M10 Family Research Articles

Determination of the role of specific amino acids in the binding of Zn(II), Ni(II), and Cu(II) to the active site of the M10 family metallopeptidase

Metallopeptidases are a group of metal-dependent enzymes that hydrolyze peptide bonds. These enzymes found in Streptococcus pneumoniae assist the pathogen in infecting the host by breaking down host tissues and extracellular matrix proteins. Considering metallopeptidases' significant role in bacterial virulence, inhibiting this enzyme represents a promising avenue for research. These enzymes are characterized by the presence of Zn(II) in the active site, proper coordination of which is essential for their catalytic function. This work aims to determine the significance of the specific amino acids in the metal binding domain of metallopeptidase from S. pneumoniae. For this purpose, we investigated the coordination chemistry of Zn(II), Ni(II), and Cu(II) ions with point-mutated peptide models of the metal-binding domain. Mutations were introduced at His-2 (L1) and Glu-1. Studies have shown that at pH 7.14 (pH of infected lungs by S. pneumoniae), point mutation on glutamic acid caused only minor effects on the binding of Zn(II) and Ni(II), while significantly improving Cu(II) binding. The stability of copper complexes is greater with the mutant Glu-1 → Gln-1 than with the original domain due to a hydrogen bonding network created by the Gln backbone with its side chain. Substituting histidine resulted in a significant reduction in metal binding for all metal ions, highlighting the crucial role of His-2 in metal coordination. Introduced mutations at neutral pH did not significantly affect the secondary structure of metal complexes. However, at alkaline pH, the peptides showed a higher percentage of antiparallel β-sheet structures upon the addition of Cu(II), Ni(II) and Zn(II).

Initial detachment of the mouse oocyte from the zona pellucida is mediated by metallopeptidase activity†.

The fully grown mammalian oocyte is tightly attached to its extracellular matrix shell, the zona pellucida (ZP), but the oocyte detaches from the ZP shortly after ovulation is signaled. The mechanism by which the oocyte detaches from the ZP is unknown. Because ZP proteins are initially secreted as transmembrane proteins, we hypothesized that attachment of the oocyte to the ZP is mediated by transmembrane ZP proteins and that detachment occurs when these proteins are cleaved by peptidases. To identify potential candidates for the type of peptidase, we used mouse oocyte transcriptome data sets to identify candidate peptidases localized to the exterior of the oocyte. Screening with a set of small molecule inhibitors that broadly target the families of peptidases represented by the candidates, we found that only inhibitors of the M10 and M12 families of metallopeptidases prevented detachment. Using more selective inhibitors indicated that detachment was prevented by an inhibitor, GI254023X, developed to be selective for ADAM10 in the M12 family but not by those considered selective for the M10 family or for other M12 metallopeptidases expressed in oocytes. Using an antibody that binds to an epitope just distal to the likely cleavage site of murine ZP3 showed that this site was gradually lost from the oocyte surface during the period when detachment occurs and that inhibiting metallopeptidase activity prevented the loss of this epitope. Taken together, these results indicate that detachment of the oocyte from the ZP is mediated by a metallopeptidase.

Evolutionary Relationships Between Dysregulated Genes in Oral Squamous Cell Carcinoma and Oral Microbiota.

Oral squamous cell carcinoma (OSCC) is one of the most prevalent cancers in the world. Changes in the composition and abundance of oral microbiota are associated with the development and metastasis of OSCC. To elucidate the exact roles of the oral microbiota in OSCC, it is essential to reveal the evolutionary relationships between the dysregulated genes in OSCC progression and the oral microbiota. Thus, we interrogated the microarray and high-throughput sequencing datasets to obtain the transcriptional landscape of OSCC. After identifying differentially expressed genes (DEGs) with three different methods, pathway and functional analyses were also performed. A total of 127 genes were identified as common DEGs, which were enriched in extracellular matrix organization and cytokine related pathways. Furthermore, we established a predictive pipeline for detecting the coevolutionary of dysregulated host genes and microbial proteomes based on the homology method, and this pipeline was employed to analyze the evolutionary relations between the seven most dysregulated genes (MMP13, MMP7, MMP1, CXCL13, CRISPO3, CYP3A4, and CRNN) and microbiota obtained from the eHOMD database. We found that cytochrome P450 3A4 (CYP3A4), a member of the cytochrome P450 family of oxidizing enzymes, was associated with 45 microbes from the eHOMD database and involved in the oral habitat of Comamonas testosteroni and Arachnia rubra. The peptidase M10 family of matrix metalloproteinases (MMP13, MMP7, and MMP1) was associated with Lacticaseibacillus paracasei, Lacticaseibacillus rhamnosus, Streptococcus salivarius, Tannerella sp._HMT_286, and Streptococcus infantis in the oral cavity. Overall, this study revealed the dysregulated genes in OSCC and explored their evolutionary relationship with oral microbiota, which provides new insight for exploring the microbiota–host interactions in diseases.

