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Review Review Structure and Function ofof HumanHuman MatrixMetalloproteinases

1,2 1,2, Helena Laronha 1,2 andand Jorge Jorge Caldeira Caldeira 1,2,* *

1 1 CentroCentro de de investigação investigação interdisci interdisciplinarplinar Egas Egas Moniz, Moniz, Instituto Instituto Univer Universitsitárioário Egas Egas Moniz, Moniz, Caparica 2829 Caparica, 2829, Portugal;Portugal; [email protected] [email protected] 2 UCIBIOUCIBIO and and LAQV LAQV Requimte Requimte Faculd Faculdadeade de de Ciências Ciências e eTecnologia, Tecnologia, Universidade Universidade Nova Nova de de Lisboa, Lisboa, 2829-516 Caparica,2829-516 Portugal Caparica, Portugal ** Correspondence:Correspondence: jcaldeira@egas [email protected];moniz.edu.pt; Tel. Tel.: +3519-1955-35-92+3519-1955-35-92

Received: 12 March 2020; Accepted: 21 April 2020; Published: date  Received: 12 March 2020; Accepted: 21 April 2020; Published: 26 April 2020  Abstract: The (ECM) is a macromolecules network, in which the most abundant Abstract: The extracellular matrix (ECM) is a macromolecules network, in which the most abundant is . This in triple helical conformation is highly resistant to proteinases molecule is collagen. This protein in triple helical conformation is highly resistant to proteinases degradation, the only capable of degrading the collagen are matrix metalloproteinases degradation, the only enzymes capable of degrading the collagen are matrix metalloproteinases (MMPs). This resistance and maintenance of collagen, and consequently of ECM, is involved in (MMPs). This resistance and maintenance of collagen, and consequently of ECM, is involved in several biological processes and it must be strictly regulated by endogenous inhibitors (TIMPs). The several biological processes and it must be strictly regulated by endogenous inhibitors (TIMPs). deregulation of MMPs activity leads to development of numerous diseases. This review shows The deregulation of MMPs activity leads to development of numerous diseases. This review shows MMPs complexity. MMPs complexity. Keywords: matrix metalloproteinases; TIMP; collagen Keywords: matrix metalloproteinases; TIMP; collagen

1. Extracellular Matrix—Collagen The extracellular matrix (ECM) is aa macromoleculesmacromolecules network,network, composedcomposed of collagen,collagen, enzymesenzymes and (Figure1 1),), that that promote promote a a structural structural and and biochemical biochemical support. support. The ECMECM hashas manymany componentscomponents [[1]:1]: Fibers (collagen,(collagen, , , and and ), fibronectin), (-1 and ), (, and entactin) and polysaccharides (hyaluronic()acid) [[2],2], thatthat regulateregulate cellcell migration,migration, growth,growth, andand didifferentiationfferentiation [ 3[3].].

Figure 1. Schematic representation of the membrane.

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The collagen is the most abundant protein in ECM which gives structural support for cells [1,3]. [1,3]. Depending of mineralization degree, the tissues can be divided in rigid () or compliant compliant (sinews) (sinews) or have a gradientgradient betweenbetween these two statesstates ()(cartilage) [[4].4]. Collagen Collagen can can come come in in two main forms: fibrillarfibrillar (type I, II, III, V, andand XI)XI) andand non-fibrillar,non-fibrillar, thethe latterlatter includesincludes facit-fibrilfacit-fibril associatedassociated collagenscollagens with interrupted triple helix (type IX, XII, XIV, XIX,XIX, andand XXI);XXI); shortshort chain (type VIII and X); basement membrane (type IV); multiplexin (type XV and XVIII);XVIII); MACIT- membrane associatedassociated with interrupted triple-helix (type XIII and XVII) and ot othershers types (Type VI, VII, and VIII). TheThe collagencollagen protein consistsconsists ofof threethree αα chains,chains, inin tripletriple helix,helix, wherewhere twotwo chainschains areare chemicallychemically similarsimilar ((αα11 and α2),), with approximate dimensions of 300 1.5 nm [3,5]. The triple helix is divided into five d-segments with approximate dimensions of 300 ×× 1.5 nm [3,5]. The triple helix is divided into five D-segments with D1–D4D1–D4 having a lengthlength ofof 6767 nmnm andand D5D5 equalequal toto 0.460.46 nmnm [[3,5].3,5]. TheThe highhigh glycineglycine contentcontent is important forfor collagen helix stabilization as it allows collagen fibers fibers to combine, facilitating hydrogen bridges and cross-link formationformation [[5].5]. The collagen synthesis involves several steps [5] [5] (Figure 22).). TheThe mRNAmRNA isis transcriptedtranscripted byby ribosome, forming pre-propeptide. pre-propeptide. The The following following thre threee stages stages occur occur in inthe the endoplasmic : reticulum: 1) (1)The The N-terminalN-terminal signal signal sequence sequence is isremoved; removed; 2) (2) hydroxylation hydroxylation of of lysine and and proline by by prolylprolyl hydroxylase andand lysyllysyl hydroxylaseshydroxylases takestakes place,place, togethertogether withwith (3)3) glycosylation of lysine, forming the pro-collagen. This This structure structure is is a a triple triple helix helix chain, chain, but but with with the the unwound unwound terminals. terminals. The The removal removal of ofthese these terminals terminals occurs occurs in extracellular in extracellular medium, medium, by collagen by collagen peptidases, peptidases, forming forming tropocollagen. tropocollagen. The Themicrofibril microfibril collagen collagen is formed is formed by lysyl by lysyloxidase oxidase that packs that packstogether together five tropocollagen five tropocollagen chain [3], chain which [3], whichhave a have characteristic a characteristic image. image. At intervals At intervals of 67 of nm, 67 nm, one one zone zone has has the the roll roll of of all all tropocollagen tropocollagen and another zonezone has has one one less—“gap”. less—“gap”. The The fibril fibril collagen collagen is composed is composed of various of various microfibril microfibril collagen collagen group andgroup the and alternating the alternating overlap overlap and gap and regions gap regions create the create characteristic the characteristic “bright and“bright dark” andd -bandingdark” D- patternbanding [ 3pattern]. [3].

