, Aug 29, 2012

The FASEB Journal, Aug 2, 2013

Matrix metalloproteinase (MMP)-13 is one of the mammalian collagenases that play key roles in tis... more Matrix metalloproteinase (MMP)-13 is one of the mammalian collagenases that play key roles in tissue remodelling and repair and in progression of diseases such as cancer, arthritis, atherosclerosis, and aneurysm. For collagenase to cleave triple helical collagens, the triple helical structure has to be locally unwound before hydrolysis, but this process is not well understood. We report crystal structures of catalytically inactive full-length human MMP-13(E223A) in complex with peptides of 14-26 aa derived from the cleaved prodomain during activation. Peptides are bound to the active site of the enzyme by forming an extended β-strand with Glu(40) or Tyr(46) inserted into the S1' specificity pocket. The structure of the N-terminal part of the peptides is variable and interacts with different parts of the catalytic domain. Those areas are designated substrate-dependent exosites, in that they accommodate different peptide structures, whereas the precise positioning of the substrate backbone is maintained in the active site. These modes of peptide-MMP-13 interactions have led us to propose how triple helical collagen strands fit into the active site cleft of the collagenase.

Journal of Medicinal Chemistry, Mar 7, 2008

Angiotensin-converting enzyme 2 (ACE2), a recently identified human homologue of angiotensin-conv... more Angiotensin-converting enzyme 2 (ACE2), a recently identified human homologue of angiotensin-converting enzyme, is a zinc metallocarboxypeptidase which may play a unique role in cardiovascular and renal function. Here we report the discovery of potent and selective inhibitors of ACE2, which have been identified by evaluating a series of phosphinic di-and tripeptides of the general formula: Z-Xaa(PO 2-CH 2)YaaOH and Ac-Zaa-Xaa(PO 2-CH 2)YaaOH. The most potent inhibitor in this series is a tripeptide that displays a K i value of 0.4 nM toward ACE2 and is 3 orders of magnitude less potent toward carboxypeptidase A. Phosphinic tripeptides exhibit high potency exclusively when the Xaa position is occupied by a pseudoproline. A model of interaction between one inhibitor of this series and ACE2 suggests that the critical role played by a proline in inhibitors, but also for substrates hydrolysis, may rely on the presence of Tyr 510 in the ACE2 active site.

Biochemistry, Jun 1, 2004

RXPA380 (Cbz-PhePsi[PO(2)CH]Pro-Trp-OH) was reported recently as the first highly selective inhib... more RXPA380 (Cbz-PhePsi[PO(2)CH]Pro-Trp-OH) was reported recently as the first highly selective inhibitor of the C-domain of somatic angiotensin-converting enzyme (ACE), able to differentiate the two active sites of somatic ACE by a selectivity factor of more than 3 orders of magnitude. The contribution of each RXPA380 residue toward this remarkable selectivity was evaluated by studying several analogues of RXPA380. This analysis revealed that both pseudo-proline and tryptophan residues in the P(1)' and P(2)' positions of RXPA380 play a critical role in the selectivity of this inhibitor for the C-domain. This selectivity is not due to a preference of the C-domain for inhibitors bearing pseudo-proline and tryptophan residues, but rather reflects the poor accommodation of these inhibitor residues by the N-domain. A model of RXPA380 in complex with the ACE C-domain, based on the crystal structure of germinal ACE, highlights residues that may contribute to RXPA380 selectivity. From this model, striking differences between the N- and C-domains of ACE are observed for residues defining the S(2)' pocket. Of the twelve residues that surround the tryptophan side chain of RXPA380 in the C-domain, five are different in the N-domain. These differences in the S(2)' composition between the N- and C-domains are suggested to contribute to RXPA380 selectivity. The structural insights provided by this study should enhance understanding of the factors controlling the selectivity of the two domains of somatic ACE and allow the design of new selective ACE inhibitors.

