Removal of Metal from Carboxypeptidase A Proceeds via a Split Pathway: Implications for the General Mechanisms of Metalloenzyme Inactivation by Chelating Agents.

The chelation of protein-bound metal ions is typically thought to follow either a dissociative (D) or an associative (A) path. While the former mechanism involves the spontaneous dissociation of the metal from the protein prior to chelation, the latter route is characterized by the formation of an intermediate protein-metal-chelator ternary complex. Using the prototypical zinc protease carboxypeptidase A (CPA) and a variety of charged and neutral chelating agents, we demonstrate that inactivation of the enzyme (and likely other metalloproteins) proceeds through a split pathway with contributions from both D- and A-type mechanisms. In the case of charged chelators such as ethylenediaminetetraacetic acid (EDTA), the proportions of both paths could be tuned over a wide range through variation of the chelator concentration and the ionic strength, I (from ∼95% A type at low I values to ∼5% at high I values). By measuring the EDTA concentration and time dependence of CPA inactivation and fitting the obtained kinetic data to a modified time-dependent inhibition model, we obtained the rate constants for the A and D paths (kinact and koff, respectively) and the inhibition constant (KI) for the formation of the CPA-Zn2+-EDTA ternary complex, indicating that the decreased contribution of the A-type mechanism at high ionic strengths originates from a large (40-fold; at I = 0.5 M) increase in KI. This observation might be related to a triarginine motif in CPA that electrostatically steers negatively charged substrates into the active site and may therefore also guide carboxylate-bearing chelators toward the Zn2+ ion.

Structure of Aedes aegypti carboxypeptidase B1-inhibitor complex uncover the disparity between mosquito and non-mosquito insect carboxypeptidase inhibition mechanism.

Metallocarboxypeptidases (MCPs) in the mosquito midgut play crucial roles in infection, as well as in mosquito dietary digestion, reproduction, and development. MCPs are also part of the digestive system of plant-feeding insects, representing key targets for inhibitor development against mosquitoes/mosquito-borne pathogens or as antifeedant molecules against plant-feeding insects. Notably, some non-mosquito insect B-type MCPs are primarily insensitive to plant protease inhibitors (PPIs) such as the potato carboxypeptidase inhibitor (PCI; MW 4 kDa), an inhibitor explored for cancer treatment and insecticide design. Here, we report the crystal structure of Aedes aegypti carboxypeptidase-B1 (CPBAe1)-PCI complex and compared the binding with that of PCI-insensitive CPBs. We show that PCI accommodation is determined by key differences in the active-site regions of MCPs. In particular, the loop regions α6-α7 (Leu242 -Ser250 ) and ß8-α8 (Pro269 -Pro280 ) of CPBAe1 are replaced by α-helices in PCI-insensitive insect Helicoverpa zea CPBHz. These α-helices protrude into the active-site pocket of CPBHz, restricting PCI insertion and rendering the enzyme insensitive. We further compared our structure with the only other PCI complex available, bovine CPA1-PCI. The potency of PCI against CPBAe1 (Ki  = 14.7 nM) is marginally less than that of bovine CPA1 (Ki  = 5 nM). Structurally, the above loop regions that accommodate PCI binding in CPBAe1 are similar to that of bovine CPA1, although observed changes in proteases residues that interact with PCI could account for the differences in affinity. Our findings suggest that PCI sensitivity is largely dictated by structural interference, which broadens our understanding of carboxypeptidase inhibition as a mosquito population/parasite control strategy.

Carboxypeptidase A6 Promotes the Proliferation and Migration of Hepatocellular Carcinoma by Up-regulating AKT Signaling Pathway.

