基质金属蛋白酶

基质金属蛋白酶是一类分解细胞外基质组分的锌蛋白酶⒚它们在有机体生长发育中的细胞外基质逆转与重塑以及疾病中的病理损害起着极为重要的作用⒚基质金属蛋白酶的表达和活性在不同细胞水平受到严密调控,如细胞因子、生长因子以及激素的调节⒚基质金属蛋白酶以酶原形式分泌,随后被其它蛋白酶如胞浆素或非蛋白酶类化学物质如有机汞所激活⒚所有基质金属蛋白酶都受到天然抑制剂 金属蛋白酶组织抑制剂所抑制⒚两者的不平衡导致许多疾病的发生,如肿瘤侵入及转移⒚合成基质金属蛋白酶组织抑制剂所抑制,如 Ｍ ａｒｉｍ ａｓｔａｔ 能控制肿瘤转移的发生及进一步扩散⒚本文将对基质金属蛋白酶的特征、分子区域结构、底物特性、激活机制、调控方式等方面进行最新概述⒚     

Crystal structures of MMPs in complex with physiological and pharmacological inhibitors

Matrix Metalloproteinases (MMPs) are a family of multidomain zinc endopeptidases that function in the extracellular space or attached to the cell membrane. Their proteolytic activity is controlled by the presence of endogenous inhibitors, the tissue inhibitors of matrix metalloproteinases (TIMPs), alpha-macroglobulin and others. Disruption of the proteinase-inhibitor balance is observed in serious diseases such as arthritis, tumor growth and metastasis, rendering the MMPs attractive targets for drug intervention by pharmacological inhibitors. The determination of MMP structures is of critical importance in order to understand their substrate preferences, dimerization events, and their association with matrix components and inhibitors. Thus, MMP structures may contribute significantly to the development of specific MMP inhibitors, which should allow precise control of individual members of the MMP family without affecting all members or the closely related metalloproteinases such as ADAMs and ADAMTSs.     

Matrix metalloproteinases and their inhibitors in connective tissue remodeling.

Matrix metalloproteinases are an important group of zinc enzymes responsible for degradation of the extracellular matrix components such as collagen and proteoglycans in normal embryogenesis and remodeling and in many disease processes such as arthritis, cancer, periodontitis, and osteoporosis. A matrixin family is defined, comprising at least seven members that range in size from Mr 28,000 to 92,000 and are related in gene sequence to collagenase. All family members are secreted as zymogens that lose peptides of about 10,000 daltons upon activation. Latency is due to a conserved cysteine that binds to zinc at the active center. Latency is overcome by physical (chaotropic agents), chemical (HOCl, mercurials), and enzymatic (trypsin, plasmin) treatments that separate the cysteine residue from the zinc. Expression of the metalloproteinases is switched on by a variety of agents acting through regulatory elements of the gene, particularly the AP-1 binding site. A family of protein inhibitors of Mr 28,500 or less binds strongly and stoichiometrically in noncovalent fashion to inhibit members of the family. The serum protein alpha 2-macroglobulin and relatives are also strongly inhibitory.     

Membrane type-matrix metalloproteinases and tumor progression

Matrix metalloproteinases (MMPs) are a family of zinc endopeptidases that process growth factors, growth factor binding proteins, cell surface proteins, degrade extracellular matrix (ECM) components and thereby play a central role in tissue remodeling and tumor progression. Membrane-type matrix metalloproteinases (MT-MMPs) are a recently discovered subgroup of intrinsic plasma membrane proteins. Their functions have been extended from pericellular proteolysis and control of cell migration to cell signaling, control of cell proliferation and regulation of multiple stages of tumor progression including growth and angiogenesis. This review sheds light on the new functions of MT-MMPs and their inhibitors in tumor development and angiogenesis, and presents recent investigations that document their influence on various cell functions.     

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.     

TIMP-2: an endogenous inhibitor of angiogenesis

Remodeling of the extracellular matrix--regulated by the matrix metalloproteinases (MMPs) and their endogenous inhibitors--is an important component of disease progression in many chronic disease states. Unchecked MMP activity can result in significant tissue damage, facilitate disease progression and is associated with host responses to pathologic injury, such as angiogenesis. The tissue inhibitors of metalloproteinases (TIMPs) have been shown to regulate MMP activity. However, recent findings demonstrate that an MMP-independent effect of TIMP-2 inhibits the mitogenic response of human microvascular endothelial cells to growth factors. This is the first demonstration of a cell-surface signaling receptor for a member of the TIMP family and suggests that TIMP-2 functions to regulate cellular responses to growth factors. These new findings are integrated in a comprehensive model of TIMP-2 function in tissue homeostasis.     