(a) (b)

Figure 2. SynthesisSynthesis of of microfibril microfibril collagen. collagen. (a ()a In) In intracellular intracellular medium, medium, the the mRNA mRNA is transcripted is transcripted by byribosome, ribosome, forming forming the thepre-peptide, pre-peptide, which which is then is then processed processed in endoplasmic in endoplasmic reticulum, reticulum, forming forming pro- pro-collagen.collagen. (b) In (b )extracellular In extracellular medium, medium, the the pro-collagen pro-collagen is isprocessed processed by by collagen collagen peptidase, peptidase, forming tropocollagen. For microfibrilmicrofibril collagen formation,formation, thethe tropocollagentropocollagen isis processedprocessed byby lysillysil oxidase.oxidase.

Its triple helix helix conformation conformation makes makes collagen collagen resi resistantstant to to many many . proteases. The The enzymes enzymes able able to tocleave cleave this this structure structure are capthesin are capthesin K and K enzymes and enzymes with collagenolytic with collagenolytic activity activity(MMPs-1, (MMPs-1, -2, -8, -13, -2, - 14, and -18) [3,4]. The collagen type I, II and III have a specific cleavage sequence: (Gln/Leu)- Gly#(Ile/Leu)-(Ala/Pro), which is located at 3/4 of N-terminal and this is crucial for collagen

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Cells 2020, 9, x FOR PEER REVIEW 3 of 19 -8, -13, -14, and -18) [3,4]. The collagen type I, II and III have a specific cleavage sequence: (Gln/Leu)-Gly#(Ile/Leu)-(Ala/Pro), which is located at 3/4 of N-terminal and this is crucial for collagen degradation [6,7] (Figure 3). The denatured collagen is called , which can be further degraded degradation [6,7] (Figure3). The denatured collagen is called gelatin, which can be further degraded by [4,6]. The collagen degradation is involved in many biological processes, such as by gelatinases [4,6]. The collagen degradation is involved in many biological processes, such as embryogenesis, , tissue remodulation, , and [4]. embryogenesis, morphogenesis, tissue remodulation, angiogenesis, and wound healing [4].

FigureFigure 3.3.Collagen Collagen degradation.degradation. TheThe enzymeenzyme withwith collagenolyticcollagenolytic activityactivity ()(collagenases) cleavescleaves thethe tripletriple helix helix at at two two fragments: fragments: 33/4/4 N N-terminal-terminal and and 1 1/4/4 C C-terminal.-terminal. EachEach chainchain ((αα11 andandα α2)2) hashas aa specificspecific cleavagecleavage sequencesequence (#(# representsrepresents the the cleavage cleavage site). site).