Hepatocellular carcinoma (HCC) has a poor treatment prognosis and high mortality worldwide. Understanding the molecular mechanism underlying HCC development would benefit the identification of diagnostic biomarkers and the improvement of the treatment strategies. The expression of carboxypeptidase A6 (CPA6) has been reported in epilepsy and febrile seizures rather than in any type of cancers. However, the function of CPA6 expression in HCC is not yet understood. In this study, we aimed to investigate the clinicopathological significance of the expression of CPA6 in HCC and the underlying mechanisms. We observed that the expression of the CPA6 protein was increased significantly in HCC tissues than in paracancerous tissues. To explore its function in HCC, both gain- and loss-of-function studies demonstrated that CPA6 played a vital role in promoting HCC growth and metastasis. When knocking down CPA6 with shRNA, HCC cell proliferation and migration could be suppressed. Meanwhile, CPA6 overexpression could promote proliferation and migration of HLF cells. Moreover, CPA6 could activate AKT serine/threonine kinase (AKT) signaling pathway as confirmed by Western blotting. In conclusion, our study revealed that CPA6 could promote HCC cell proliferation and migration via AKT-mediated signaling pathway. These findings suggest that CPA6 is a promising diagnostic biomarker and therapeutic target to improve the prognosis of HCC.

Synthesis and Structural/Functional Characterization of Selective M14 Metallocarboxypeptidase Inhibitors Based on Phosphinic Pseudopeptide Scaffold: Implications on the Design of Specific Optical Probes.

Metallocarboxypeptidases (MCPs) of the M14 family are Zn2+-dependent exoproteases present in almost every tissue or fluid in mammals. These enzymes perform a large variety of physiological functions and are involved in several pathologies, such as pancreatic diseases, inflammation, fibrinolysis, and cancer. Here, we describe the synthesis and functional/structural characterization of a series of reversible tight-binding phosphinic pseudopeptide inhibitors that show high specificity and potency toward these proteases. Characterization of their inhibitory potential against a large variety of MCPs, combined with high-resolution crystal structures of three selected candidates in complex with human carboxypeptidase A (CPA)1, allowed to decipher the structural determinants governing selectivity for type-A of the M14A MCP family. Further, the phosphinic pseudopeptide framework was exploited to generate an optical probe selectively targeting human CPAs. The phosphinic pseudopeptides presented here constitute the first example of chemical probes useful to selectively report on type-A MCPs activity in complex media.

Modeling association detection in order to discover compounds to inhibit oral cancer.

In the past, algorithms exploiting varying semantics in interactions between biological objects such as genes and diseases have been used in bioinformatics to uncover latent relationships within biological datasets. In this paper, we consider the algorithm Medusa in parallel with binary classification in order to find potential compounds to inhibit oral cancer. Oral cancer affects the mouth and pharynx and has a high mortality rate due to its late discovery. Current methods of oral cancer treatment, such as chemoradiation and surgery, fail to provide better chances for survival, warranting an alternative approach. By running Medusa on a data fusion graph consisting of biological objects, we incorporate binary classification to model the algorithm's association detection to discover compounds with the potential to mitigate the effects of oral cancer.

Morphologic, Phylogenetic and Chemical Characterization of a Brackish Colonial Picocyanobacterium (Coelosphaeriaceae) with Bioactive Properties.

Despite their cosmopolitan distribution, knowledge on cyanobacteria in the family Coelosphaeriaceae is limited. In this study, a single species culture of a coelosphaeran cyanobacterium isolated from a brackish rock pool in the Baltic Sea was established. The strain was characterized by morphological features, partial 16S rRNA sequence and nonribosomal oligopeptide profile. The bioactivity of fractionated extracts against several serine proteases, as well as protein-serine/threonine phosphatases was studied. Phylogenetic analyses of the strain suggested a close relationship with Snowella litoralis, but its morphology resembled Woronichinia compacta. The controversial morphologic and phylogenetic results demonstrated remaining uncertainties regarding species division in this cyanobacteria family. Chemical analyses of the strain indicated production of nonribosomal oligopeptides. In fractionated extracts, masses and ion fragmentation spectra of seven possible anabaenopeptins were identified. Additionally, fragmentation spectra of cyanopeptolin-like peptides were collected in several of the fractions. The nonribosomal oligopeptide profile adds another potential identification criterion in future inter- and intraspecies comparisons of coelosphaeran cyanobacteria. The fractionated extracts showed significant activity against carboxypeptidase A and trypsin. Inhibition of these important metabolic enzymes might have impacts at the ecosystem level in aquatic habitats with high cyanobacteria densities.

