Structure, expression, and developmental function of early divergent forms of metalloproteinases in Hydra

Metalloproteinases have a critical role in a broad spectrum of cellular processes ranging from the break-down of extracellulax matrix to the processing of signal transduction-related proteins. These hydrolyticfunctions underlie a variety of mechanisms related to developmental processes as well as disease states.Structural analysis of metalloproteinases from both invertebrate and vertebrate species indicates that theseenzymes are highly conserved and arose early during metazoan evolution. In this regard, studies from vari-ous laboratories have reported that a number of classes of metalloproteinases are found in hydra, a memberof Cnidaria, the second oldest of existing animal phyla. These studies demonstrate that the hydra genomecontains at least three classes of metalloproteinases to include members of the 1) astacin class, 2) matrix met-alloproteinase class, and 3) neprilysin class. Functional studies indicate that these metalloproteinases playdiverse and important roles in hydra morphogenesis and cell differentiation as well as specialized functionsin adult polyps. This article will review the structure, expression, and function of these metalloproteinasesin hydra.     

Structure,expression, and developmental function of early divergent forms of metalloproteinases in hydra

Metalloproteinases have a critical role in a broad spectrum of cellular processes ranging from the breakdown of extracellular matrix to the processing of signal transduction-related proteins. These hydrolytic functions underlie a variety of mechanisms related to developmental processes as well as disease states. Structural analysis of metalloproteinases from both invertebrate and vertebrate species indicates that these enzymes are highly conserved and arose early during metazoan evolution. In this regard, studies from various laboratories have reported that a number of classes of metalloproteinases are found in hydra, a member of Cnidaria, the second oldest of existing animal phyla. These studies demonstrate that the hydra genome contains at least three classes of metalloproteinases to include members of the 1) astacin class, 2) matrix metalloproteinase class, and 3) neprilysin class. Functional studies indicate that these metalloproteinases play diverse and important roles in hydra morphogenesis and cell differentiation as well as specialized functions in adult polyps. This article will review the structure, expression, and function of these metalloproteinases in hydra.     

A novel hydra matrix metalloproteinase (HMMP) functions in extracellular matrix degradation, morphogenesis and the maintenance of differentiated cells in the foot process

As a member of Cnidaria, the body wall of hydra is structurally reduced to an epithelial bilayer with an intervening extracellular matrix (ECM). Biochemical and cloning studies have shown that the molecular composition of hydra ECM is similar to that seen in vertebrates and functional studies have demonstrated that cell-ECM interactions are important to developmental processes in hydra. Because vertebrate matrix metalloproteinases (MMPs) have been shown to have an important role in cell-ECM interactions, the current study was designed to determine whether hydra has homologues of these proteinases and, if so, what function these enzymes have in morphogenesis and cell differentiation in this simple metazoan. Utilizing a PCR approach, a single hydra matrix metalloproteinase, named HMMP was identified and cloned. The structure of HMMP was similar to that of vertebrate MMPs with an overall identity of about 35%. Detailed structural analysis indicated some unique features in (1) the cysteine-switch region of the prodomain, (2) the hinge region preceding the hemopexin domain, and (3) the hemopexin domain. Using a bacterial system, HMMP protein was expressed and folded to obtain an active enzyme. Substrate analysis studies indicated that recombinant HMMP could digest a number of hydra ECM components such as hydra laminin. Using a fluorogenic MMP substrate assay, it was determined that HMMP was inhibited by peptidyl hydroxamate MMP inhibitors, GM6001 and matlistatin, and by human recombinant TIMP-1. Whole-mount in situ studies indicated that HMMP mRNA was expressed in the endoderm along the entire longitudinal axis of hydra, but at relatively high levels at regions where cell-transdifferentiation occurred (apical and basal poles). Functional studies using GM6001 and TIMP-1 indicated that these MMP inhibitors could reversibly block foot regeneration. Blockage of foot regeneration was also observed using antisense thio-oligo nucleotides to HMMP introduced into the endoderm of the basal pole using a localized electroporation technique. Studies with adult intact hydra found that GM6001 could also cause the reversible de-differentiation or inhibition of transdifferentiation of basal disk cells of the foot process. Basal disk cells are adjacent to those endoderm cells of the foot process that express high levels of HMMP mRNA. In summary, these studies indicate that hydra has at least one MMP that is functionally tied to morphogenesis and cell transdifferentiation in this simple metazoan.     

