Metalloproteinase - an overview | ScienceDirect Topics (2023)

Tetracyclines and metalloproteinases

A major target for non-infective indications of the tetracyclines is inhibition of metalloproteinases. The following is a brief summary of what is known about tetracyclines and metalloproteinases, followed by some comments about possible adverse reactions.

The matrix metalloproteinases (MMPs) are a family of calcium- and/or zinc-dependent endopeptidases involved in degradation of extracellular matrix and tissue remodelling [67]. At least 21 mammalian MMPs have been described. They participate in various biological processes, such as embryonic development, ovulation, angiogenesis, apoptosis, wound healing, and nerve growth.

Under normal conditions, the activity of MMPs is very low and is strongly regulated by natural tissue inhibitors (TIMPs). The TIMPS are a family of four structurally related proteins (TIMP-1, 2, 3, and 4), exerting dual control of the MMPs by inhibiting both their active forms and their activation. In addition, the proteolytic activity of MMPs is inhibited by non-specific protease inhibitors, such as α₂-macroglobulin and α₁-antiprotease.

In the presence of specific stimuli, exemplified by cytokines and growth factors, MMPs can be up-regulated. Chronic activation of MMPs, due to an imbalance between the activity of MMPs and TIMPs, can result in excessive degradation of the extracellular matrix and is believed to contribute to the pathogenesis of several diseases, such as rheumatoid arthritis, osteoarthritis, periodontal disease, emphysema, atherosclerosis, skin ulceration, and cancer.

The physiological and pathophysiological roles of MMPs and TIMPs have been extensively studied in knockout mice [67]. For most of the MMPs and TIMPs there seem to be significant overlaps in functions, and a deficiency of one enzyme can be compensated for by the presence of others [67,68]. Each MMP is encoded by a distinct gene, and about half of the human MMP genes so far discovered are on chromosome 11.

The mere fact that MMPs might be involved in the pathogenesis of several chronic disorders has made this field attractive to numerous pharmaceutical companies. One major approach for controlling abnormal MMP activity has been the use of small molecular weight inhibitors, and several excellent reviews of the design of such inhibitors have been published [67].

Structural Sites

Metalloproteases frequently use either disulfides or calcium ions to aid in stabilizing the structure of the enzyme. Structural metal sites have four protein ligands and no metal-bound water molecule. Cys ligands, followed by His, are the preferred ligands in such sites in zinc metalloenzymes. However, only one Cys-containing zinc site is observed in the class of metalloproteases. Human picornavirus endoprotease 2A binds one zinc to three Cys and one His in the sequence Cx₁Cx₅₇Hx₁C. One other metal site that fits the criteria for a structural zinc site has been observed in the matrix metalloproteases and has a signature of Hx₁Dx₁₂Hx₁₂H (Figure 1). This site may indirectly affect the activity of the enzyme. The amino acids adjacent to the third and fourth His ligands to the structural zinc site provide a number of hydrophobic residues that border a catalytic Glu residue. These residues could provide a hydrophobic environment for the Glu carboxylate group and thus raise its pK_a and allow it to play a role in stabilizing the transition state in catalysis. The zymogen form of the MMPs has a Cys ligand in place of the zinc-bound water in the catalytic zinc site, thus having the properties of a structural zinc site.

2.12 MMP-14

MMP-14 (also known as MT1-MMP) is inhibited by TIMP-2, but not TIMP-1, and activates MMP-2 and MMP-13.^{29,74–77,164}

MMP-14 mRNA was increased in carotid plaques compared to healthy arteries and was also significantly increased in vulnerable compared to stable looking plaques³¹ and found within the vulnerable shoulder regions of these plaques.^165,166 MMP-14 is the dominant MT-MMP in both monocytes and macrophages and promotes monocyte invasion and recruitment.^167,168 Interestingly, carrying at least one allele of +7096T>C polymorphism in the MMP-14 gene has been associated with lower risk of a vulnerable plaque in the carotid artery, implying a role for this MMP in plaque stability.¹⁶⁹