Crystal structure of novel metallocarboxypeptidase inhibitor from marine mollusk Nerita versicolor in complex with human carboxypeptidase A4.

NvCI is a novel exogenous proteinaceous inhibitor of metallocarboxypeptidases from the marine snail Nerita versicolor. The complex between human carboxypeptidase A4 and NvCI has been crystallized and determined at 1.7 Å resolution. The NvCI structure defines a distinctive protein fold basically composed of a two-stranded antiparallel ß-sheet connected by three loops and the inhibitory C-terminal tail and stabilized by three disulfide bridges. NvCI is a tight-binding inhibitor that interacts with the active site of the enzyme in a substrate-like manner. NvCI displays an extended and novel interface with human carboxypeptidase A4, responsible for inhibitory constants in the picomolar range for some members of the M14A subfamily of carboxypeptidases. This makes NvCI the strongest inhibitor reported so far for this family. The structural homology displayed by the C-terminal tails of different carboxypeptidase inhibitors represents a relevant example of convergent evolution.

Discovery and synthesis of namalide reveals a new anabaenopeptin scaffold and peptidase inhibitor.

The discovery, structure elucidation, and solid-phase synthesis of namalide, a marine natural product, are described. Namalide is a cyclic tetrapeptide; its macrocycle is formed by only three amino acids, with an exocyclic ureido phenylalanine moiety at its C-terminus. The absolute configuration of namalide was established, and analogs were generated through Fmoc-based solid phase peptide synthesis. We found that only natural namalide and not its analogs containing l-Lys or l-allo-Ile inhibited carboxypeptidase A at submicromolar concentrations. In parallel, an inverse virtual screening approach aimed at identifying protein targets of namalide selected carboxypeptidase A as the third highest scoring hit. Namalide represents a new anabaenopeptin-type scaffold, and its protease inhibitory activity demonstrates that the 13-membered macrolactam can exhibit similar activity as the more common hexapeptides.

Nerve injury evoked loss of latexin expression in spinal cord neurons contributes to the development of neuropathic pain.

Nerve injury leads to sensitization mechanisms in the peripheral and central nervous system which involve transcriptional and post-transcriptional modifications in sensory nerves. To assess protein regulations in the spinal cord after injury of the sciatic nerve in the Spared Nerve Injury model (SNI) we performed a proteomic analysis using 2D-difference gel electrophoresis (DIGE) technology. Among approximately 2300 protein spots separated on each gel we detected 55 significantly regulated proteins after SNI whereof 41 were successfully identified by MALDI-TOF MS. Out of the proteins which were regulated in the DIGE analyses after SNI we focused on the carboxypeptidase A inhibitor latexin because protease dysfunctions contribute to the development of neuropathic pain. Latexin protein expression was reduced after SNI which could be confirmed by Western Blot analysis, quantitative RT-PCR and in-situ hybridisation. The decrease of latexin was associated with an increase of the activity of carboxypeptidase A indicating that the balance between latexin and carboxypeptidase A was impaired in the spinal cord after peripheral nerve injury due to a loss of latexin expression in spinal cord neurons. This may contribute to the development of cold allodynia because normalization of neuronal latexin expression in the spinal cord by AAV-mediated latexin transduction or administration of a small molecule carboxypeptidase A inhibitor significantly reduced acetone-evoked nociceptive behavior after SNI. Our results show the usefulness of proteomics as a screening tool to identify novel mechanisms of nerve injury evoked hypernociception and suggest that carboxypeptidase A inhibition might be useful to reduce cold allodynia.

The X-ray structure of carboxypeptidase A inhibited by a thiirane mechanism-based inhibitor.

The three-dimensional X-ray crystal structure of carboxypeptidase A, a zinc-dependent hydrolase, covalently modified by a mechanism-based thiirane inactivator, 2-benzyl-3,4-epithiobutanoic acid, has been solved to 1.38 A resolution. The interaction of the thiirane moiety of the inhibitor with the active site zinc ion promotes its covalent modification of Glu-270 with the attendant opening of the thiirane ring. The crystal structure determination at high resolution allowed for the clear visualization of the covalent ester bond to the glutamate side chain. The newly generated thiol from the inhibitor binds to the catalytic zinc ion in a monodentate manner, inducing a change in the zinc ion geometry and coordination, while its benzyl group fits into the S1' specificity pocket of the enzyme. The inhibitor molecule is distorted at the position of the carbon atom that is involved in the ester bond linkage on one side and the zinc coordination on the other. This particular type of thiirane-based metalloprotease inhibitor is for the first time analyzed in complex to the target protease at high resolution and may be used as a general model for zinc-dependent proteases.

