Hemopexin Domain Research Articles

Abstract 4473: Targeting matrix metalloproteinase-3 (MMP-3) in high-grade serous ovarian cancer

Abstract High-Grade Serous Ovarian Cancer (HGSOC) is the most aggressive and lethal gynecological malignancy. Surgery and chemotherapy (with platinum compounds) are the main treatments. Many patients relapse and develop chemotherapy resistance, limiting the therapeutic utility of these therapies. This persistent resistance makes HGSOC treatment difficult. Cisplatin-resistant ovarian cancer cells upregulate Matrix Metalloproteinase-3. MMP3 is an enzyme involved in active tissue remodeling and extracellular matrix protein breakdown during dynamic physiological processes. The structure of MMP3 includes the prodomain, the catalytic domain and the hemopexin domain. We showed that MMP3 protein levels are higher in cisplatin resistant as compared with cisplatin sensitive HGSOC cells. Our research team showed that small interference RNA (siRNA)-mediated MMP3 knockdown reduced cisplatin-resistant ovarian cancer cell growth and invasion. This data indicates that MMP3 may be a promising HGSOC treatment target. Targeting MMP3 with small chemical inhibitors (SCI) could represent a better option than siRNA because SCI are more stable and easier to deliver. The purpose of this project is to design SCI of MMP3 in the propeptide, catalytic, and hemopexin domains of MMP3. We used in silico methods to identify MMP3 inhibitors in these domains. The ZINC20 database of commercially compounds was used for virtual screening using Python Prescription (PyRx) software. We identified 25 ligands out of 370,000 with the greatest affinity for each MMP3 functional domains. SwissADME was then used to evaluate in silico absorption, distribution, metabolism, pharmacokinetics, drug-likeliness, and medicinal chemistry. This research lays the groundwork for HGSOC therapeutic strategies to improve treatment efficacy, especially for chemotherapy-resistant patients. Citation Format: Víctor G. Reyes-Burgos, Eliud Hernández-O'Farrill, Luis E. Vazquez Quiñones, Marienid Flores-Colón, Fatima Valiyeva, Pablo E. Vivas-Mejía. Targeting matrix metalloproteinase-3 (MMP-3) in high-grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4473.

Binding and Activation of LRP1-Dependent Cell Signaling in Schwann Cells Using a Peptide Derived from the Hemopexin Domain of MMP-9.

Schwann cells (SCs) undergo phenotypic transformation and then orchestrate nerve repair following a peripheral nervous system injury. The low-density lipoprotein receptor-related protein-1 (LRP1) is significantly upregulated in SCs in response to acute injury, activating cJun and promoting SC survival. Matrix-metalloproteinase-9 (MMP-9) is an LRP1 ligand that binds LRP1 through its hemopexin domain (PEX) and activates SC survival signaling and migration. To identify novel peptide mimetics within the hemopexin domain of MMP-9, we examined the crystal structure of PEX, synthesized four peptides, and examined their potential to bind and activate LRP1. We demonstrate that a 22 amino acid peptide, peptide 2, was the only peptide that activated Akt and ERK1/2 signaling in SCs, similar to a glutathione s-transferase (GST)-fused holoprotein, GST-PEX. Intraneural injection of peptide 2, but not vehicle, into crush-injured sciatic nerves activated cJun greater than 2.5-fold in wild-type mice, supporting that peptide 2 can activate the SC repair signaling in vivo. Peptide 2 also bound to Fc-fusion proteins containing the ligand-binding motifs of LRP1, clusters of complement-like repeats (CCRII and CCRIV). Pulldown and computational studies of alanine mutants of peptide 2 showed that positively charged lysine and arginine amino acids within the peptide are critical for stability and binding to CCRII. Collectively, these studies demonstrate that a novel peptide derived from PEX can serve as an LRP1 agonist and possesses qualities previously associated with LRP1 binding and SC signaling in vitro and in vivo.