Matrix metalloproteinases and their expression in mammary gland

The matrix metalloproteinases (MMPs) are a family of zine-dependent endopeptidases that play a key role in both normal and pathological processes involving tissue remodeling events.The expression of these proteolytic enzymes is highly regulated by a balance between extracellular matrix (ECM) deposition and its degradation,and is controlled by growth factors,cytokines,hormones,as well as interactions with the ECM macromolecules.Furthermore,the activity of the MMPs is regulated by their natural endogenous inhibitors,which are members of the tissue inhibitor of metalloproteinases (TIMP) family.In the normal mammary gland,MMPs are expressed during ductal development,lobulo-alveolar development in pregnancy and involution after lactation.Under pathological conditions,such as tumorigenesis,the dysregulated expression of MMPs play a role in tumor initiation,progression and malignant conversion as well as facilitating invasion and metastasis of malignant cells through degradation of the ECM and basement membranes.     

Epithelial morphogenesis in hydra requires de novo expression of extracellular matrix components and matrix metalloproteinases

As a member of the phylum Cnidaria, the body wall of hydra is organized as an epithelium bilayer (ectoderm and endoderm) with an intervening extracellular matrix (ECM). Previous studies have established the general molecular structure of hydra ECM and indicate that it is organized as two subepithelial zones that contain basement membrane components such as laminin and a central fibrous zone that contains interstitial matrix components such as a unique type I fibrillar collagen. Because of its simple structure and high regenerative capacity, hydra has been used as a developmental model to study cell-ECM interaction during epithelial morphogenesis. The current study extends previous studies by focusing on the relationship of ECM biogenesis to epithelial morphogenesis in hydra, as monitored during head regeneration or after simple incision of the epithelium. Histological studies indicated that decapitation or incision of the body column resulted in an immediate retraction of the ECM at the wound site followed by a re-fusion of the bilayer within 1 hour. After changes in the morphology of epithelial cells at the regenerating pole, initiation of de novo biogenesis of an ECM began within hours while full reformation of the mature matrix required approximately 2 days. These processes were monitored using probes to three matrix or matrix-associated components: basement membrane-associated hydra laminin beta1 chain (HLM-beta1), interstitial matrix-associated hydra fibrillar collagen (Hcol-I) and hydra matrix metalloproteinase (HMMP). While upregulation of mRNA for both HLM-beta1 and Hcol-I occurred by 3 hours, expression of the former was restricted to the endoderm and expression of the latter was restricted to the ectoderm. Upregulation of HMMP mRNA was also associated with the endoderm and its expression paralleled that for HLM-beta1. As monitored by immunofluorescence, HLM-beta1 protein first appeared in each of the two subepithelial zones (basal lamina) at about 7 hours, while Hcol-I protein was first observed in the central fibrous zone (interstitial matrix) between 15 and 24 hours. The same temporal and spatial expression pattern for these matrix and matrix-associated components was observed during incision of the body column, thus indicating that these processes are a common feature of the epithelium in hydra. The correlation of loss of the ECM, cell shape changes and subsequent de novo biogenesis of matrix and matrix-associated components were all functionally coupled by antisense experiments in which translation of HLM-beta1 and HMMP was blocked and head regeneration was reversibly inhibited. In addition, inhibition of translation of HLM-beta1 caused an inhibition in the appearance of Hcol-I into the ECM, thus suggesting that binding of HLM-beta1 to the basal plasma membrane of ectodermal cells signaled the subsequent discharge of Hcol-I from this cell layer into the newly forming matrix. Given the early divergence of hydra, these studies point to the fundamental importance of cell-ECM interactions during epithelial morphogenesis.     

Activation and silencing of matrix metalloproteinases

Matrix metalloproteinases (MMPs) were first described as proteases that act on protein components of the extracellular matrix. However, subsequent studies of MMP function in vivo have revealed that these proteinases also cleave numerous non-ECM protein substrates. Because their substrates are diverse in functions, MMPs are involved in variety of homeostatic functions, such as tissue repair and immunity, as well as pathological processes, including cancer, fibroses and inflammation. Essential steps in regulating MMP proteolysis are conversion of the zymogen into an active proteinase and subsequent inactivation. A number of mechanisms including proteolysis, allosteric interactions, oxidative modification, pericellular compartmentalization, interaction with tissue inhibitor of metalloproteinases (TIMPs), endocytosis, and more have been proposed to control the activation and inactivation of MMPs. In this paper, we discuss these and other mechanisms, and their relevance to in vivo control of MMP-mediated functions.     

Pyrimidine-2,4,6-Triones: a new effective and selective class of matrix metalloproteinase inhibitors

Matrix metalloproteinases (MMPs) are a family of zinc endopeptidases that have been implicated in various disease processes. Different classes of MMP inhibitors, including hydroxamic acids, phosphinic acids and thiols, have been previously described. Most of these mimic peptides and most likely bind in a similar way to the corresponding peptide substrates. Here we describe pyrimidine-triones as a completely new class of metalloprotease inhibitors. While the pyrimidine-trione template is used as the zinc-chelating moiety, the substituents have been optimized to yield inhibitors comparable in their inhibition efficiency of matrix metalloproteinases to hydroxamic acid derivatives such as batimastat. However, they are much more specific for a small subgroup of MMPs, namely the gelatinases (MMP-2 and MMP-9).     