Membrane type-1 matrix metalloproteinases and tissue inhibitor of metalloproteinases-2 RNA levels mimic each other during Xenopus laevis metamorphosis

Matrix metalloproteinases (MMPs) and their endogenous inhibitors TIMPs (tissue inhibitors of MMPs), are two protein families that work together to remodel the extracellular matrix (ECM). TIMPs serve not only to inhibit MMP activity, but also aid in the activation of MMPs that are secreted as inactive zymogens. Xenopus laevis metamorphosis is an ideal model for studying MMP and TIMP expression levels because all tissues are remodeled under the control of one molecule, thyroid hormone. Here, using RT-PCR analysis, we examine the metamorphic RNA levels of two membrane-type MMPs (MT1-MMP, MT3-MMP), two TIMPs (TIMP-2, TIMP-3) and a potent gelatinase (Gel-A) that can be activated by the combinatory activity of a MT-MMP and a TIMP. In the metamorphic tail and intestine the RNA levels of TIMP-2 and MT1-MMP mirror each other, and closely resemble that of Gel-A as all three are elevated during periods of cell death and proliferation. Conversely, MT3-MMP and TIMP-3 do not have similar RNA level patterns nor do they mimic the RNA levels of the other genes examined. Intriguingly, TIMP-3, which has been shown to have anti-apoptotic activity, is found at low levels in tissues during periods of apoptosis.     

IgLON Cell Adhesion Molecules Are Shed from the Cell Surface of Cortical Neurons to Promote Neuronal Growth

Matrix metalloproteinases and a disintegrin and metalloproteinases are members of the zinc endopeptidases, which cleave components of the extracellular matrix as well as cell surface proteins resulting in degradation or release of biologically active fragments. Surface ectodomain shedding affects numerous biological processes, including survival, axon outgrowth, axon guidance, and synaptogenesis. In this study, we evaluated the role of metalloproteinases in regulating cortical neurite growth. We found that treatment of mature cortical neurons with pan-metalloproteinase inhibitors or with tissue inhibitors of metalloproteinase-3 reduced neurite outgrowth. Through mass spectrometry, we characterized the metalloproteinase-sensitive cell surface proteome of mature cortical neurons. Members of the IgLON family of glycosylphosphatidylinositol-anchored neural cell adhesion molecules were identified and validated as proteins that were shed from the surface of mature cortical neurons in a metalloproteinase-dependent manner. Introduction of two members of the IgLON family, neurotrimin and NEGR1, in early embryonic neurons was sufficient to confer sensitivity to metalloproteinase inhibitors in neurite outgrowth assays. Outgrowth experiments on immobilized IgLON proteins revealed a role for all IgLON family members in promoting neurite extension from cortical neurons. Together, our findings support a role for metalloproteinase-dependent shedding of IgLON family members in regulating neurite outgrowth from mature cortical neurons.     

Recombinant interferon-γ secreted by chinese hamster ovary-320 cells cultivated in suspension in protein-free media is protected against extracellular proteolysis by the expression of natural protease inhibitors and by the addition of plant protein hydrolysates to the culture medium

Summary Biosafety requirements increasingly restrict the cultivation of mammalian cells producing therapeutic glycoproteins to conditions that are devoid of any compound of animal origin. On cultivation in serum-free media, the proteases inhibitors, usually found in serum, cannot protect secreted recombinant proteins against unwanted endogenous proteolysis. Chinese hamster ovary (CHO) cells, secreting recombinant human interferon-γ (CHO-320 cell line) and cultivated in suspension in an original protein-free medium, expressed at least two members of the matrix metalloproteinases (MMP), either at the cell surface (proMMP-14 and MMP-14) or secreted (proMMP-9). In addition, tissue- and urinary-type plasminogen activators were also secreted in such culture conditions. At the cell surface, dipeptidyl peptidase IV and tripeptidyl peptidase II (TPPII) activities were also detected, and their activities decreased during time course of batch cultures. The proteolytic activities of these proteins were counterbalanced by (1) their expression as zymogens (proMMP-9, proMMP-14), (2) the expression of their natural inhibitors, tissue inhibitors of metalloproteinases-1 and-2 and plasminogen activator inhibitor-1 (PAI-1), or (3) the addition of plant protein hydrolysates to the culture medium, acting as a nonspecific source of TPPII inhibitors. This study points out that, even in protein-free media, recombinant proteins secreted by CHO cells are actively protected against physiological and unwanted extracellular proteolysis either by endogenous or by exogenous inhibitors.     