Biochemical Society Transactions, Jun 1, 2000

The recent elucidation of the three-dimensional structure of human placental alkaline phosphatase... more The recent elucidation of the three-dimensional structure of human placental alkaline phosphatase (PLAP) has enabled me to perform structural studies aimed at characterizing the properties of human PLAP and tissue-nonspecific AP (TNAP) as paradigms for mammalian APs in general, using site-directed mutagenesis, protein expression, kinetic analysis and computer modeling. In Paper I, we found that a single critical E429G substitution explains the difference in stability and kinetics between the common allelic variants of PLAP and the D allozyme. In Paper II, we demonstrated the role of residue E429 in PLAP in stabilizing the active site metals, elucidated the distinct roles of residues H153 and H317 in catalysis, and the relative importance of five Cys residues in PLAP. We also discovered the significance of Y367, a unique feature of mammalian APs, for enzyme stability and specific inhibition by amino acids. Paper III focused on the identification and mutagenesis analysis of a novel, non-catalytic peripheral binding site of PLAP that appears to mediate a mitogenic effect of PLAP. This site provides indications that PLAP may function as a fetal growth factor. The last two papers focus on the TNAP isozyme as paradigm. A deficiency in TNAP activity is the cause of the human disease hypophosphatasia, characterized by rickets, osteomalacia and occasionally epileptic seizures. Paper IV has been able to partially explain the variable expressivity of hypophosphatasia traits by examining site-directed mutants of TNAP and performing kinetic analysis using natural substrates PPi and PLP. Finally, Paper V has clarified the mechanism of inhibition of TNAP by uncompetitive inhibitors L-homoarginine, levamisole and theophylline. We identified residues that confer to TNAP its distinct inhibitory properties. These data have significance for future drug design of specific TNAP inhibitors to therapeutically target TNAP as a way of elevating PPi extracellular level and alleviating pathological bone hypermineralization conditions.

A many-body model to study proteins. I. Applications to MLnm+ complexes, Mm+=Li+, Na+, K+, Mg2+, Ca2+, and Zn2+, L=H2O, CH3OH, HCONH2, n=1–6, and to small hydrogen bonded systems

Journal of Chemical Physics, Jul 2, 2003

A new model to study proteinic systems including a many-body polarization and a hydrogen bond ene... more A new model to study proteinic systems including a many-body polarization and a hydrogen bond energy contribution is presented. This model represents an extension of an earlier water many-body model [M. Masella and J.-P. Flament, J. Chem. Phys. 107 9105 (1997)]. As in this earlier model, the new model is developed to reproduce quantum computations on small molecular aggregates, and, in this first paper, we focus our efforts in developing an accurate potential to describe interactions among all nonbonded atoms occurring in proteins, and among those atoms and six cations of biological interest: Li+, Na+, K+, Mg2+, Ca2+, and Zn2+. Intramolecular degrees of freedom are described as in classical two-body force fields. In the present paper, the new model is applied to investigate the properties of small ion–neutral [M,Ln]m+ complexes and of small hydrogen-bonded systems. The results showed that this model is able to reproduce most of the theoretical quantum predictions and experimental data published until now regarding those systems.

Structure, 2015

Membrane type 1 metalloprotease (MT1-MMP) is a membrane-anchored, zinc-dependent protease. MT1-MM... more Membrane type 1 metalloprotease (MT1-MMP) is a membrane-anchored, zinc-dependent protease. MT1-MMP is an important mediator of cell migration and invasion, and overexpression of this enzyme has been correlated with the malignancy of various tumor types. Therefore, modulators of MT1-MMP activity are proposed to possess therapeutic potential in numerous invasive diseases. Here we report the inhibition mode of MT1-MMP by LEM-2/15 antibody, which targets a surface epitope of MT1-MMP. Specifically, the crystal structures of Fab LEM-2/15 in complex with the MT1-MMP surface antigen suggest that conformational swiveling of the enzyme surface loop is required for effective binding and consequent inhibition of MT1-MMP activity on the cell membrane. This inhibition mechanism appears to be effective in controlling active MT1-MMP in endothelial cells and at the leading edge of migratory cancer cells. D = 2.32 3 10 À9 M).