MMP-14-deficient mice die by 3 weeks of age, so in order to study the effect of MMP-14 loss in macrophages Schneider et al.¹⁷⁰ created a mouse model using LDL-deficient mice that were irradiated and the bone marrow repopulated with either normal or MMP-14-deficient bone marrow. Deletion of macrophage MMP-14 in this way did not alter aortic root plaque size after 16 weeks of high-fat feeding. It did, however, increase plaque interstitial collagen. The deficient macrophages were found to have less collagenase activity compared to those with MMP-14. The aortic plaques with MMP-14-deficient macrophages showed less activation of MMP-13, but there was no change in activation of MMP-2 or MMP-8. On the other hand, increasing the amount of MMP-14 in the macrophages using anti-microRNA-24 promoted macrophage invasion and increased plaque size and markers of instability.¹⁷¹

Meanwhile, in hypercholesterolemic rabbit, aortic atherosclerotic lesions the amount of MMP-14 detected were correlated to the severity of the atherosclerotic plaques present.^81,164 Although conversely Liu and coworkers found that MMP-14 decreased as aortic atherosclerotic lesions developed in rabbits,⁹⁵ MMP-14 was found in vulnerable, macrophage-rich areas of the atherosclerotic plaques, where it correlated with levels of MMP-2 and COX-2.⁸¹ MMP-14 interacts with LOX-1 to activate signaling pathways in the presence of oxidized LDL. MMP-14 can act via a wide range of downstream pathways, including RhoA/Rac1, ROS generation, and Akt signaling.¹⁷² MMP-14 can cleave ApoA-IV, and therefore remove its antioxidant effect.¹⁰³ Together, these studies imply a proatherogenic role for MMP-14.

Endocytosis of ECM-Degrading Metalloproteinases

For MT-MMPs, endocytosis is a crucial regulatory mechanism. Some soluble enzymes are also known to be endocytosed via interaction with membrane proteins. In general, endocytosis is considered as a step of down regulation, but in some cases it is necessary for the enzyme to express their biologic function. It has been shown that MT1-MMP is endocytosed via both clathrin- and caveolae-dependent pathways.^60,61 The clathrin-dependent pathway is faster than the caveolae-dependent pathway, but it was also shown that clathrin-dependent endocytosis is required for MT1-MMP to promote cellular migration and invasion.⁶⁰ In the cytoplasmic domain of MT1-MMP, there is the LLY⁵⁷³ motif that is recognized by adaptor protein 2, a subunit of clathrin, and it is crucial for the enzyme to be endocytosed in this mechanism.⁶⁰ Just downstream of LLY⁵⁷³, there is a Cys⁵⁷⁴, which is post-translationally palmitoylated.⁶² Interestingly, this palmitoylation was found to be crucial for the enzyme to be endocytosed via clathrin-dependent mechanism as well as for its cell migration promoting effect.⁶² MT1-, MT3-, and MT5-MMP are endocytosed and recycled back to cell surface, and three amino acid motifs of DKV⁵⁸² (MT1-MMP); EWV⁶⁰⁷ (MT3-MMP); and EWV⁶⁴⁵ (MT5-MMP) at their C-terminus were identified as recycling motifs.^63,64