Molecular and functional evidence for early divergence of an endothelin-like system during metazoan evolution: analysis of the Cnidarian, hydra

A novel putative endothelin-converting enzyme (ECE) has been cloned from hydra, a freshwater invertebrate that belongs to the second oldest phylum of the animal kingdom. As an integral component of the endothelin system, vertebrate ECE functions in the activation of endothelin (ET) peptides. Vertebrate ETs are (1) the most potent vasoconstrictors known in mammals; and (2) function as essential signaling ligands during development of tissues derived from neural crest cells. To date, only a limited number of immunocytochemical studies have suggested the presence of endothelin-like peptides in invertebrates. Based on structural and functional analyses, we present evidence for a functional endothelin-like system in hydra that is involved in both muscle contraction and developmental processes. These findings indicate the broad use of endothelin systems in metazoans and also indicate that this type of signaling system arose early in evolution even before divergence of protostomes and deuterostomes.     

The cell surface: the stage for matrix metalloproteinase regulation of migration

Matrix metalloproteinases are important for the turnover of extracellular matrix in tissue. Recent studies have expanded their roles well beyond extracellular matrix degradation - they also cleave many growth factors, cytokines and cell adhesion molecules in the extracellular milieu, modulating their functions irreversibly. In particular, some matrix metalloproteinases that associate with the cell surface have arisen as intriguing regulators of cellular functions, including migration.     

基质金属蛋白酶及其组织抑制剂研究进展

基质金属蛋白酶家族是细胞外基质降解过程中的重要酶类,组织金属蛋白酶抑制剂是基质金属蛋白酶的天然抑制物。研究证实,细胞外基质中基质金属蛋白酶及其组织抑制剂的失衡与多种病理机制有关,尤其与肿瘤的侵袭和转移密切相关。本就基质金属蛋白酶及其组织抑制剂的性质、结构以及功能进行了综述。     

Matrix Metalloproteinases of Normal Human Tissues

This review considers biochemical properties of the family of matrix metalloproteinases (MMPs) of normal human tissues and the involvement of these enzymes in morphogenesis. Four main MMP subfamilies are characterized, and a group of other MMPs is described. Data on mechanisms of activation and inhibition of MMPs in certain tissues during various physiological processes (embryogenesis, angiogenesis, tissue growth and involution) are considered. Information about tissue inhibitors of MMP is presented, and the ability of these inhibitors to regulate the activity of MMPs is analyzed.     

植物基质金属蛋白酶的研究进展

基质金属蛋白酶(MMPs)是高度保守的锌依赖型内肽酶家族.医学研究表明,人体MMPs不仅在一系列生理过程中发挥关键作用,而且与很多重大疾病关联.例如,MMPs在恶性肿瘤组织中的表达量大幅度上升,和肿瘤的侵袭转移密切相关.MMPs也广泛存在于高等植物,它可能参与植物发育调控、免疫应答及非生物逆境胁迫响应等多个方面.该文对近年来国内外有关植物MMPs的分布、结构特点、活性调节以及生物学功能等方面的研究进展进行综述,并对该领域的研究趋势和重点问题进行了讨论.     

Proteomic identification of protease cleavage sites: cell-biological and biomedical applications

Proteolysis shapes proteomes by protein degradation or restricted proteolysis, which generates stable cleavage products. Proteolytic (in-)activation of enzymes and cytokines is an essential aspect of the functional proteome status. Proteome-wide identification and quantification of proteolytic processing is accessible by complementary techniques for the focused analysis of protein termini. These innovative strategies are now widely applied and have transformed protease research. Pioneering studies portrayed apoptotic and caspase-dependent cleavage events. Protease-centric investigations focused predominantly on matrix metalloproteinases (MMPs), granzymes and aspartyl and cysteine cathepsins. The first in vivo degradomic studies were performed with mice lacking either cysteine cathepsins or matrix metalloproteinases. Process-centric degradomic analyses investigated infectious processes and mitochondrial import. Peptidomic analyses yielded disease biomarkers representing cleavage fragments from bodily fluids. The diversity of degradomic endeavors illustrates the importance of portraying proteolytic processing in health and disease. The present review provides an overview of the current status of degradomic studies.     