Tissue inhibitors of metalloproteinases

Orchestration of the growth and remodeling of tissues and responses of cells to their extracellular environment is mediated by metalloproteinases of the Metzincin clan. This group of proteins comprises several families of endopeptidases in which a zinc atom is liganded at the catalytic site to three histidine residues and an invariant methionine residue. Tissue inhibitors of metalloproteinases (TIMPs) are endogenous protein regulators of the matrix metalloproteinase (MMPs) family, and also of families such as the disintegrin metalloproteinases (ADAM and ADAMTS). TIMPs therefore have a pivotal role in determining the influence of the extracellular matrix, of cell adhesion molecules, and of many cytokines, chemokines and growth factors on cell phenotype. The TIMP family is an ancient one, with a single representative in lower eukaryotes and four members in mammals. Although much is known about their mechanism of action in proteinase regulation in mammalian cells, less is known about their functions in lower organisms. Recently, non-inhibitory functions of TIMPs have been identified in mammalian cells, including signaling roles downstream of specific receptors. There are clearly still questions to be answered with regard to their overall roles in biology.     

Tissue inhibitors of metalloproteinases

Orchestration of the growth and remodeling of tissues and responses of cells to their extracellular environment is mediated by metalloproteinases of the Metzincin clan. This group of proteins comprises several families of endopeptidases in which a zinc atom is liganded at the catalytic site to three histidine residues and an invariant methionine residue. Tissue inhibitors of metalloproteinases (TIMPs) are endogenous protein regulators of the matrix metalloproteinase (MMPs) family, and also of families such as the disintegrin metalloproteinases (ADAM and ADAMTS). TIMPs therefore have a pivotal role in determining the influence of the extracellular matrix, of cell adhesion molecules, and of many cytokines, chemokines and growth factors on cell phenotype. The TIMP family is an ancient one, with a single representative in lower eukaryotes and four members in mammals. Although much is known about their mechanism of action in proteinase regulation in mammalian cells, less is known about their functions in lower organisms. Recently, non-inhibitory functions of TIMPs have been identified in mammalian cells, including signaling roles downstream of specific receptors. There are clearly still questions to be answered with regard to their overall roles in biology.     

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.     

The low density lipoprotein receptor-related protein modulates levels of matrix metalloproteinase 9 (MMP-9) by mediating its cellular catabolism

Members of the matrix metalloproteinase (MMP) family of enzymes participate in matrix remodeling and share a number of structural and functional features. The activity of this family of proteinases is carefully regulated at the level of zymogen activation and by a family of specific inhibitors termed tissue inhibitors of metalloproteinases (TIMP). It is now becoming clear that levels of certain MMPs are modulated by their association with cellular receptors that mediate their rapid internalization and degradation. In the current investigation we report that the amount of MMP-9 in conditioned cell culture medium is significantly increased when mouse embryonic fibroblasts are grown in the presence of the 39-kDa receptor-associated protein (RAP), an antagonist of ligand binding to low density lipoprotein receptor-related protein (LRP). In vitro assays reveal that the MMP-9.TIMP-1 complex binds to LRP with high affinity and that the binding determinant for LRP appears to reside on MMP-9. Cell lines expressing LRP mediate the internalization of 125I-labeled MMP-9.TIMP-1 complexes, whereas cell lines genetically deficient in LRP show a diminished capacity to mediate the cellular catabolism of MMP-9.TIMP-1 complexes. The results demonstrate that LRP is a functional receptor for MMP-9 and suggest a major role for LRP in modulating remodeling of the extracellular matrix by regulating extracellular proteinase activity.     