MMP14high macrophages orchestrate progressive pulmonary fibrosis in SR-Ag-induced hypersensitivity pneumonitis

Fibrotic hypersensitivity pneumonitis (FHP) is a fatal interstitial pulmonary disease with limited treatment options. Lung macrophages are a heterogeneous cell population that exhibit distinct subsets with divergent functions, playing pivotal roles in the progression of pulmonary fibrosis. However, the specific macrophage subpopulations and underlying mechanisms involved in the disease remain largely unexplored. In this study, a decision tree model showed that matrix metalloproteinase-14 (MMP14) had higher scores for important features in the up-regulated genes in macrophages from mice exposed to the Saccharopolyspora rectivirgula antigen (SR-Ag). Using single-cell RNA sequencing (scRNA-seq) analysis of hypersensitivity pneumonitis (HP) mice profiles, we identified MMP14high macrophage subcluster with a predominant M2 phenotype that exhibited higher activity in promoting fibroblast-to myofibroblast transition (FMT). We demonstrated that suppressing toll-like receptor 2 (TLR2) and nuclear factor kappa-B (NF-κB) could attenuate MMP14 expression and exosome secretion in macrophages stimulation with SR-Ag. The exosomes derived from MMP14-overexpressing macrophages were found to be more effective in regulating the transition of fibroblasts through exosomal MMP14. Importantly, it was observed that the transfer of MMP14-overexpressing macrophages into mice promoted lung inflammation and fibrosis induced by SR-Ag. NSC-405020 binding to the hemopexin domain (PEX) of MMP-14 ameliorated lung inflammation and fibrosis induced by SR-Ag in mice. Thus, MMP14-overexpressing macrophages may be an important mechanism contributing to the exacerbation of allergic reactions. Our results indicated that MMP14 in macrophages has the potential to be a therapeutic target for HP.

Degradomic Identification of Membrane Type 1-Matrix Metalloproteinase as an ADAMTS9 and ADAMTS20 Substrate

The secreted metalloproteases ADAMTS9 and ADAMTS20 are implicated in extracellular matrix proteolysis and primary cilium biogenesis. Here, we show that clonal gene-edited RPE-1 cells in which ADAMTS9 was inactivated, and which constitutively lack ADAMTS20 expression, have morphologic characteristics distinct from parental RPE-1 cells. To investigate underlying proteolytic mechanisms, a quantitative terminomics method, terminal amine isotopic labeling of substrates was used to compare the parental and gene-edited RPE-1 cells and their medium to identify ADAMTS9 substrates. Among differentially abundant neo-amino (N) terminal peptides arising from secreted and transmembrane proteins, a peptide with lower abundance in the medium of gene-edited cells suggested cleavage at the Tyr314-Gly315 bond in the ectodomain of the transmembrane metalloprotease membrane type 1-matrix metalloproteinase (MT1-MMP), whose mRNA was also reduced in gene-edited cells. This cleavage, occurring in the MT1-MMP hinge, that is, between the catalytic and hemopexin domains, was orthogonally validated both by lack of an MT1-MMP catalytic domain fragment in the medium of gene-edited cells and restoration of its release from the cell surface by reexpression of ADAMTS9 and ADAMTS20 and was dependent on hinge O-glycosylation. A C-terminally semitryptic MT1-MMP peptide with greater abundance in WT RPE-1 medium identified a second ADAMTS9 cleavage site in the MT1-MMP hemopexin domain. Consistent with greater retention of MT1-MMP on the surface of gene-edited cells, pro-MMP2 activation, which requires cell surface MT1-MMP, was increased. MT1-MMP knockdown in gene-edited ADAMTS9/20-deficient cells restored focal adhesions but not ciliogenesis. The findings expand the web of interacting proteases at the cell surface, suggest a role for ADAMTS9 and ADAMTS20 in regulating cell surface activity of MT1-MMP, and indicate that MT1-MMP shedding does not underlie their observed requirement in ciliogenesis.

Abstract 5823: Uncovering the mechanisms of matrix metalloproteinase-3 (MMP-3) in the cisplatin resistance of ovarian cancer