Human matrix metalloproteinases: Characteristics and pathologic role in altering mesangial homeostasis

Matrix metalloproteinases are zinc dependent endopeptidases belonging to the M10 family of the metalloproteinase superfamily. They are ubiquitous enzymes, structurally and functionally related, with a high degree of sequence homology. They are primarily involved in extracellular matrix (ECM) turn-over and cell migration through their expanding repertoire of substrate affinities. Twenty three different forms of human MMPs have been described to be arranged in eight distinct structural classes. Their interactions with tissue inhibitors of metalloproteinases (TIMPs), and other indigenous inhibitors have been well documented. This manuscript reviews pertinent information available on matrix metalloproteinases and TIMPs in the literature. Light chain-mediated glomerular injury represents an excellent example of how metalloproteinases participate in altering mesangial homeostasis. Investigations regarding these conditions have shown that the physico-chemical characteristics of the light chains govern the pattern of renal damage that will ensue with the mesangium representing the critical site where pathological alterations are centered. The mesangium is either replaced or expanded depending on the light chains involved in the pathologic process.

Molecular characterization of vomeronasal sensory neurons responding to a male-specific peptide in tear fluid: Sexual communication in mice

Abstract Pheromonal signals received by the vomeronasal organ (VNO) have been shown to elicit various behavioral and physiological responses that are typically stereotyped and preprogrammed. Recently, we found a novel male-specific peptide, named exocrine gland-secreting peptide 1 (ESP1), that is secreted in tear fluid and stimulates the VNO in mice. Excreted ESP1 appears to be transferred to the female VNO, where it induces c-Fos expression and elicits an electrical response in a small subset of vomeronasal sensory neurons (VSNs). We report here the identification of molecular components expressed in ESP1-stimulated VSNs by double-staining with c-Fos. We found that the c-Fos-induced cells were localized amongst the Gαo-expressing VSNs. Furthermore, the ESP1 signal was received by VSNs expressing a single type of vomeronasal receptor type 2 (V2Rp5). Finally, double in situ hybridization of the V2Rp5 and various members of the M1 and M10 families of major histocompatibility complex (MHC) class Ib molecules revealed that V2Rp5-expressing VSNs can express multiple MHC molecules. These results suggest that a V2R rather than MHC molecule is mainly responsible for the recognition of ESP1. The identification of the putative sex-pheromone ESP1 and its cognate receptor therefore will help clarify the molecular mechanisms underlying pheromone recognition in the mouse vomeronasal system.

ADAMs, cell migration and cancer

The ADAMs are modular type I transmembrane proteins that contain a metalloprotease and a disintegrin-like domain. In fact, ADAM stands for “A Disintegrin And Metalloprotease” and has become the most widespread name to designate this group of proteins. Alternative terms such as metalloprotease disintegrins or MDC (metalloprotease/disintegrin/cysteinerich) are currently less used. Although ADAMs are frequently referred to as a family, they constitute a subfamily of the M10 family that, in turn, belongs to the metzincin clan of metalloproteases [4]. To date, 23 ADAMs have been identified in the human genome: ADAM1-3, 6–12, 15, 17–23, 28–30, 32 and 33 (for an up-to-date list of ADAMs in different organisms, see http://www.people.virginia.edu/%7Ejw7 g/Table of the ADAMs.html). ADAM9, 10, 12, 15, 17, 19, and 33 are widely expressed, the rest show a restricted tissue distribution. Analysis of their primary structure shows that, in addition to the disintegrin and metalloprotease, all ADAMs contain the following domains: signal peptide, prodomain, cysteine-rich, epidermal growth factor (EGF)-like, transmembrane and cytoplasmic (Fig. 1). The prodomain keeps the metalloprotease domain inactive until its removal by furin-like proprotein convertases or autocatalytic processing. The related group of ADAMTS are secreted soluble proteins that contain a variable number of thrombospondin-like repeats (Fig. 1). A particularly intriguing feature of the ADAMs is that they support both proteolytic activity and cell adhesion, making

Structure of a Pheromone Receptor-Associated MHC Molecule with an Open and Empty Groove

Neurons in the murine vomeronasal organ (VNO) express a family of class Ib major histocompatibility complex (MHC) proteins (M10s) that interact with the V2R class of VNO receptors. This interaction may play a direct role in the detection of pheromonal cues that initiate reproductive and territorial behaviors. The crystal structure of M10.5, an M10 family member, is similar to that of classical MHC molecules. However, the M10.5 counterpart of the MHC peptide-binding groove is open and unoccupied, revealing the first structure of an empty class I MHC molecule. Similar to empty MHC molecules, but unlike peptide-filled MHC proteins and non-peptide–binding MHC homologs, M10.5 is thermally unstable, suggesting that its groove is normally occupied. However, M10.5 does not bind endogenous peptides when expressed in mammalian cells or when offered a mixture of class I–binding peptides. The F pocket side of the M10.5 groove is open, suggesting that ligands larger than 8–10-mer class I–binding peptides could fit by extending out of the groove. Moreover, variable residues point up from the groove helices, rather than toward the groove as in classical MHC structures. These data suggest that M10s are unlikely to provide specific recognition of class I MHC–binding peptides, but are consistent with binding to other ligands, including proteins such as the V2Rs.