Endocytosis is the regulatory mechanism not only for MT-MMPs, but also for soluble enzymes. It was discovered that several ECM-degrading metalloproteinases and inhibitors are endocytosed via low-density lipoprotein receptor-related protein 1 (LRP1). LRP-1 is synthesized as a 600 kDa precursor that is processed by proprotein convertases during secretion to the cell surface, resulting in a 515 kDa α chain and an 85 kDa β chain associated noncovalently. The α chain contains four extra-cellular binding regions for various molecules. It binds to extra-cellular molecules, rapidly endocytosed through clathrin-dependent mechanisms, and molecules bound to LRP-1 are degraded in lysosome. LRP-1 is widely expressed in different cell types and controls extra-cellular levels of numerous biologically active molecules to maintain tissue homeostasis.⁶⁵ Currently, more than 50 ligands have been characterized, including lipoproteins, ECM proteins, growth factors, cell surface receptors, proteinases, proteinase inhibitors, and secreted intra-cellular proteins.⁶⁵ In cartilage, it was shown that LRP-1 controls the Wnt/b-catenin signaling pathway by interacting with frizzled-1⁶⁶ and connective tissue growth factor (CCN2), and both regulate endochondral ossification and articular cartilage regeneration.⁶⁷ It has been shown that LRP-1 is also responsible for clearing extra-cellular MMP-2, MMP-9, MMP-13, ADAMTS5, TIMP-1, and TIMP-3.^68–71 LRP-1 was also reported to endocytose tPA, PAI-1, and uPA, and cathepsin D also binded to LRP-1 ectodomain.⁶⁹ Endocytic regulation of ECM-degrading enzymes in arthritic tissues is becoming an important area that directly influences disease progression, and further investigation is required.

Distinguishing Features

hMP1 cleaves dynorphin B-29 N-terminal to Arg in the monobasic processing site as evidenced by MALDI-TOF mass spectrometry. However, the enzyme does not exhibit strict monobasic cleavage specificity since peptide substrates with amino acid substitutions around the monobasic site are also efficiently cleaved by hMP1. The hMP1 mRNA is expressed in a number of human tissues; expression is higher in muscle and heart compared to other tissues. hMP1 exhibits neutral pH optimum and high sensitivity to thiol reagents. It is inactivated by 1,10-phenanthroline, a specific inhibitor of Zn²⁺-dependent metalloproteases. These results suggest that hMP1 is a novel member of the metalloendoprotease superfamily with ubiquitous distribution that could play a broad role in general cellular regulation.

7.2.1.4.2 Pro-enzyme activation

Metalloproteinases are secreted as inactive zymogens with a structural propiece; the activation of these zymogens requires double proteolytic cleavage of the pro-domain at the N-terminal of the MMP. Initial MMP cleavage, for example, by plasmin or other serine proteases, releases a small peptide and unveils a new sequence for the second cleavage to occur. A second small peptide is subsequently removed and the enzyme is fully activated with the catalytic site free to interact with the targets. Discussed in the next section, MMP activity is regulated by interactions with members of the TIMP family. Remarkably, TIMPs may also play a role in the activation process of MMPs by binding to the hemopexin-like domains of adjacent MMPs thus favoring the reciprocal activation. This interaction is of particular importance in the case of a soluble MMP activated by a MT-MMP. In this case, the soluble MMP is brought on the membrane of the cell and this localization can favor the cell migration through the newly degraded ECM [15,18,21,37].

2.2.1 Structure/Regulation

ADAMs contain a propeptide and catalytic domain, similar to MMPs. In addition, ADAMs have a disintegrin-binding region, which can interact with integrins and mediate cell–ECM interactions; a cysteine-rich domain; an epidermal growth factor (EGF)-like region; a transmembrane region (similar to MT-MMPs), which anchors the ADAM to the cell membrane; and a cytoplasmic tail, which for some ADAMs has been found to mediate intracellular signaling.⁸

Similar to MMPs, ADAMs are regulated by gene expression, pro-ADAM activation, and enzyme inactivation. For most ADAMs, the prodomain is removed intracellularly by the proprotein convertase furin.⁸ The prodomain of ADAM8 and -28, however, is thought to be removed by autocatalysis due to mutations in the binding site of the catalytic domain.²³ ADAM activity, as well as localization, has also been found to be induced by a variety of stimuli, including G protein-coupled receptors and protein kinase C (PKC) signaling, likely through phosphorylation of the cytoplasmic domain.^8,24