Abstract Seventy percent of women with ovarian cancer will develop chemotherapy-resistance to cisplatin. Regardless of the advancements in technology, mortality rates for ovarian cancer have declined only slightly in the last forty years, reiterating the need to generate novel treatment options targeting cisplatin resistance. In RNA-seq and microarrays experiments we observed that Matrix-metalloproteinase-3 (MMP3) was highly abundant in cisplatin-resistant when compared to cisplatin-sensitive cells. MMP3 is a member of the matrix metalloproteinases (MMPs) family of proteolytic enzymes, involved in the degradation of proteins in the extracellular matrix (ECM). Deregulation of MMPs alters the extracellular matrix and contributes to proliferation, invasion, and metastasis of cancerous cells. A typical MMP structure consists of a pro-domain, a catalytic, and a hemopexin domain. The catalytic domain is responsible for MMPs proteolytic activity, while the hemopexin domain contributes to substrate and ligand specificity. Inhibitors targeting the catalytic domain of MMPs were unsuccessful in clinical trials due to a lack of selectivity and specificity. When we used a chemical inhibitor, we did not observe reduced cell-proliferation or invasiveness. However, MMP3-targeted small interference RNA (siRNA) was able to reduce cell proliferation and invasive capabilities in cisplatin resistant ovarian cancer cells. Additionally, MMP3-targeted siRNA significantly reduced the sensitivity of ovarian cancer cells to cisplatin treatment. Cisplatin also increased MMP3 mRNA levels in ovarian cancer mice models and human cell lines. This evidence suggests that the MMP3 levels correlate with the sensitivity of ovarian cancer cells to cisplatin treatment. We hypothesize that the hemopexin domain will bind to proteins promoting cisplatin-resistance. We validated our expression profile studies at mRNA and protein levels via western blot, ELISA and qRT-PCR. Additionally, survival analysis using the Kaplan-Meier plotter database showed that ovarian cancer patients with low MMP3 levels live longer than patients with higher MMP3 levels. Furthermore, when we measured MMP3 activity, we observed decreased activity in cisplatin-resistant versus cisplatin-sensitive cells. Taken together, these data suggest that other domains of MMP3 (i.e., hemopexin domain) may have a function in the cisplatin resistance of ovarian cancer cells. Citation Format: Mariela Rivera-Serrano, Pablo E. Vivas-Mejia. Uncovering the mechanisms of matrix metalloproteinase-3 (MMP-3) in the cisplatin resistance of ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5823.

Detecting the PEX Like Domain of Matrix Metalloproteinase-14 (MMP-14) with Therapeutic Conjugated CNTs.

Matrix metalloproteinases (MMPs) are essential proteins acting directly in the breakdown of the extra cellular matrix and so in cancer invasion and metastasis. Given its impact on tumor angiogenesis, monitoring MMP-14 provides strategic insights on cancer severity and treatment. In this work, we report a new approach to improve the electrochemical interaction of the MMP-14 with the electrode surface while preserving high specificity. This is based on the detection of the hemopexin (PEX) domain of MMP-14, which has a greater availability with a stable and low-cost commercial molecule, as a recognition element. This molecule, called NSC-405020, is specific of the PEX domain of MMP-14 within the binding pocket. Through the covalent grafting of the NSC-405020 molecule on carbon nanotubes (CNTs), we were able to detect and quantify MMP-14 using electrochemical impedance spectroscopy with a linear range of detection of 10 ng⋅mL−1 to 100 ng⋅mL−1, and LOD of 7.5 ng⋅mL−1. The specificity of the inhibitory small molecule was validated against the PEX domain of MMP-1. The inhibitor loaded CNTs system showed as a desirable candidate to become an alternative to the conventional recognition bioelements for the detection of MMP-14.

Identification of amino acid residues in the MT-loop of MT1-MMP critical for its ability to cleave low-density lipoprotein receptor

Low-density lipoprotein receptor (LDLR) mediates clearance of plasma LDL cholesterol, preventing the development of atherosclerosis. We previously demonstrated that membrane type 1-matrix metalloproteinase (MT1-MMP) cleaves LDLR and exacerbates the development of atherosclerosis. Here, we investigated determinants in LDLR and MT1-MMP that were critical for MT1-MMP-induced LDLR cleavage. We observed that deletion of various functional domains in LDLR or removal of each of the five predicted cleavage sites of MT1-MMP on LDLR did not affect MT1-MMP-induced cleavage of the receptor. Removal of the hemopexin domain or the C-terminal cytoplasmic tail of MT1-MMP also did not impair its ability to cleave LDLR. On the other hand, mutant MT1-MMP, in which the catalytic domain or the MT-loop was deleted, could not cleave LDLR. Further Ala-scanning analysis revealed an important role for Ile at position 167 of the MT-loop in MT1-MMP’s action on LDLR. Replacement of Ile167 with Ala, Thr, Glu, or Lys resulted in a marked loss of the ability to cleave LDLR, whereas mutation of Ile167 to a non-polar amino acid residue, including Leu, Val, Met, and Phe, had no effect. Therefore, our studies indicate that MT1-MMP does not require a specific cleavage site on LDLR. In contrast, an amino acid residue with a hydrophobic side chain at position 167 in the MT-loop is critical for MT1-MMP-induced LDLR cleavage.