Journal of Bone and Mineral Research, Jun 28, 2004

Recent data have pointed to TNALP as a therapeutic target for soft-tissue ossification abnormalit... more Recent data have pointed to TNALP as a therapeutic target for soft-tissue ossification abnormalities. Here, we used mutagenesis, kinetic analysis, and computer modeling to identify the residues important for the binding of known ALP inhibitors to the TNALP active site. These data will enable drug design efforts aimed at developing improved specific TNALP inhibitors for therapeutic use. Introduction: We have shown previously that the genetic ablation of tissue-nonspecific alkaline phosphatase (TNALP) function leads to amelioration of soft-tissue ossification in mouse models of osteoarthritis and ankylosis (i.e., Enpp1 Ϫ/Ϫ and ank/ank mutant mice). We surmise that the pharmacologic inhibition of TNALP activity represents a viable therapeutic approach for these diseases. As a first step toward developing suitable TNALP therapeutics, we have now clarified the residues involved in binding well-known uncompetitive inhibitors to the TNALP active site. Materials and Methods: We compared the modeled 3D structure of TNALP with the 3D structure of human placental alkaline phosphatase (PLALP) and identified the residues that differ between these isozymes within a 12 Å radius of the active site, because these isozymes differ significantly in inhibitor specificity. We then used site-directed mutagenesis to substitute TNALP residues to their respective homolog in PLALP. In addition, we mutagenized most of these residues in TNALP to Ala and the corresponding residues in PLALP to their TNALP homolog. All mutants were characterized for their sensitivity toward the uncompetitive inhibitors L-homoarginine (L-hArg), levamisole, theophylline, and L-phenylalanine. Results and Conclusions: We found that the identity of residue 108 in TNALP largely determines the specificity of inhibition by L-hArg. The conserved Tyr-371 is also necessary for binding of L-hArg. In contrast, the binding of levamisole to TNALP is mostly dependent on His-434 and Tyr-371, but not on residues 108 or 109. The main determinant of sensitivity to theophylline is His-434. Thus, we have clarified the location of the binding sites for all three TNALP inhibitors, and we have also been able to exchange inhibitor specificities between TNALP and PLALP. These data will enable drug design efforts aimed at developing improved, selective, and drug-like TNALP inhibitors for therapeutic use.

FEBS Journal, Apr 1, 2006

Somatic angiotensin‐converting enzyme (ACE) contains two homologous domains, each bearing a funct... more Somatic angiotensin‐converting enzyme (ACE) contains two homologous domains, each bearing a functional active site. Studies on the selectivity of these ACE domains towards either substrates or inhibitors have mostly relied on the use of mutants or isolated domains of ACE. To determine directly the selectivity properties of each ACE domain, working with wild‐type enzyme, we developed an approach based on the combined use of N‐domain‐selective and C‐domain‐selective ACE inhibitors and fluorogenic substrates. With this approach, marked differences in substrate selectivity were revealed between rat, mouse and human somatic ACE. In particular, the fluorogenic substrate Mca‐Ala‐Ser‐Asp‐Lys‐DpaOH was shown to be a strict N‐domain‐selective substrate of mouse ACE, whereas with rat ACE it displayed marked C‐domain selectivity. Similar differences in selectivity between these ACE species were also observed with a new fluorogenic substrate of ACE, Mca‐Arg‐Pro‐Pro‐Gly‐Phe‐Ser‐Pro‐DpaOH. In support of these results, changes in amino‐acid composition in the binding site of these three ACE species were pinpointed. Together these data demonstrate that the substrate selectivity of the N‐domain and C‐domain depends on the ACE species. These results raise concerns about the interpretation of functional studies performed in animals using N‐domain and C‐domain substrate selectivity data derived only from human ACE.