Immunohistochemistry of matrix metalloproteinases (MMP), their substrates, and their inhibitors (TIMP) during trophoblast invasion in the human placenta

The invasion of extravillous trophoblast cells into the maternal endometrium is one of the key events in human placentation. The ability of these cells to infiltrate the uterine wall and to anchor the placenta to it as well as their ability to infiltrate and to adjust utero-placental vessels to pregnancy depends, among other things, on their ability to secrete enzymes that degrade the extracellular matrix. Most of the latter enzymes belong to the family of matrix metalloproteinases. Their activity is regulated by the tissue inhibitors of matrix metalloproteinases. We have studied the distribution patterns of matrix metalloproteinases-1, -2, -3, and -9 and their inhibitors TIMP-1 and TIMP-2 as compared to the distribution of their substrates along the invasive pathway of extravillous trophoblast of 1st, 2nd, and 3rd trimester placentas by means of light microscopy on paraffin and cryostat sections as well as at the ultrastructural level (only 3rd trimester placenta). The comparison of different methods proved to be necessary, since the immunohistochemical distribution patterns of these soluble enzymes are considerably influenced by the pretreatment of tissues. All three methods revealed immunoreactivities of both, proteinases and their inhibitors, not only intracellularly in the extravillous trophoblast but also extracellularly in its surrounding matrix, the distribution patterns depending on the stage of pregnancy and on the degree of differentiation of trophoblast cells along their invasive pathway. Within the extracellular matrix, immunolocalization of matrix metalloproteinases as well as their inhibitors showed a specific relation to certain extracellular matrix molecules.     

Matrix metalloproteinases (MMPs): chemical-biological functions and (Q)SARs

Matrix metalloproteinases (MMPs) are a large family of calcium-dependent zinc-containing endopeptidases, which are responsible for the tissue remodeling and degradation of the extracellular matrix (ECM), including collagens, elastins, gelatin, matrix glycoproteins, and proteoglycan. They are regulated by hormones, growth factors, and cytokines, and are involved in ovarian functions. MMPs are excreted by a variety of connective tissue and pro-inflammatory cells including fibroblasts, osteoblasts, endothelial cells, macrophages, neutrophils, and lymphocytes. These enzymes are expressed as zymogens, which are subsequently processed by other proteolytic enzymes (such as serine proteases, furin, plasmin, and others) to generate the active forms. Matrix metalloproteinases are considered as promising targets for the treatment of cancer due to their strong involvement in malignant pathologies. Clinical/preclinical studies on MMP inhibition in tumor models brought positive results raising the idea that the development of strategies to inhibit MMPs may be proved to be a powerful tool to fight against cancer. However, the presence of an inherent flexibility in the MMP active-site limits dramatically the accurate modeling of MMP-inhibitor complexes. The interest in the application of quantitative structure-activity relationships (QSARs) has steadily increased in recent decades and we hope it may be useful in elucidating the mechanisms of chemical-biological interactions for this enzyme. In the present review, an attempt has been made to explore the in-depth knowledge from the classification of this enzyme to the clinical trials of their inhibitors. A total number of 92 QSAR models (44 published and 48 new formulated QSAR models) have also been presented to understand the chemical-biological interactions. QSAR results on the inhibition of various compound series against MMP-1, -2, -3, -7, -8, -9, -12, -13, and -14 reveal a number of interesting points. The most important of these are hydrophobicity and molar refractivity, which are the most important determinants of the activity.     

Temporospatial expression of tissue inhibitors of matrix metalloproteinases-1, -2 and -3 during development, growth and aging of the mouse skeleton

Proteolytic degradation of collagen-rich extracellular matrices is a key feature in the development, growth and aging of skeleton. Matrix metalloproteinases (MMPs) are a family of enzymes capable of performing this function, whereas tissue inhibitors of MMPs (TIMPs) are believed to play an important role in regulating their activity. To better understand the roles of TIMP-1, -2 and -3, we have studied their mRNA levels in several different mouse tissues with special emphasis on the skeleton and the developing eye. A systematic analysis of TIMP-1, -2 and -3 mRNA levels in mouse knee joints during growth and aging demonstrated markedly different expression patterns for each TIMP. Immunohistochemical analysis revealed several time-dependent changes in the distribution of TIMP-1 and -2 in articular and growth cartilages, synovial tissue and bone. The data suggest that upon aging synovial tissue becomes the major source of synovial fluid TIMPs. In articular cartilage these inhibitors were mainly found in the deep layer and in subchondral bone. Compared with epiphyseal growth plate, the amounts of TIMP-1 and -2 in articular cartilage were quite low. These findings suggest that the capacity of articular cartilage chondrocytes to inhibit MMP activities by local production of TIMPs is limited, which may be of consequence during osteoarthritic cartilage degeneration.