The Catalytic Domain Mediates Homomultimerization of MT1-MMP and the Prodomain Interferes with MT1-MMP Oligomeric Complex Assembly.

Homomultimerization of MT1-MMP (membrane type 1 matrix metalloproteinase) through the hemopexin, transmembrane, and cytoplasmic domains plays a very important role in the activation of proMMP-2 and the degradation of pericellular collagen. MT1-MMP is overexpressed in many types of cancers, and it is considered to be a key enzyme in facilitating cancer cell migration. Since the oligomerization of MT1-MMP is important for its proteolytic activity in promoting cancer invasion, we have further investigated the multimerization by using heterologously expressed MT1-MMP ectodomains in insect cells to gain additional mechanistic insight into this process. We show that the whole ectodomain of MT1-MMP can form dimers and higher-order oligomeric complexes. The enzyme is secreted in its active form and the multimeric complex assembly is mediated by the catalytic domain. Blocking the prodomain removal determines the enzyme to adopt the monomeric structure, suggesting that the prodomain prevents the MT1-MMP oligomerization process. The binding affinity of MT1-MMP to type I collagen is dependent on the oligomeric state. Thus, the monomers have the weakest affinity, while the binding strength increases proportionally with the complexity of the multimers. Collectively, our experimental results indicate that the catalytic domain of MT1-MMP is necessary and sufficient to mediate the formation of multimeric structures.

Cloning of Metalloproteinase 17 Genes from Oriental Giant Jellyfish Nemopilema nomurai (Scyphozoa: Rhizostomeae).

We previously demonstrated that Nemopilema nomurai jellyfish venom metalloproteinases (JVMPs) play a key role in the toxicities induced by N. nomurai venom (NnV), including dermotoxicity, cytotoxicity, and lethality. In this study, we identified two full-length JVMP cDNA and genomic DNA sequences: JVMP17-1 and JVMP17-2. The full-length cDNA of JVMP17-1 and 17-2 contains 1614 and 1578 nucleotides (nt) that encode 536 and 525 amino acids, respectively. Putative peptidoglycan (PG) binding, zinc-dependent metalloproteinase, and hemopexin domains were identified. BLAST analysis of JVMP17-1 showed 42, 41, 37, and 37% identity with Hydra vulgaris, Acropora digitifera, Megachile rotundata, and Apis mellifera venom metalloproteinases, respectively. JVMP17-2 shared 38 and 36% identity with H. vulgaris and A. digitifera, respectively. Alignment results of JVMP17-1 and 17-2 with other metalloproteinases suggest that the PG domain, the tissue inhibitor of metalloproteinase (TIMP)-binding surfaces, active sites, and metal (ion)-binding sites are highly conserved. The present study reports the gene cloning of metalloproteinase enzymes from jellyfish species for the first time. We hope these results can expand our knowledge of metalloproteinase components and their roles in the pathogenesis of jellyfish envenomation.