TheThe ECMECM degradationdegradation isis alsoalso anan importantimportant processprocess inin development,development, morphogenesis,morphogenesis, tissuetissue repairrepair andand remodulationremodulation [[8],8], andand cancan aaffectffect the the cellular cellular behavior behavior and and phenotype phenotype [ 6[6,9].,9]. TheThe excessiveexcessive degradationdegradation ofof ECMECM leadsleads toto metabolicmetabolic andand immuneimmune diseases,diseases, cancercancer andand cardiovascularcardiovascular disordersdisorders (hypertension,(hypertension, atherosclerosis atherosclerosis and and aneurysm) aneurysm) [ 2[2,3,6].,3,6]. 2. Metzincs Superfamily—Matrix Metalloproteinases (MMPs) 2. Metzincs Superfamily—Matrix Metalloproteinases (MMPs) The metzincs are a family of multidomain -dependent , where the The metzincs are a family of multidomain zinc-dependent endopeptidases, where the metalloproteinases such as matrix metalloproteinases (MMPs) [10], the desintegrins and metalloproteinases such as matrix metalloproteinases (MMPs) [10], the desintegrins and (ADAMS), the ADAMs with thrombospondin motif (ADAMTS), were snapalysins, metalloproteins (ADAMS), the ADAMs with thrombospondin motif (ADAMTS), were snapalysins, leishmanolysins, pappalysins belong [1,6,8,11–14]. All of them have a common conserved domain: leishmanolysins, pappalysins belong [1,6,8,11–14]. All of them have a common conserved domain: HExxHxxGxxH [6,11–13,15–19] and, in case for MMPs, this sequence is HEBGHxLGLxHSBMxP [13]. HExxHxxGxxH [6,11–13,15–19] and, in case for MMPs, this sequence is HEBGHxLGLxHSBMxP [13]. The MMPs were first described in 1949 [20], as depolymerizing enzymes that facilitate tumor The MMPs were first described in 1949 [20], as depolymerizing enzymes that facilitate tumor growth, by making connective tissue stroma, including small blood vessels that are more fluid [16,20,21]. growth, by making connective tissue stroma, including small blood vessels that are more fluid In 1962, a MMP was isolated and characterized as an responsible for tadpole tail [16,20,21]. In 1962, a MMP collagenase was isolated and characterized as an enzyme responsible for resorption [1,2,6,13,14,16,21,22], by Gross and Lapiere [22]. Over the next 20 years, several enzymes tadpole tail resorption [1,2,6,13,14,16,21,22], by Gross and Lapiere [22]. Over the next 20 years, several were purified, but it was in 1985 that the area has developed more significantly [16,21]. Taken together enzymes were purified, but it was in 1985 that the area has developed more significantly [16,21]. it has been demonstrated that MMPs are present in virus, archeabacteria, bacteria, plants, , Taken together it has been demonstrated that MMPs are present in virus, archeabacteria, bacteria, and animals [6,13]. plants, nematodes, and animals [6,13]. The MMPs are a family of proteolytic enzyme that have different substrates, but which share similar The MMPs are a family of proteolytic enzyme that have different substrates, but which share structural characteristics [1,2,11,13,23]. The is zinc-dependent [1,2,6,11,13,15–18,24,25] and is similar structural characteristics [1,2,11,13,23]. The active site is zinc-dependent [1,2,6,11,13,15– highly conserved, with three residues bound to catalytic zinc [6,11,12,15] (Figure4). They are 18,24,25] and is highly conserved, with three histidine residues bound to catalytic zinc [6,11,12,15] functional at neutral pH [9,18]. (Figure 4). They are functional at neutral pH [9,18].