ADAMs are inhibited by TIMPs; however, this inhibition is mediated primarily by TIMP3.²⁵ For example, TIMP3 is thought to be the physiological inhibitor of both ADAM10 and ADAM17.¹⁵ Further, TIMP2 and TIMP4 are capable of inhibiting some ADAMs, but no evidence exists of a role for TIMP1 in ADAM inhibition.²⁵ It has been suggested that TIMPs may bind ADAMs via multiple interactions, using both inhibitory N-terminus and the C-terminus, which has been found to be involved in protein–protein interactions.²⁶

2 Classification, Structure, and Function of MMP

Based on substrate preference, MMP are grouped into collagenases, gelatinases, stromelysins, matrilysins, membrane-type (MT)-MMP, and others (Table 1.1).

Collagenases (MMP-1, -8, -13, and -18 in Xenopus) cleave interstitial collagens I, II, and III into characteristic 3/4 and 1/4 fragments and can digest other ECM molecules and soluble proteins. Recent studies indicate that MMP-1 activates protease-activated receptor 1 by cleaving the same thrombin-sensitive Arg-Ser bond promoting growth and invasion of breast carcinoma cells [11]. Two other matrixins, MMP-2 and MMP-14 (MT1-MMP), have collagenolytic activity, but they are classified into other subgroups because of their domain composition.

Gelatinases (MMP-2 and -9) readily digest gelatin with the help of their three fibronectin type II repeats that bind to gelatin/collagen. They also digest a number of ECM molecules including type IV, V, and XI collagens, laminin, aggrecan core protein, etc. MMP-2, but not MMP-9, digests collagens I, II, and III in a manner similar to collagenases [12,13]. The collagenolytic activity of MMP-2 is much weaker than MMP-1 in solution. Interestingly, proMMP-2 is recruited to the cell surface and activated by the membrane-bound MT-MMP. As such, it may express reasonable collagenolytic activity on or near the cell surface.

Stromelysins (MMP-3, -10, and -11) have a domain arrangement similar to collagenases but do not cleave interstitial collagens. MMP-3 and -10 are similar in structure and substrate specificity, but MMP-11 (stromelysin 3) is distantly related. The MMP-11 gene is located on chromosome 22, whereas MMP-3 and -10 genes are located on chromosome 11, along with MMP-1, -7, -8, -12, -20, -26, and -27. MMP-3 and -10 digest a number of ECM molecules and participate in proMMP activation. MMP-11, on the other hand, has very weak activity toward ECM molecules [14], but cleaves serpins more readily [15]. MMP-11 has a furin recognition motif RX[R/K]R at the C-terminal end of the propeptide, and therefore, it is activated intracellularly [16]. An intracellular 40-kDa MMP-11 isoform (h-stromelysin 3) is found in cultured cells and placenta [9]. This transcript results from alternative splicing and promoter usage thus lacking the signal peptide and prodomain. The function of this isoenzyme is not known.

Matrilysins (MMP-7 and -26) lack a hemopexin domain. MMP-7 is synthesized by epithelial cells and is secreted apically. Besides ECM components, it processes cell surface molecules such as pro-a-defensin, Fas-ligand, protumor necrosis factor a, and E-cadherin. MMP-26 is expressed in normal cells such as those of the endometrium and in some carcinomas. It digests several ECM molecules, and unlike most other MMP, it is largely stored intracellularly [17].

MT-MMP in mammals includes four type I transmembrane proteins (MMP-14, -15, -16, and -24) and two glycosylphosphatidylinositol-anchored proteins (MMP-17 and -25). They all possess a furin recognition sequence RX[R/K]R at the propeptide C-terminus. They are activated intracellularly, and active enzymes are likely to be expressed on the cell surface. All MT-MMP, except MT4-MMP (MMP-17) [18], can activate proMMP-2. MT1-MMP (MMP-14) has collagenolytic activity on collagens I, II, and III [19]. MT1-MMP null mice exhibit skeletal abnormalities during postnatal development attributed to the lack of collagenolytic activity [20].