Inhibiting ADAM10‐MMP14 Complex Blocks Cystogenesis of Kidney Epithelial Cells

BackgroundPolycystic kidney disease (PKD) is the most common genetic kidney disorder. It is characterized by the aberrant renal tubule epithelial cells proliferation leads to the formation of multiple fluid‐filled cysts, and it causes renal failure. Currently, kidney transplant or dialysis is the only effective treatment. A disintegrin‐like metalloproteinase 10 (ADAM10) and matrix metalloproteinase 14 (MMP14) are two major members in the superfamily of metalloproteinases that primarily cleave several cell membrane proteins and growth factors. Inhibition of ADAM10 activity reduces cystic growth of renal epithelial cells, and inhibition of MMP14 also reduces the number of cysts in the kidneys of rats with PKD. In our study, we propose to elucidate the unclear relationship between ADAM10 and MMP14 in the development of cystogenesis in kidney.MethodsMadin‐Darby Canine Kidney (MDCK) cells with stable expression of MMP14 and its mutants were developed and used in our study. MTT proliferation assay was used to measure cell growth. Immunoprecipitation and Western blots were used to determine physical association and proteinase activity. 3‐dimensional (3D) culture of MDCK cells was used to examine cell growth patterns.ResultsWe found that a unique ADAM10‐MMP14 complex forms in cell membrane. The hemopexin domain of MMP14 is essential for its association with ADAM10. Their interaction causes differential cleavage of cell membrane proteins, E‐cadherin and L1 cell adhesion molecule protein (L1CAM). In MDCK cells, only ADAM10 cleaves E‐cadherin but its cleavage is dependent on the active form of MMP14. In addition, both ADAM10 and MMP14 cleave L1CAM but inactive MMP14 blocks the ADAM10 cleavage of L1CAM. We tested the effects of ADAM10 and MMP14 interaction on cell proliferation and cystic growth. Ectopic expression of MMP14 increases cell proliferation and cystic growth of MDCK cells in a 3D culture matrix. Further, use of MMP14‐specific inhibitor NSC405020 or ectopic expression of a dominant‐negative form of MMP14 mutant also results in proper contact inhibition, and further promotes tubular growth of MDCK cells in 3D culture. Inhibition of ADAM10 with siRNA or enzymatic inhibitor GI254023X significantly reduces cell proliferation and promotes tubular growth. Combined use of GI254023X and NSC405020 results in a 73.4 ±8.23% decrease in cell proliferation compared to GI254023X, and 68.6 ± 6.84% decrease compared to NSC405020 alone. In 3D culture, MDCK‐MMP14 (ectopic expression of MMP14) cells form cystic growth only, but addition of both ADAM10 and MMP14 inhibitors leads to tubular growth only.ConclusionOur data identified the solid physical association of ADAM10 and MMP14. Their functional interaction causes differential cleavage of cell membrane proteins, E‐cadherin and L1CAM. Inhibition of ADAM10‐MMP14 complex blocks proliferation and the cystogenesis of of kidney epithelial cells. Individual ADAM10 or MMP14 inhibitors have been used in clinical trials for certain tumors, however, they have never been used together nor have been used in PKD treatments. Our results suggest the combination of ADAM10 and MMP14 inhibitors could be served as a novel effective therapeutic strategy of PKD.

Identification of Novel Natural Product Inhibitors against Matrix Metalloproteinase 9 Using Quantum Mechanical Fragment Molecular Orbital-Based Virtual Screening Methods.

Matrix metalloproteinases (MMPs) are calcium-dependent zinc-containing endopeptidases involved in multiple cellular processes. Among the MMP isoforms, MMP-9 regulates cancer invasion, rheumatoid arthritis, and osteoarthritis by degrading extracellular matrix proteins present in the tumor microenvironment and cartilage and promoting angiogenesis. Here, we identified two potent natural product inhibitors of the non-catalytic hemopexin domain of MMP-9 using a novel quantum mechanical fragment molecular orbital (FMO)-based virtual screening workflow. The workflow integrates qualitative pharmacophore modeling, quantitative binding affinity prediction, and a raw material search of natural product inhibitors with the BMDMS-NP library. In binding affinity prediction, we made a scoring function with the FMO method and applied the function to two protein targets (acetylcholinesterase and fibroblast growth factor 1 receptor) from DUD-E benchmark sets. In the two targets, the FMO method outperformed the Glide docking score and MM/PBSA methods. By applying this workflow to MMP-9, we proposed two potent natural product inhibitors (laetanine 9 and genkwanin 10) that interact with hotspot residues of the hemopexin domain of MMP-9. Laetanine 9 and genkwanin 10 bind to MMP-9 with a dissociation constant (KD) of 21.6 and 0.614 μM, respectively. Overall, we present laetanine 9 and genkwanin 10 for MMP-9 and demonstrate that the novel FMO-based workflow with a quantum mechanical approach is promising to discover potent natural product inhibitors of MMP-9, satisfying the pharmacophore model and good binding affinity.