Nucleic Acids Research, Mar 21, 2021

Barrier-to-autointegration factor (BAF), encoded by the BANF1 gene, is an abundant and ubiquitous... more Barrier-to-autointegration factor (BAF), encoded by the BANF1 gene, is an abundant and ubiquitously expressed metazoan protein that has multiple functions during the cell cycle. Through its ability to cross-bridge two double-stranded DNA (dsDNA), it favours chromosome compaction, participates in post-mitotic nuclear envelope reassembly and is essential for the repair of large nuclear ruptures. BAF forms a ternary complex with the nuclear envelope proteins lamin A/C and emerin, and its interaction with lamin A/C is defective in patients with recessive accelerated aging syndromes. Phosphorylation of BAF by the vaccinia-related kinase 1 (VRK1) is a key regulator of BAF localization and function. Here, we demonstrate that VRK1 successively phosphorylates BAF on Ser4 and Thr3. The crystal structures of BAF before and after phosphorylation are extremely similar. However, in solution, the extensive flexibility of the N-terminal helix ␣1 and loop ␣1␣2 in BAF is strongly reduced in di-phosphorylated BAF, due to interactions between the phosphorylated residues and the positively charged C-terminal helix ␣6. These regions are involved in DNA and lamin A/C binding. Consistently, phosphorylation causes a 5000-fold loss of affinity for dsDNA. However, it does not impair binding to lamin A/C Igfold domain and emerin nucleoplasmic region, which leaves open the question of the regulation of these interactions.

Nucleic Acids Research, May 5, 2009

culaire des Proté ines (SIMOPRO) and 2 Groupe informatique pour les scientifiques d'

Journal of Biological Chemistry, 1998

The influence of the substrate P 1 position on the specificity of two zinc matrix metalloprotease... more The influence of the substrate P 1 position on the specificity of two zinc matrix metalloproteases, membrane type-1 matrix metalloprotease (MT1-MMP) and stromelysin-3 (ST3), was evaluated by synthesizing a series of fluorogenic substrates of general formula dansyl-Pro-Leu-Ala-Xaa-Trp-Ala-Arg-NH 2 , where Xaa in the P 1 position represents unusual amino acids containing either long arylalkyl or alkyl side chains. Our data demonstrate that both MT1-MMP and ST3 cleave substrates containing in their P 1 position unusual amino acids with extremely long side chains more efficiently than the corresponding substrates with natural phenylalanine or leucine amino acids. In this series of substrates, the replacement of leucine by S-para-methoxybenzyl cysteine increased the k cat /K m ratio by a factor of 37 for MT1-MMP and 9 for ST3. The substrate with a S-paramethoxybenzyl cysteine residue in the P 1 position displayed a k cat /K m value of 1.59 10 6 M ؊1 s ؊1 and 1.67 10 4 M ؊1 s ؊1 , when assayed with MT1-MMP and ST3, respectively. This substrate is thus one of the most rapidly hydrolyzed substrates so far reported for matrixins, and is the first synthetic peptide efficiently cleaved by ST3. These unexpected results for these two matrixins suggest that extracellular proteins may be cleaved by matrixins at sites containing amino acids with unusual long side chains, like those generated in vivo by some post-translational modifications.

Journal of Medicinal Chemistry, Dec 17, 2003

Phosphinic peptides were previously reported to be potent inhibitors of several matrixins (MMPs).... more Phosphinic peptides were previously reported to be potent inhibitors of several matrixins (MMPs). To identify more selective inhibitors of MMP-11, a matrixin overexpressed in breast cancer, a series of phosphinic pseudopeptides bearing a variety of P(1)'-side chains has been synthesized, by parallel diversification of a phosphinic template. The potencies of these compounds were evaluated against a set of seven MMPs (MMP-2, MMP-7, MMP-8, MMP-9, MMP-11, MMP-13, and MMP-14). The chemical strategy applied led to the identification of several phosphinic inhibitors displaying high selectivity toward MMP-11. One of the most selective inhibitors of MMP-11 in this series, compound 22, exhibits a K(i) value of 0.23 microM toward MMP-11, while its potency toward the other MMPs tested is 2 orders of magnitude lower. This remarkable selectivity may rely on interactions of the P(1)'-side chain atoms of these inhibitors with residues located at the entrance of the S(1)'-cavity of MMP-11. The design of inhibitors able to interact with residues located at the entrance of MMPs' S(1)'-cavity might represent an alternative strategy to identify selective inhibitors that will fully differentiate one MMP among the others.