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Figure 4.4. Active site of matrixmatrix metalloproteinasemetalloproteinase (MMP)-1.(MMP)-1. The zinc catalytic is represented by grey ball and the three histidinehistidine residuesresidues areare representedrepresented byby sticks.sticks. MMPs have been recently recognized as biomarkers in several fields (diagnosis, monitoring, and MMPs have been recently recognized as biomarkers in several fields (diagnosis, monitoring, and treatment efficacy) [19], since their overexpression in diseases conditions is specific and elevated [19]. treatment efficacy) [19], since their overexpression in diseases conditions is specific and elevated [19]. Huang et al. [26] related that MMP-9 represents a potential biomarker which is overexpressed in several Huang et al. [26] related that MMP-9 represents a potential biomarker which is overexpressed in types of tumors (colarectal carcinoma, breast, pancreatic, ovaria, cervical, osteosarcoma non-small several types of tumors (colarectal carcinoma, breast, pancreatic, ovaria, cervical, osteosarcoma non- cell cancer (NSCLC), and giant cell tumor of (GCTB)), which makes MMP-9 a preferential small cell lung cancer (NSCLC), and giant cell tumor of bone (GCTB)), which makes MMP-9 a candidate for the early detection of these diseases [26]. The elevated levels of MMP-9 can be detected preferential candidate for the early detection of these diseases [26]. The elevated levels of MMP-9 can in plasma or blood, show triple protein levels compared to healthy patients [19,27,28]. The MMP-9 in be detected in plasma or blood, show triple protein levels compared to healthy patients [19,27,28]. contrast with other proposed biomarkers (MMP-1 and -3), also presents increased levels in specific The MMP-9 in contrast with other proposed biomarkers (MMP-1 and -3), also presents increased risk group to atherosclerosis [19], as demonstrated by Rohde et al. [29]. Nilsson et al. [30] studied the levels in specific risk group to atherosclerosis [19], as demonstrated by Rohde et al. [29]. Nilsson et MMPs-1, -3, -7, -10, and -12 in plasma and demonstrated that MMP-7 and -12 were elevated in type 2 al. [30] studied the MMPs-1, -3, -7, -10, and -12 in plasma and demonstrated that MMP-7 and -12 were diabetes, which is related to atherosclerosis and coronary events [30]. elevated in type 2 diabetes, which is related to atherosclerosis and coronary events [30]. 3. MMPs Functions 3. MMPs Functions The principal biologic function of MMPs is degradation of ECM proteins and The principal biologic function of MMPs is degradation of ECM proteins and glycoproteins glycoproteins [1,2,6,8,11,12,14,16,18,19,24,31],membrane receptors [13,14,24,31], [11,13,14,31], [1,2,6,8,11,12,14,16,18,19,24,31], membrane receptors[13,14,24,31], cytokines [11,13,14,31], and growth and growth factors [11,13,14,24,31]. The MMPs are involved in many biologic processes, such as, tissue factors [11,13,14,24,31]. The MMPs are involved in many biologic processes, such as, tissue repair and repair and remodulation [1,2,9,13,14,16,18,24], [1,2,18], embryogenesis [2,6,9,13,14], remodulation [1,2,9,13,14,16,18,24], cellular differentiation [1,2,18], embryogenesis [2,6,9,13,14], morphogenesis [2,24], cell mobility [9,18], angiogenesis [1,2,6,9,14,18,24], cell proliferation [1,2], and morphogenesis [2,24], cell mobility [9,18], angiogenesis [1,2,6,9,14,18,24], cell proliferation [1,2], and migration [1,2], wound healing [1,2,6,9,13–15,18], [1,2,18], and main reproductive events migration [1,2], wound healing [1,2,6,9,13–15,18], apoptosis [1,2,18], and main reproductive events such as ovulation [13,14] and endometrial proliferation [18]. such as ovulation [13,14] and endometrial proliferation [18]. The deregulation of MMP activity leads to the progression of various pathologies, that can The deregulation of MMP activity leads to the progression of various pathologies, that can be be grouped into [1,6,14,18,19,24,25]: (1) Tissue destruction, (2) fibrosis, and (3) matrix weakening. grouped into [1,6,14,18,19,24,25]: 1) Tissue destruction, 2) fibrosis, and 3) matrix weakening. The The overexpression of MMPs is involves in several diseases (Table1). overexpression of MMPs is involves in several diseases (Table 1). The MMPs are involved in the development and progression of atherosclerosis, which is correlated with cardiovascular diseases [19]. Their activity promotes the loss of collagen, elastin and other ECM proteins, inducing the necrotic core of atherosclerotic plaque that leads to myocardial infarction or a stroke [19].

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Table 1. Examples of diseases caused by deregulation of MMPs.

Pathologies Diseases Cancer invasion and Tissue destruction Ulcers Periodontal diseases Brain degenerative diseases Liver Fibrotic lung disease Fibroses Otosclerosis Atherosclerosis Multiple sclerosis Dilated cardiomyopathy Weakening of matrix Aortic aneurysm Varicose veins

Some studies show intracellular localization of MMP-2 in cardiac myocytes and colocalization of MMP-2 with troponin I in cardiac myofilaments [32]. The MMP-2 activity has also been detected in nuclear extracts from and rat liver [32]. Poly ADP-ribose polymerase is a nuclear matrix enzyme involved in DNA repair and is susceptible to cleavage by MMP-2, in vitro its cleavage being blocked by MMPs inhibitors [32]. The presence of MMP-2 in nucleus could play a role in poly ADP-ribose polymerase degradation and affect DNA repair [32].