Seven MMP are not grouped in the above categories although MMP-12, -20, and -27 have similar structures and chromosomal location as stromelysins. Metalloelastase (MMP-12) is not only expressed primarily in macrophages but also found in hypertrophic chondrocytes [21] and osteoclasts [22]. It digests elastin and a number of ECM molecules. MMP-19 digests many ECM molecules including the components of basement membranes [23]. Enamelysin (MMP-20) is expressed in newly formed tooth enamel and digests amelogenin [24]. MMP-21 was originally found in Xenopus [25] and more recently in mice and humans [26]. It is expressed in various fetal and adult tissues and in basal and squamous cell carcinomas [27]. It digests gelatin, but information on its action on ECM components is not known. MMP-23 is a unique member as it has unique a C-terminal cysteine-rich immunoglobulin-like region instead of a hemopexin domain [28]. The propeptide lacks a cysteine switch. It is proposed to be a type II membrane protein having a transmembrane domain at the N-terminal of the propeptide, but the enzyme is released from the cell as the membrane-anchored propeptide cleaved by proprotein convertase [29].

MMP-27 was first found in chicken embryo fibroblasts [30]. MMP-27 digests gelatin and casein and causes autolysis of the enzyme, but little information is available on the activity of mammalian enzyme.

Epilysin (MMP-28) is expressed in many tissues such as lung, placenta, heart, gastrointestinal tract, and testis. The enzyme expressed in basal keratinocytes in skin is considered to function in wound repair [31]. MMP-21, -23, and -28 have a furin recognition sequence that precedes the catalytic domain and are therefore likely activated intracellularly and secreted as active enzymes.

A disintegrin and metalloproteinases (ADAM) family of transmembrane proteins also belong to the zinc protease superfamily. Members have a modular design, characterized by the presence of metalloprotease and integrin receptor-binding activities, and a cytoplasmic domain that in many family members specifies binding sites for various signal transducing proteins. The ADAM family has been implicated in the control of membrane fusion, cytokine and growth factor shedding, and cell migration, as well as processes such as muscle development, fertilization, and cell fate determination. Pathology such as inflammation and cancer also involve ADAM family members [32].

II.C.1 Proteases and Their Inhibitors

Matrix metalloproteases (MMPs) are proteases whose expression may be regulated in function of cell or tissue behavior. The expression of most MMPs is transcriptionally regulated by growth factors and cellular transformation. They contribute to tissue remodeling and have a key role in the migration of normal and malignant cells through the body. Their functions are associated with degradation of the extracellular matrix and of the basement membrane, which is a specialized matrix barrier essentially composed of type IV collagen, laminins, entactin glycosaminoglycans, and proteolycans. The breakdown of this lamina by MMPs is required for cell invasion and for metastatic process. MMP2 and MMP9, the so-called gelatinase-A and -B, are secreted type IV collagenases, mainly involved in the latter process. These MMPs may be produced by the tumor cells themselves, but there is growing evidence that the normal host stromal cells respond to tumor cells by inducing MMPs.

MMP activity is regulated at transcriptional and processing levels and can also be blocked by direct association of the protein and physiological MMP inhibitors, termed tissue inhibitor of metalloproteases (TIMP), generating an inactive MMP/TIMP complex. The degradative potential is the result of the balanced or imbalanced production of these molecules.

Evidence has shown that cells focus proteolytic activities at their cell surface and essentially at their leading edge to help remove extracellular matrix barriers for migration during their invasion. Membrane-bound MMPs, termed Mt-MMP, have been identified, and Mt1-MMP was shown to be predominantly localized in the invadopodia or lamellipodia of invading cells. Furthermore, Mt1-MMP could form cell surface complexes with TIMP that behave as MMP2 receptors.