bioRxiv (Cold Spring Harbor Laboratory), Dec 9, 2021

Microbial Cell Factories, 2013

Background: Disulfide-rich proteins or DRPs are versatile bioactive compounds that encompass a wi... more Background: Disulfide-rich proteins or DRPs are versatile bioactive compounds that encompass a wide variety of pharmacological, therapeutic, and/or biotechnological applications. Still, the production of DRPs in sufficient quantities is a major bottleneck for their complete structural or functional characterization. Recombinant expression of such small proteins containing multiple disulfide bonds in the bacteria E. coli is considered difficult and general methods and protocols, particularly on a high throughput scale, are limited. Results: Here we report a high throughput screening approach that allowed the systematic investigation of the solubilizing and folding influence of twelve cytoplasmic partners on 28 DRPs in the strains BL21 (DE3) pLysS, Origami B (DE3) pLysS and SHuffle® T7 Express lysY (1008 conditions). The screening identified the conditions leading to the successful soluble expression of the 28 DRPs selected for the study. Amongst 336 conditions tested per bacterial strain, soluble expression was detected in 196 conditions using the strain BL21 (DE3) pLysS, whereas only 44 and 50 conditions for soluble expression were identified for the strains Origami B (DE3) pLysS and SHuffle® T7 Express lysY respectively. To assess the redox states of the DRPs, the solubility screen was coupled with mass spectrometry (MS) to determine the exact masses of the produced DRPs or fusion proteins. To validate the results obtained at analytical scale, several examples of proteins expressed and purified to a larger scale are presented along with their MS and functional characterization. Conclusions: Our results show that the production of soluble and functional DRPs with cytoplasmic partners is possible in E. coli. In spite of its reducing cytoplasm, BL21 (DE3) pLysS is more efficient than the Origami B (DE3) pLysS and SHuffle® T7 Express lysY trxB-/gorstrains for the production of DRPs in fusion with solubilizing partners. However, our data suggest that oxidation of the proteins occurs ex vivo. Our protocols allow the production of a large diversity of DRPs using DsbC as a fusion partner, leading to pure active DRPs at milligram scale in many cases. These results open up new possibilities for the study and development of DRPs with therapeutic or biotechnological interest whose production was previously a limitation.

F1000Research, Aug 29, 2012

The FASEB Journal, Aug 2, 2013

Matrix metalloproteinase (MMP)-13 is one of the mammalian collagenases that play key roles in tis... more Matrix metalloproteinase (MMP)-13 is one of the mammalian collagenases that play key roles in tissue remodelling and repair and in progression of diseases such as cancer, arthritis, atherosclerosis, and aneurysm. For collagenase to cleave triple helical collagens, the triple helical structure has to be locally unwound before hydrolysis, but this process is not well understood. We report crystal structures of catalytically inactive full-length human MMP-13(E223A) in complex with peptides of 14-26 aa derived from the cleaved prodomain during activation. Peptides are bound to the active site of the enzyme by forming an extended β-strand with Glu(40) or Tyr(46) inserted into the S1' specificity pocket. The structure of the N-terminal part of the peptides is variable and interacts with different parts of the catalytic domain. Those areas are designated substrate-dependent exosites, in that they accommodate different peptide structures, whereas the precise positioning of the substrate backbone is maintained in the active site. These modes of peptide-MMP-13 interactions have led us to propose how triple helical collagen strands fit into the active site cleft of the collagenase.