Functional Expression of Mammalian Odorant Receptors

Functional Expression of Mammalian Odorant Receptors

Matching accessories.

Heterodimerization enhances the complexity of ligand recognition and diversity of signaling responses of heterotrimeric guanine nucleotide-binding protein-coupled receptors (GPCRs). Many accessory proteins (for ion channels or GPCRs) appear to associate with their partners relatively early in the process whereby proteins are transported to the cell surface; their roles in modulating function may have evolved out of simple proximity to a protein that once upon a time they either facilitated or accompanied through the maturation process. The receptor activity-modifying proteins (RAMPs) are a family of single-transmembrane accessory proteins that heterodimerize with GPCRs and, thereby, allow individual GPCRs to recognize multiple ligands and to activate various signaling pathways in response to ligand binding. The M10 family of major histocompatibility complex (MHC) class 1b proteins has recently been shown to associate with murine vomeronasal V2R receptors, as well as to escort them to the cell surface. The exact role of M10 in modulating V2R function (or vice versa) remains to be determined.

Making Scents of Atypical MHC Genes

In mammals, pheromones--chemical cues that trigger mating and social behaviors--are detected through two large families of heterotrimeric guanine nucleotide-binding protein-coupled receptors (GPCRs), V1R and V2R, expressed in sensory neurons in discreet regions of the vomeronasal organ (VNO). The molecular mechanisms through which diversity and specificity of pheromone responses are achieved, however, have remained unclear. Loconto et al. used subtractive differential screening of cDNA libraries from individual rat VNO neurons and isolated a nonclassical major histocompatibility complex (MHC) class Ib gene. The authors used a combination of techniques including in situ hybridization, single-cell reverse transcription polymerase chain reaction (RT-PCR), immunocytochemistry, and transgenic analysis to investigate the association of M10.7 , the mouse homolog of this gene, with pheromone receptors. M10.7 and the five other presumably functional members of the M10 gene family were expressed exclusively in V2R-expressing neurons in the basal neuroepithelium of the VNO. Particular V2Rs specifically associated with one or a few members of the M10 family. Western analysis in combination with immunoaffinity chromatography indicated that V2R, M10, and β2-microglobulin (β2m), which generally associates with MHC molecules, formed a multimolecular complex. In vitro experiments indicated that this association was required for V2R localization to the plasma membrane. Moreover, transgenic mice lacking β2m showed V2R mislocalization and lacked typical male-male aggressive behavior. The authors speculated that M10, in addition to escorting V2R to the cell surface, may alter the mechanism and specificity of pheromone recognition by heterodimerizing with the V2R. J. Loconto, F. Papes, E. Chang, L. Stowers, E. P. Jones, T. Takada, A. Kumánovics, K. F. Lindahl, C. Dulac, Functional expression of murine V2R pheromone receptors involves selective association with the M10 and M1 families of MHC class Ib molecules. Cell 112 , 607-618 (2003). [Online Journal]

Functional Expression of Murine V2R Pheromone Receptors Involves Selective Association with the M10 and M1 Families of MHC Class Ib Molecules

The vomeronasal organ (VNO) of the mouse has two neuronal compartments expressing distinct families of pheromone receptors, the V1Rs and the V2Rs. We report here that two families of major histocompatibility complex (MHC) class Ib molecules, the M10 and the M1 families, show restricted expression in V2R-expressing neurons. Our data suggest that neurons expressing a given V2R specifically co-express one or a few members of the M10 family. Biochemical and immunocytochemical analysis demonstrates that in VNO sensory dendrites M10s belong to large multi-molecular complexes that include pheromone receptors and β2-microglobulin (β2m). In cultured cells, M10s appear to function as escort molecules in transport of V2Rs to the cell surface. Accordingly, β2m-deficient mice exhibit mislocalization of V2Rs in the VNO and a specific defect in male-male aggressive behavior. The functional characterization of M10 highlights an unexpected role for MHC molecules in pheromone detection by mammalian VNO neurons.

Mhc class I and non-class I gene organization in the proximal H2-M region of the mouse.

A bacterial artificial chromosome (BAC) contig was constructed across the proximal part of the H2-M region from the major histocompatibility complex (Mhc) of mouse strain 129 (H2bc). The contig is composed of 28 clones that span approximately 1 megabasepair (Mb), from H2-T1 to Mog, and contains three H2-T genes and 18 H2-M genes. We report the fine mapping of the H2-M class I gene cluster, which includes the previously reported M4-M6, the M1 family, the M10 family, and four additional class I genes. All but two of the H2-M class I genes are conserved among haplotypes H2k, H2b, and H2bc, and only two genes are found in polymorphic HindIII fragments. Six evolutionarily conserved non-class I genes were mapped to a 180 kilobase interval in the distal part of the class I region in mouse, and their order Znf173-Rfb30-Tctex5-Tctex6- Tctex4-Mog was found conserved between human and mouse. In this Znf173-Mog interval, three mouse class I genes, M6, M4, and M5, which are conserved among haplotypes, occupy the same map position as the human HLA-A class I cluster, which varies among haplotypes and is diverged in sequence from the mouse genes. These results further support the view that class I gene diverge and evolve independently between species.