Insights into the two-domain architecture of the metallocarboxypeptidase inhibitor from the Ascaris parasite inferred from the mechanism of its oxidative folding.

The metallocarboxypeptidase inhibitor identified in the intestinal parasite Ascaris (ACI) comprises 67 amino acid residues and a novel fold consisting of two structurally similar modules, an N-terminal (NTD) and a C-terminal domain (CTD), each stabilized by two disulfide bonds. Both domains are linked via a connecting segment (CS) that includes a fifth disulfide bond. Here, we investigated the oxidative folding and reductive unfolding of ACI. It folds through a sequential formation of disulfide bonds that finally leads to the accumulation of a heterogeneous population of 5-disulfide non-native scrambled isomers. The reshuffling of these species into the native form constitutes the major kinetic trap of the folding reaction, being efficiently enhanced by the presence of reducing agent or protein disulfide isomerase. The analysis of ACI variants lacking the NTD reveals that this domain is indispensable for the correct folding of such inhibitor, most likely acting as a pro-segment that helps in the acquisition of a CTD native structure, the fundamental inhibitory piece. In addition to the CTD, both the NTD and the CS play a significant role in the function of ACI, as derived from the diminished inhibitory capacity of the truncated ACI variants. Finally, the reductive unfolding and disulfide scrambling analyses reveal that ACI displays an extremely high disulfide and conformational stability, which is consistent with its physiological function in a hostile environment. Altogether, the results provide important clues about the two-domain nature of ACI and may pave the way for its further engineering and development of a minimized inhibitor.

Characterization of alpha-nitromethyl ketone as a new zinc-binding group based on structural analysis of its complex with carboxypeptidase A.

Zinc-binding groups (ZBGs) are exhaustively applied in the development of the new inhibitors against a wide variety of physiologically and pathologically important zinc proteases. Here the alpha-nitro ketone was presented as a new ZBG, which is a transition-state analog featured by the unique bifurcated hydrogen bonds at the active site of carboxypeptidase A based on the structural analysis. Introduction of a nitro group at the alpha-position of the ketone could provide more non-covalent interactions without loss of the abilities to form a tetrahedral transition-state analog.

Monocyte chemoattractant protein-1 (MCP-1), not MCP-3, is the primary chemokine required for monocyte recruitment in mouse peritonitis induced with thioglycollate or zymosan A.

MCP-1/CCL2 plays a critical role in monocyte recruitment into sites of immune responses and cancer. However, the role of other MCPs remains unclear. In this study, we generated a novel MCP-1-deficient (designated as MCP-1(Delta/Delta)) mouse model by deleting a 2.3-kb DNA fragment from the mouse genome using the Cre/loxP system. MCP-1 was not produced by LPS-activated MCP-1(Delta/Delta) macrophages; however, the production of MCP-3, coded by the immediate downstream gene, was significantly increased. In contrast, macrophages from another mouse line with a neo-gene cassette in intron 2 produced a significantly lower level of MCP-1 and MCP-3. Decreased MCP-3 production was also detected in previously generated MCP-1-deficient mice in which a neo-gene cassette was inserted in exon 2 (designated as MCP-1 knockout (KO)). Altered MCP-1 and/or MCP-3 production was also observed in vivo in each mouse model in response to i.p. injection of thioglycolate or zymosan. The up- and down-regulation of MCP-3 production in MCP-1(Delta/Delta) and MCP-1 KO mice, respectively, provided us with a unique opportunity to evaluate the role for MCP-3. Despite the increased MCP-3 production in MCP-1(Delta/Delta) mice, thioglycolate- or zymosan-induced monocyte/macrophage accumulation was still reduced by approximately 50% compared with wild-type mice, similar to the reduction detected in MCP-1 KO mice. Thus, up-regulated MCP-3 production did not compensate for the loss of MCP-1, and MCP-3 appears to be a less effective mediator of monocyte recruitment than MCP-1. Our results also indicate the presence of other mediators regulating the recruitment of monocytes in these models.