Journal of Medicinal Chemistry, Mar 7, 2008

Angiotensin-converting enzyme 2 (ACE2), a recently identified human homologue of angiotensin-conv... more Angiotensin-converting enzyme 2 (ACE2), a recently identified human homologue of angiotensin-converting enzyme, is a zinc metallocarboxypeptidase which may play a unique role in cardiovascular and renal function. Here we report the discovery of potent and selective inhibitors of ACE2, which have been identified by evaluating a series of phosphinic di-and tripeptides of the general formula: Z-Xaa(PO 2-CH 2)YaaOH and Ac-Zaa-Xaa(PO 2-CH 2)YaaOH. The most potent inhibitor in this series is a tripeptide that displays a K i value of 0.4 nM toward ACE2 and is 3 orders of magnitude less potent toward carboxypeptidase A. Phosphinic tripeptides exhibit high potency exclusively when the Xaa position is occupied by a pseudoproline. A model of interaction between one inhibitor of this series and ACE2 suggests that the critical role played by a proline in inhibitors, but also for substrates hydrolysis, may rely on the presence of Tyr 510 in the ACE2 active site.

Biochemistry, Jun 1, 2004

RXPA380 (Cbz-PhePsi[PO(2)CH]Pro-Trp-OH) was reported recently as the first highly selective inhib... more RXPA380 (Cbz-PhePsi[PO(2)CH]Pro-Trp-OH) was reported recently as the first highly selective inhibitor of the C-domain of somatic angiotensin-converting enzyme (ACE), able to differentiate the two active sites of somatic ACE by a selectivity factor of more than 3 orders of magnitude. The contribution of each RXPA380 residue toward this remarkable selectivity was evaluated by studying several analogues of RXPA380. This analysis revealed that both pseudo-proline and tryptophan residues in the P(1)' and P(2)' positions of RXPA380 play a critical role in the selectivity of this inhibitor for the C-domain. This selectivity is not due to a preference of the C-domain for inhibitors bearing pseudo-proline and tryptophan residues, but rather reflects the poor accommodation of these inhibitor residues by the N-domain. A model of RXPA380 in complex with the ACE C-domain, based on the crystal structure of germinal ACE, highlights residues that may contribute to RXPA380 selectivity. From this model, striking differences between the N- and C-domains of ACE are observed for residues defining the S(2)' pocket. Of the twelve residues that surround the tryptophan side chain of RXPA380 in the C-domain, five are different in the N-domain. These differences in the S(2)' composition between the N- and C-domains are suggested to contribute to RXPA380 selectivity. The structural insights provided by this study should enhance understanding of the factors controlling the selectivity of the two domains of somatic ACE and allow the design of new selective ACE inhibitors.

Biochemical Society Transactions, Jun 1, 2000

The recent elucidation of the three-dimensional structure of human placental alkaline phosphatase... more The recent elucidation of the three-dimensional structure of human placental alkaline phosphatase (PLAP) has enabled me to perform structural studies aimed at characterizing the properties of human PLAP and tissue-nonspecific AP (TNAP) as paradigms for mammalian APs in general, using site-directed mutagenesis, protein expression, kinetic analysis and computer modeling. In Paper I, we found that a single critical E429G substitution explains the difference in stability and kinetics between the common allelic variants of PLAP and the D allozyme. In Paper II, we demonstrated the role of residue E429 in PLAP in stabilizing the active site metals, elucidated the distinct roles of residues H153 and H317 in catalysis, and the relative importance of five Cys residues in PLAP. We also discovered the significance of Y367, a unique feature of mammalian APs, for enzyme stability and specific inhibition by amino acids. Paper III focused on the identification and mutagenesis analysis of a novel, non-catalytic peripheral binding site of PLAP that appears to mediate a mitogenic effect of PLAP. This site provides indications that PLAP may function as a fetal growth factor. The last two papers focus on the TNAP isozyme as paradigm. A deficiency in TNAP activity is the cause of the human disease hypophosphatasia, characterized by rickets, osteomalacia and occasionally epileptic seizures. Paper IV has been able to partially explain the variable expressivity of hypophosphatasia traits by examining site-directed mutants of TNAP and performing kinetic analysis using natural substrates PPi and PLP. Finally, Paper V has clarified the mechanism of inhibition of TNAP by uncompetitive inhibitors L-homoarginine, levamisole and theophylline. We identified residues that confer to TNAP its distinct inhibitory properties. These data have significance for future drug design of specific TNAP inhibitors to therapeutically target TNAP as a way of elevating PPi extracellular level and alleviating pathological bone hypermineralization conditions.