Identification of allosteric fingerprints of alpha-synuclein aggregates in matrix metalloprotease-1 and substrate-specific virtual screening with single molecule insights

Alpha-synuclein (aSyn) has implications in pathological protein aggregations in neurodegeneration. Matrix metalloproteases (MMPs) are broad-spectrum proteases and cleave aSyn, leading to aggregation. Previous reports showed that allosteric communications between the two domains of MMP1 on collagen fibril and fibrin depend on substrates, activity, and ligands. This paper reports quantification of allostery using single molecule measurements of MMP1 dynamics on aSyn-induced aggregates by calculating Forster Resonance Energy Transfer (FRET) between two dyes attached to the catalytic and hemopexin domains of MMP1. The two domains of MMP1 prefer open conformations that are inhibited by a single point mutation E219Q of MMP1 and tetracycline, an MMP inhibitor. A two-state Poisson process describes the interdomain dynamics, where the two states and kinetic rates of interconversion between them are obtained from histograms and autocorrelations of FRET values. Since a crystal structure of aSyn-bound MMP1 is unavailable, binding poses were predicted by molecular docking of MMP1 with aSyn using ClusPro. MMP1 dynamics were simulated using predicted binding poses and compared with the experimental interdomain dynamics to identify an appropriate pose. The selected aSyn-MMP1 binding pose near aSyn residue K45 was simulated and analyzed to define conformational changes at the catalytic site. Allosteric residues in aSyn-bound MMP1 exhibiting strong correlations with the catalytic motif residues were compared with allosteric residues in free MMP1, and aSyn-specific residues were identified. The allosteric residues in aSyn-bound MMP1 are K281, T283, G292, G327, L328, E329, R337, F343, G345, N346, Y348, G353, Q354, D363, Y365, S366, S367, F368, P371, R372, V374, K375, A379, F391, A394, R399, M414, F419, V426, and C466. Shannon entropy was defined to quantify MMP1 dynamics. Virtual screening was performed against a site on selected aSyn-MMP1 binding poses, which showed that lead molecules differ between free MMP1 and substrate-bound MMP1. Also, identifying aSyn-specific allosteric residues in MMP1 enabled further selection of lead molecules. In other words, virtual screening needs to take substrates into account for potential substrate-specific control of MMP1 activity in the future. Molecular understanding of interactions between MMP1 and aSyn-induced aggregates may open up the possibility of degrading aggregates by targeting MMPs.

Inhibitors of gelatinases (MMP-2 and MMP-9) for the management of hematological malignancies.

Matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) are collectively known as gelatinases whereas MMP-2 is gelatinase-A and MMP-9 is termed as gelatinase-B. Gelatinases and other matrix metalloproteinases (MMPs) have long been associated with solid tumor invasion, metastasis and angiogenesis. However, there is paucity of data available regarding the role of gelatinases in hematological malignancies. Recent studies have shown that gelatinases activities or functions are correlated with hematological malignancies. Strategies for designing more specific gelatinase inhibitors like catalytic (CAT) domain inhibitors and hemopexin (PEX) domain inhibitors as well as signaling pathway based or gelatinase expression inhibitors had been reported against hematologic malignant cells. Several substrate based non-selective to non-substrate based relatively selective synthetic matrix metalloproteinase inhibitors (MMPIs) had been developed. Few MMPIs had reached in clinical trials during the period of 1990s-2000s. Unfortunately the anti-tumor and anti-metastatic efficacies of these MMPIs were not justified with patients having several advanced stage solid tumor cancers in any substantial number of clinical trials. Till date not a single MMPI passed phase III clinical trials designed for advanced metastatic cancers due to adverse events as well as lack of ability to show uniformity in disease prolongation. With the best of our knowledge no clinical trial study has been reported with small molecule synthetic inhibitors against hematological malignancies. This review looks at the outcome of clinical trials of MMPIs for advanced stage solid tumors. This can therefore, act as a learning experience for future development of successful gelatinase inhibitors for the management of hematological malignancies.

PsEND1 Is a Key Player in Pea Pollen Development Through the Modulation of Redox Homeostasis.