Cytokine regulation of metalloproteinase gene expression

Matrix metalloproteinases belong to a family of zinc-dependent enzymes capable of degrading extracellular matrix and basement membrane components. Their expression is greatly modulated by cytokines and growth factors and involves the gene products of the Fos and Jun families of oncogenes. After extra(peri)cellular activation, their activity can be further controlled by specific tissue inhibitors of metalloproteinases. A correct balance between these regulatory mechanisms is necessary to ensure matrix remodeling in normal physiological processes such as embryonic development, but the overexpression of these enzymes may initiate or contribute to pathological situations such as cartilage degradation in rheumatoid arthritis or to tumor progression and metastasis. Delineation of the mechanisms of metalloproteinase and metalloproteinase inhibitors gene expression, understanding of their mode of interactions, and characterization of their patterns of expression in various tissues in normal and pathological states will lead to new therapeutic strategies to counteract the deleterious effects of matrix metalloproteinases in human disease.     

Mapping von Willebrand factor A domain binding sites on a snake venom metalloproteinase cysteine-rich domain

The PIII class of the snake venom metalloproteinases (SVMPS) are acknowledged to be one of the major hemorrhage producing toxins in crotalid venoms. This class of SVMPS are structurally distinguished by the presence of disintegrin-like and cysteine-rich domains carboxy to the metalloproteinase domain and thus share structural homology with many of the ADAMs proteins. It has been suggested that the presence of the carboxy domain are the key structural determinants for potent hemorrhagic activity in that they may serve to target the proteinases to specific key extracellular matrix and cell surface substrates for proteolysis leading to hemorrhage production at the capillaries. Following from previous studies in our laboratory in this investigation we scanned the cysteine-rich domain of the PIII hemorrhagic SVMP jararhagin using synthetic peptides in an attempt to identify regions which could bind to von Willebrand factor (vWF), a known binding partner for jararhagin. From these studies we identified two such peptide, Jar6 and Jar7 that could support binding to vWF as well as block the recombinant cysteine-rich domain of jararhagin binding to vWF. Using the coordinates for the recently solved crystal structure of the PIII SVMP VAP1, we modeled the structure of jararhagin and attempted to dock the modeled cysteine-rich structure of that protein to the A1 domain of vWF. These studies indicated that effective protein-protein interaction between the two ligands was possible and supported the data indicating that the Jar6 peptide was involved, whereas the Jar7 peptide was observed to be sterically blocked from interaction. In summary, our studies have identified a region on the cysteine-rich domain of a PIII SVMP that interacts with vWF and based on molecular modeling could be involving in the interaction of the cysteine-rich domain of the SVMP with the A1 domain of vWF thus serving to target the toxin to the protein for subsequent proteolytic degradation.     

Multiple roles of matrix metalloproteinases during apoptosis

Structural, molecular and biochemical approaches have contributed to piecing together the puzzle of how matrix metalloproteinases (MMPs) work and contribute to various disease processes. However, MMPs have many unexpected substrates other than components of the extracellular matrix which profoundly influence cell behaviour, survival and death. With the current understanding of diverse/novel roles of matrix metalloproteinases—particularly their direct or indirect relevance for the early steps during programmed cell death—some seemingly contrasting results seem less surprising. To better target MMPs an appreciation of their many extracellular, intracellular and intranuclear functions, often acting in opposing directions with paradoxical roles in cell death, is carefully required.     

HyBMP5-8b, a BMP5-8 orthologue, acts during axial patterning and tentacle formation in hydra

Developmental gradients play a central role in axial patterning in hydra. As part of the effort towards elucidating the molecular basis of these gradients as well as investigating the evolution of the mechanisms underlying axial patterning, genes encoding signaling molecules are under investigation. We report the isolation and characterization of HyBMP5-8b, a BMP5-8 orthologue, from hydra. Processes governing axial patterning are continuously active in adult hydra. Expression patterns of HyBMP5-8b in normal animals and during bud formation, hydra's asexual form of reproduction, were examined. These patterns, coupled with changes in patterns of expression in manipulated tissues during head regeneration, foot regeneration as well as under conditions that alter the positional value gradient indicate that the gene is active in two different processes. The gene plays a role in tentacle formation and in patterning the lower end of the body axis.     

基质金属蛋白酶在心肌缺血再灌注损伤中的作用

肌缺血再灌注损伤是指缺血心肌组织在恢复血流供给后,其细胞代谢功能障碍及结构破坏反而加重的现象,主要表现在心肌收缩与舒张功能障碍、血管内皮功能障碍、微循环血流紊乱、细胞代谢失调、电解质平衡紊乱、细胞凋亡与坏死等,并伴随着氧自由基的大量产生和毒性损伤以及炎症反应的激活,是一个极其复杂的病理过程。基质金属蛋白酶（MMPs）及其组织抑制物（TIMPs）是心肌组织中多种细胞分泌的内源性细胞因子,其作用涵盖了细胞外基质降解、炎症反应激活、调节血管功能、影响细胞凋亡与存活等众多病理生理过程,而这些过程均在心肌缺血再灌注损伤中发挥着重要的作用。