A many-body model to study proteins. I. Applications to MLnm+ complexes, Mm+=Li+, Na+, K+, Mg2+, Ca2+, and Zn2+, L=H2O, CH3OH, HCONH2, n=1–6, and to small hydrogen bonded systems

Journal of Chemical Physics, Jul 2, 2003

A new model to study proteinic systems including a many-body polarization and a hydrogen bond ene... more A new model to study proteinic systems including a many-body polarization and a hydrogen bond energy contribution is presented. This model represents an extension of an earlier water many-body model [M. Masella and J.-P. Flament, J. Chem. Phys. 107 9105 (1997)]. As in this earlier model, the new model is developed to reproduce quantum computations on small molecular aggregates, and, in this first paper, we focus our efforts in developing an accurate potential to describe interactions among all nonbonded atoms occurring in proteins, and among those atoms and six cations of biological interest: Li+, Na+, K+, Mg2+, Ca2+, and Zn2+. Intramolecular degrees of freedom are described as in classical two-body force fields. In the present paper, the new model is applied to investigate the properties of small ion–neutral [M,Ln]m+ complexes and of small hydrogen-bonded systems. The results showed that this model is able to reproduce most of the theoretical quantum predictions and experimental data published until now regarding those systems.

Structure, 2015

Membrane type 1 metalloprotease (MT1-MMP) is a membrane-anchored, zinc-dependent protease. MT1-MM... more Membrane type 1 metalloprotease (MT1-MMP) is a membrane-anchored, zinc-dependent protease. MT1-MMP is an important mediator of cell migration and invasion, and overexpression of this enzyme has been correlated with the malignancy of various tumor types. Therefore, modulators of MT1-MMP activity are proposed to possess therapeutic potential in numerous invasive diseases. Here we report the inhibition mode of MT1-MMP by LEM-2/15 antibody, which targets a surface epitope of MT1-MMP. Specifically, the crystal structures of Fab LEM-2/15 in complex with the MT1-MMP surface antigen suggest that conformational swiveling of the enzyme surface loop is required for effective binding and consequent inhibition of MT1-MMP activity on the cell membrane. This inhibition mechanism appears to be effective in controlling active MT1-MMP in endothelial cells and at the leading edge of migratory cancer cells. D = 2.32 3 10 À9 M).

Journal of Bone and Mineral Research, Jun 28, 2004

Recent data have pointed to TNALP as a therapeutic target for soft-tissue ossification abnormalit... more Recent data have pointed to TNALP as a therapeutic target for soft-tissue ossification abnormalities. Here, we used mutagenesis, kinetic analysis, and computer modeling to identify the residues important for the binding of known ALP inhibitors to the TNALP active site. These data will enable drug design efforts aimed at developing improved specific TNALP inhibitors for therapeutic use. Introduction: We have shown previously that the genetic ablation of tissue-nonspecific alkaline phosphatase (TNALP) function leads to amelioration of soft-tissue ossification in mouse models of osteoarthritis and ankylosis (i.e., Enpp1 Ϫ/Ϫ and ank/ank mutant mice). We surmise that the pharmacologic inhibition of TNALP activity represents a viable therapeutic approach for these diseases. As a first step toward developing suitable TNALP therapeutics, we have now clarified the residues involved in binding well-known uncompetitive inhibitors to the TNALP active site. Materials and Methods: We compared the modeled 3D structure of TNALP with the 3D structure of human placental alkaline phosphatase (PLALP) and identified the residues that differ between these isozymes within a 12 Å radius of the active site, because these isozymes differ significantly in inhibitor specificity. We then used site-directed mutagenesis to substitute TNALP residues to their respective homolog in PLALP. In addition, we mutagenized most of these residues in TNALP to Ala and the corresponding residues in PLALP to their TNALP homolog. All mutants were characterized for their sensitivity toward the uncompetitive inhibitors L-homoarginine (L-hArg), levamisole, theophylline, and L-phenylalanine. Results and Conclusions: We found that the identity of residue 108 in TNALP largely determines the specificity of inhibition by L-hArg. The conserved Tyr-371 is also necessary for binding of L-hArg. In contrast, the binding of levamisole to TNALP is mostly dependent on His-434 and Tyr-371, but not on residues 108 or 109. The main determinant of sensitivity to theophylline is His-434. Thus, we have clarified the location of the binding sites for all three TNALP inhibitors, and we have also been able to exchange inhibitor specificities between TNALP and PLALP. These data will enable drug design efforts aimed at developing improved, selective, and drug-like TNALP inhibitors for therapeutic use.