Redox homeostasis has been linked to proper anther and pollen development. Accordingly, plant cells have developed several Reactive Oxygen Species (ROS)-scavenging mechanisms to maintain the redox balance. Hemopexins constitute one of these mechanisms preventing heme-associated oxidative stress in animals, fungi, and plants. Pisum sativum ENDOTHECIUM 1 (PsEND1) is a pea anther-specific gene that encodes a protein containing four hemopexin domains. We report the functional characterization of PsEND1 and the identification in its promoter region of cis-regulatory elements that are essential for the specific expression in anthers. PsEND1 promoter deletion analysis revealed that a putative CArG-like regulatory motif is necessary to confer promoter activity in developing anthers. Our data suggest that PsEND1 might be a hemopexin regulated by a MADS-box protein. PsEND1 gene silencing in pea, and its overexpression in heterologous systems, result in similar defects in the anthers consisting of precocious tapetum degradation and the impairment of pollen development. Such alterations were associated to the production of superoxide anion and altered activity of ROS-scavenging enzymes. Our findings demonstrate that PsEND1 is essential for pollen development by modulating ROS levels during the differentiation of the anther tissues surrounding the microsporocytes.

Sperm MMP-2 is indispensable for fast electrical block to polyspermy at fertilization in Xenopus tropicalis.

Sperm matrix metalloproteinase-2 (MMP-2) is necessary for frog fertilization. Monospermy is ensured by a fast, electrical block to polyspermy mediated by a positive fertilization potential. To determine the role of the MMP-2 hemopexin domain (HPX) in a fast block to polyspermy during fertilization of the frog, Xenopus tropicalis, we prepared mutant frogs deficient in mmp2 gene using the transcription activator-like effector nuclease method. mmp2 ΔHPX (-/-) sperm without MMP-2 protein were able to fertilize wild-type (WT; +/+) eggs. However, polyspermy occurred in some eggs. The mutant sperm generated a normal fertilization potential amounting to 10 mV, and were able to fertilize eggs at 10 mV, at which WT sperm never fertilized. Sensitivity during voltage-dependent fertilization decreased in mutant sperm. This study demonstrates for the first time that the genetic alteration of the MMP-2 molecule in sperm causes polyspermy during fertilization of a monospermic species. Our findings provide reliable evidence that sperm MMP-2 is indispensable for the fast, electrical block to polyspermy during Xenopus fertilization.

Matrix metalloproteinase contribution in management of cancer proliferation, metastasis and drug targeting.

Matrix metalloproteinases (MMPs) or matrixins, are members of a zinc-dependent endopeptidase family. They cause remodeling of the extracellular matrix (ECM) leading to numerous diseases. MMPs subfamilies possess: collagenases, gelatinases, stromelysins and membrane-type MMPs (MT-MMP). They consist of several domains; pro-peptide, catalytic, linker peptide and the hemopexin (Hpx) domains. MMPs are involved in initiation, proliferation and metastasis of cancer through the breakdown of ECM physical barriers. Overexpression of MMPs is associated with poor prognosis of cancer. This review will discuss both types of MMPs and current inhibitors, which target them in different aspects, including, biosynthesis, activation, secretion and catalytic activity. Several synthetic and natural inhibitors of MMPs (MMPIs) that can bind the catalytic domain of MMPs have been designed including; peptidomimetic, non-peptidomimetic, tetracycline derivatives, off-target MMPI, natural products, microRNAs and monoclonal antibodies.

Structural and functional role of invariant water molecules in matrix metalloproteinases: a data-mining approach

Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases known to degrade extracellular matrix (ECM). Being involved in many biological and physiological processes of tissue remodeling, MMPs play a crucial role in many pathological conditions such as arthritis, cancer, cardiovascular diseases, etc. Typically, MMPs possess a propeptide, a zinc-containing catalytic domain, a hinge region and a hemopexin domain. Based on their structural domain organization and substrates, MMPs are classified into six different classes, viz. collagenases, stromelysins, gelatinases, matrilysins, membrane-type and other MMPs. As per previous studies, a set of invariant water (IW) molecules of MMP-1 (a collagenase) play a significant role in stabilizing their catalytic domain. However, a functional role of IW molecule in other classes of MMPs has not been reported yet. Thus, in this study, IW molecules of MMPs from different classes were located and their plausible role(s) have been assigned. The results suggest that IW molecules anchor the structurally and functionally essential metal ions present in the vicinity of the active site of MMPs. Further, they (in)directly interlink different structural features and bridge the active site metal ions of MMPs. This study provides the key IW molecules that are structurally and functionally relevant to MMPs and hence, in turn, might facilitate the development of potent generalized inhibitor(s) against different classes of MMPs. Communicated by Ramaswamy H. Sarma

Simultaneous targeting of CD44 and MMP9 catalytic and hemopexin domains as a therapeutic strategy.