A new type of five-membered heterocyclic inhibitors of basic metallocarboxypeptidases.

A structure-based virtual screening survey was used to identify potential inhibitors of the human M14 family of metallocarboxypeptidases. A good correlation between docking energy scores and measured K(i) values was observed, indicating an efficient performance of the screening procedure. Among various compounds displaying K(i) values in the low micromolar range, N-(3-chlorophenyl)-4-((5-(3-methoxybenzylthio)-1,3,4-oxadiazol-2-yl)methyl)thiazol-2-amine emerged as the most powerful inhibitor for human carboxypeptidase B (CPB). According to molecular docking, this compound fits into CPB active site cleft through coordination of the catalytic zinc ion with the 1,3,4-oxadiazole moiety. This represents a novel five-membered heterocyclic type of inhibitor for disease-linked metallocarboxypeptidases and an interesting lead for further development.

Synthesis and structure-activity correlation of a brunsvicamide-inspired cyclopeptide collection.

Cyanobacterial cyclopeptides: A series of analogues of the cyanobacterial cyclopeptide brunsvicamide A was prepared by effective solid-support-based total synthesis. Variations in stereochemistry revealed the importance of the D-lysine and the L-isoleucine residues for the substrate-competitive inhibitory activity of brunsvicamide A against carboxypeptidase A. The brunsvicamides are modified cyclopeptides from cyanobacteria, cyclised through the epsilon-amino group of a D-lysine unit. They are functionalised with urea groups and show potent carboxypeptidase inhibitory activities. In order to unravel the structural parameters that determine their activities, a collection of brunsvicamide analogues with varied amino acid structures and stereochemistries was synthesised by a combined solution- and solid-phase approach. Biochemical investigation of the compound collection for carboxypeptidase A inhibition revealed that the presence of D-lysine and L-isoleucine in the urea section is important for inhibition. It was found that brunsvicamide A is a substrate-competitive inhibitor of carboxypeptidase A. These findings are in agreement with the substrate specificity of the enzyme and were rationalised by computational studies, which showed the high relevance of the lysine stereochemistry for inhibitory activity.

Distinct roles for angiotensin-converting enzyme 2 and carboxypeptidase A in the processing of angiotensins within the murine heart.

Angiotensin-converting enzyme 2 (ACE2), a homologue of angiotensin-converting enzyme (ACE), converts angiotensin (Ang) I to Ang(1-9) and Ang II to Ang(1-7), but does not directly process Ang I to Ang II. Cardiac function is compromised in ACE2 null mice; however, the importance of ACE2 in the processing of angiotensin peptides within the murine heart is not known. We determined the metabolism of angiotensins in wild-type (WT), ACE (ACE(-/-)) and ACE2 null mice (ACE2(-/-)). Angiotensin II was converted almost exclusively to Ang(1-7) in the cardiac membranes of WT and ACE(-/-) strains, although generation of Ang(1-7) was greater in the ACE(-/-) mice (27.4 +/- 4.1 versus 17.5 +/- 3.2 nmol(-1) mg h(-1) for WT). The ACE2 inhibitor MLN4760 significantly attenuated Ang II metabolism and the subsequent formation of Ang(1-7) in both strains. In the ACE2(-/-) hearts, Ang II metabolism and the generation of Ang(1-7) were significantly attenuated; however, the ACE2 inhibitor reduced the residual Ang(1-7)-forming activity in this strain. Angiotensin I was primarily converted to Ang(1-9) (WT, 28.9 +/- 3.1 nmol(-1) mg h(-1); ACE(-/-), 49.8 +/- 5.3 nmol(-1) mg h(-1); and ACE2(-/-), 35.9 +/- 5.4 nmol(-1) mg h(-1)) and to smaller quantities of Ang(1-7) and Ang II. Although the ACE2 inhibitor had no effect on Ang(1-9) formation, the carboxypeptidase A inhibitor benzylsuccinate essentially abolished the formation of Ang(1-9) and increased the levels of Ang I in cardiac membranes. In conclusion, our studies in the murine heart suggest that ACE2 is the primary pathway for the metabolism of Ang II and the subsequent formation of Ang(1-7), a peptide that, in contrast to Ang II, exhibits both antifibrotic and antiproliferative actions.