FEBS Journal, Apr 1, 2006

Somatic angiotensin‐converting enzyme (ACE) contains two homologous domains, each bearing a funct... more Somatic angiotensin‐converting enzyme (ACE) contains two homologous domains, each bearing a functional active site. Studies on the selectivity of these ACE domains towards either substrates or inhibitors have mostly relied on the use of mutants or isolated domains of ACE. To determine directly the selectivity properties of each ACE domain, working with wild‐type enzyme, we developed an approach based on the combined use of N‐domain‐selective and C‐domain‐selective ACE inhibitors and fluorogenic substrates. With this approach, marked differences in substrate selectivity were revealed between rat, mouse and human somatic ACE. In particular, the fluorogenic substrate Mca‐Ala‐Ser‐Asp‐Lys‐DpaOH was shown to be a strict N‐domain‐selective substrate of mouse ACE, whereas with rat ACE it displayed marked C‐domain selectivity. Similar differences in selectivity between these ACE species were also observed with a new fluorogenic substrate of ACE, Mca‐Arg‐Pro‐Pro‐Gly‐Phe‐Ser‐Pro‐DpaOH. In support of these results, changes in amino‐acid composition in the binding site of these three ACE species were pinpointed. Together these data demonstrate that the substrate selectivity of the N‐domain and C‐domain depends on the ACE species. These results raise concerns about the interpretation of functional studies performed in animals using N‐domain and C‐domain substrate selectivity data derived only from human ACE.

Nucleic Acids Research, Mar 21, 2021

Barrier-to-autointegration factor (BAF), encoded by the BANF1 gene, is an abundant and ubiquitous... more Barrier-to-autointegration factor (BAF), encoded by the BANF1 gene, is an abundant and ubiquitously expressed metazoan protein that has multiple functions during the cell cycle. Through its ability to cross-bridge two double-stranded DNA (dsDNA), it favours chromosome compaction, participates in post-mitotic nuclear envelope reassembly and is essential for the repair of large nuclear ruptures. BAF forms a ternary complex with the nuclear envelope proteins lamin A/C and emerin, and its interaction with lamin A/C is defective in patients with recessive accelerated aging syndromes. Phosphorylation of BAF by the vaccinia-related kinase 1 (VRK1) is a key regulator of BAF localization and function. Here, we demonstrate that VRK1 successively phosphorylates BAF on Ser4 and Thr3. The crystal structures of BAF before and after phosphorylation are extremely similar. However, in solution, the extensive flexibility of the N-terminal helix ␣1 and loop ␣1␣2 in BAF is strongly reduced in di-phosphorylated BAF, due to interactions between the phosphorylated residues and the positively charged C-terminal helix ␣6. These regions are involved in DNA and lamin A/C binding. Consistently, phosphorylation causes a 5000-fold loss of affinity for dsDNA. However, it does not impair binding to lamin A/C Igfold domain and emerin nucleoplasmic region, which leaves open the question of the regulation of these interactions.