Crosstalk of the oncogenic matrix metalloproteinase-9 (MMP9) and one of its ligands, CD44, involves cleavage of CD44 by the MMP9 catalytic domain, with the CD44–MMP9 interaction on the cell surface taking place through the MMP9 hemopexin domain (PEX). This interaction promotes cancer cell migration and invasiveness. In concert, MMP9-processed CD44 induces the expression of MMP9, which degrades ECM components and facilitates growth factor release and activation, cancer cell invasiveness, and metastasis. Since both MMP9 and CD44 contribute to cancer progression, we have developed a new strategy to fully block this neoplastic process by engineering a multi-specific inhibitor that simultaneously targets CD44 and both the catalytic and PEX domains of MMP9. Using a yeast surface display technology, we first obtained a high-affinity inhibitor for the MMP9 catalytic domain, which we termed C9, by modifying a natural non-specific MMP inhibitor, N-TIMP2. We then conjugated C9 via a flexible linker to PEX, thereby creating a multi-specific inhibitor (C9-PEX) that simultaneously targets the MMP9 catalytic and PEX domains and CD44. It is likely that, via its co-localization with CD44, C9-PEX may compete with MMP9 localization on the cell surface, thereby inhibiting MMP9 catalytic activity, reducing MMP9 cellular levels, interfering with MMP9 homodimerization, and reducing the activation of downstream MAPK/ERK pathway signaling. The developed platform could be extended to other oncogenic MMPs as well as to other important target proteins, thereby offering great promise for creating novel multi-specific therapeutics for cancer and other diseases.

Activity-dependent interdomain dynamics of matrix metalloprotease-1 on fibrin

The roles of protein conformational dynamics and allostery in function are well-known. However, the roles that interdomain dynamics have in function are not entirely understood. We used matrix metalloprotease-1 (MMP1) as a model system to study the relationship between interdomain dynamics and activity because MMP1 has diverse substrates. Here we focus on fibrin, the primary component of a blood clot. Water-soluble fibrinogen, following cleavage by thrombin, self-polymerize to form water-insoluble fibrin. We studied the interdomain dynamics of MMP1 on fibrin without crosslinks using single-molecule Forster Resonance Energy Transfer (smFRET). We observed that the distance between the catalytic and hemopexin domains of MMP1 increases or decreases as the MMP1 activity increases or decreases, respectively. We modulated the activity using (1) an active site mutant (E219Q) of MMP1, (2) MMP9, another member of the MMP family that increases the activity of MMP1, and (3) tetracycline, an inhibitor of MMP1. We fitted the histograms of smFRET values to a sum of two Gaussians and the autocorrelations to an exponential and power law. We modeled the dynamics as a two-state Poisson process and calculated the kinetic rates from the histograms and autocorrelations. Activity-dependent interdomain dynamics may enable allosteric control of the MMP1 function.

Interaction between matrix metalloproteinase-9 (MMP-9) and neutrophil gelatinase-associated lipocalin (NGAL): A recent evolutionary event in primates

Matrix metalloproteases are known to represent an early step in the evolution of the immune system. Similarly, neutrophil gelatinase-associated lipocalin is known to be a key effector in immune response. MMP-9 interacts with NGAL, but their interaction mechanisms remain unclear. Functional interaction between proteins is ensured by coevolution. Protein coevolution was inferred by calculating the linear correlation coefficients between inter-protein distance matrices using MirrorTree. Among examined mammal species, we found a robust signal of MMP-9/NGAL coevolution exclusively within Primates (R = 0.96, p < 1e-06). Owing to the high conservation of these proteins among Mammals, we chose to utilize a recent version of Blocks in Sequences (BIS2) algorithm implemented in BIS2Analyzer webserver. Coevolution clusters between the two proteins were identified in MMP-9 fibronectin and hemopexin domains. Our results suggest that MMP-9/NGAL interaction is a recent evolutionary acquisition in Primates. Furthermore, MMP-9 hemopexin domain would represent a promising target for drug design against these molecules.