DNA-encoded chemical libraries for the discovery of MMP-3 inhibitors.

Encoded self-assembling chemical (ESAC) libraries are characterized by the covalent display of chemical moieties at the extremity of self-assembling oligonucleotides carrying a unique DNA sequence for the identification of the corresponding chemical moiety. We have used ESAC library technology in a two-step selection procedure for the identification of novel inhibitors of stromelysin-1 (MMP-3), a matrix metalloproteinase involved in both physiological and pathological tissue remodeling processes, yielding novel inhibitors with micromolar potency.

Chitosan citrate as multifunctional polymer for vaginal delivery. Evaluation of penetration enhancement and peptidase inhibition properties.

In the present work the employment of chitosan citrate (Chs citrate) as multifunctional polymer in vaginal applications was evaluated. Potential properties of penetration enhancement and protease inhibition could be expected because of the capability of citrate to bind divalent cations such as calcium, that is involved in the regulation of gap and tight junctions, and zinc, that is essential co-factor for some proteases. A comparison was performed with chitosan HCl (Chs HCl). Ex vivo drug permeation experiments were performed on pig vaginal mucosa, by application of 3.0% (w/w) chitosan gels. Acyclovir (5.0%, w/w) and ciprofloxacin HCl (0.3%, w/w) were used as low molecular weight model drugs. Fluorescein isothiocyanate dextran MW 4400 (FD4) was used as hydrophilic high molecular weight fluorescent probe (0.2%, w/w). In the case of low MW drugs the amount penetrated into pig vaginal mucosa was measured by extraction from tissue slices and HPLC detection. From the samples maintained in contact with FD4, slices were cut perpendicularly to the surface and observed by means of confocal laser scanning microscopy (CLSM). FD4 permeation was also measured in in-vitro cell culture model (Caco-2). The penetration enhancing capacity of Chs citrate was comparable to that of Chs HCl. Both Chs citrate and Chs HCl were tested for the inhibition of the proteolytic enzymes carboxypeptidase A and leucine aminopeptidase. In both cases Chs citrate showed a significantly higher inhibition of enzymatic activity with respect to Chs HCl.

Nitro as a novel zinc-binding group in the inhibition of carboxypeptidase A.

2-Substituted 3-nitropropanoic acids were designed and synthesized as inhibitors against carboxypeptidase A (CPA). (R)-2-Benzyl- 3-nitropropanoic acid showed a potent inhibition against CPA (K(i)=0.15 microM). X-ray crystallography discloses that the nitro group well mimics the transition state occurred in the hydrolysis catalyzed by CPA, that is, an O,O'-bidentate coordination to the zinc ion and the two respective hydrogen bonds with Glu-270 and Arg-127. Because the nitro group is a planar species, we proposed (R)-2-benzyl-3-nitropropanoic acid as a pseudo-transition-state analog inhibitor against CPA.

Evaluating protein:protein complex formation using synchrotron radiation circular dichroism spectroscopy.

Circular dichroism (CD) spectroscopy beamlines at synchrotrons produce dramatically higher light flux than conventional CD instruments. This property of synchrotron radiation circular dichroism (SRCD) results in improved signal-to-noise ratios and allows data collection to lower wavelengths, characteristics that have led to the development of novel SRCD applications. Here we describe the use of SRCD to study protein complex formation, specifically evaluating the complex formed between carboxypeptidase A and its protein inhibitor latexin. Crystal structure analyses of this complex and the individual proteins reveal only minor changes in secondary structure of either protein upon complex formation (i.e., it involves only rigid body interactions). Conventional CD spectroscopy reports on changes in secondary structure and would therefore not be expected to be sensitive to such interactions. However, in this study we have shown that SRCD can identify differences in the vacuum ultraviolet CD spectra that are significant and attributable to complex formation.