4. Types of MMPs In , there are 28 different types of MMPs [1,2,8,9,11–13,16,17], at least 23 are expressed in human tissue [1]. MMPs can be subdivided according to bioinformatic analysis, in 5 types [23]: 1. Non- regulated MMPs (MMP-1, -3, -7, -8, -10, -12, -13, -20, and -27); 2. MMPs bearing three fibronectin-like inserts in the catalytic domain (MMP-2 and -9); 3. MMPs anchored to the cellular membrane by a C-terminal glycosylphosphatidylinositol (GPI) moiety (MMP-11, -17 and -25); 4. MMPs bearing a transmembrane domain (MMP-14, -15, -16, and -24) and 5. All the other MMPs (MMP-19, -21, -23, -26 and -28). But, MMPs can be also subdivided according to specificity, sequential similarity and domain organization (AppendixA) into: Collagenases, gelatinases, stromelysins, metrilysins, membrane-type MMPs, and other MMPs [1,2,8,9,12–14,16–19,25]. Collagenases (AppendixA, Table A1) cleave some ECM proteins and other soluble proteins, but the most important role of this type of MMPs is the cleavage of fibrillar collagen type I, II, III, IV and XI into two characteristic fragments, 1/4 C-terminal and 3/4 N -terminal [1,6,8,9,12,14,17,21]. This process takes place in two steps: first MMP unwind triple helical collagen and then hydrolyze the peptide bonds [1]. The domain is essential for cleaving native fibrillar collagen while the catalytic domain can cleave non-collagen substrates [1]. Gelatinases (AppendixA, Table A2) play an important role in many physiological processes, such as ECM degradation and remodeling, osteogenesis and wound healing [9]. Gelatinases degrade gelatin [12,17], collagen type IV [8,17,21], V [8,17], VIII, X, XI [8,17], and XIV, elastin [21], core proteins [8,17], fibronectin [21], laminin [17,21], fibrilin-1, and TNF-α and IL-1b precursor [9], due to the existence of three repeated fibronectin type II domain [1,8,12,17,18], which binds gelatin [1], collagen, and laminin [1]. MMP-2 is primarily a gelatinases, but can acts like collagenase, albeit in a weaker manner [1]. MMP-2 degrades collagen in two steps: first by inducing a weak -like collagen degradation and then by promoting gelatinolysis using the fibronectin-like domain [1]. MMP-9 can act as collagenases and [1]. Cells 2020, 9, 1076 6 of 18

Gelatinases are involved in physiological and pathological states, such as, embryonic growth and development, angiogenesis, vascular diseases, inflammatory, infective diseases, degenerative diseases of the brain and tumor progression [1]. Tumor metastasis is a process that involves the release of tumor cells, their migration through blood vessels, penetration into the blood and lymphatic system and their adhesion into the endothelial vessel and extravasation into tissue [11]. The activity of gelatinases is crucial for metastatic cell output and metastasis site entry [11]. Increased expression and activity of gelatinases have been described in malignant diseases such as breast, urogenital, brain, lung, skin and colorectal cancer [11]. Stromelysines (AppendixA, Table A3) have the same domain arrangement as collagenases, but do not cleave interstitial collagen [1]. MMP-3 and -10 are closely related by their structure and substrate specificity [1,8,9,17], while MMP-11 is distantly related [1]. The intracellular activation of MMP-1 is regulated by 10 amino acids insert, localized between the pro- and catalytic domains (RXRXKR), which is recognition by Golgi-associated proteinase furin. The main characteristic of the matrilysins (AppendixA, Table A4) is the lack of hemopexin domain, present in the other MMPs [9,12,17,18]. This MMP group has a specific feature in the sequence with a residue adjacent to the Zn2+- [1]. Membrane-type metalloproteinases (MT-MMP; AppendixA, Table A5) contain a furin-like pro-protein convertase recognition site (RX[R/K]R) in their pro-domain C-terminal [1,8,17,18], allowing pro-enzyme activation by proteolytic removal of this domain. They are activated intracellularly and the active enzymes are expressed on the cell surface [1]. This group can be subdivided into: type I transmembrane proteins (MMPs-14, -15, -16, and -24) [1,8,9,17,18] and glycosylphosphatidylinositol (GPI) anchored proteins (MMPs-17 and -25) [1,8,9,17,18]. The type I have about 20 amino acids long cytoplasmic tail following the transmembrane domain [1]. MT-MMPs have the insert of eight amino acids in the catalytic domain, which in case of MMP-14 consists of PYAYIREG and this sequence can influence on conformation of the active site cleft [1].

5. Structure Lovejoy et al. reported the first structure of MMP-inhibitor complex [33]. This structure reveals that the active site of MMP is a deep cavity and moreover that the catalytic domains of MMPs share a sequential similarity, where the percentage of similarity ranges between 33% (between MMP-21 and MMP23) and 86% (between MMP-3 and MMP-10) [33]. 3D structures of the catalytic domains of MMP-1 and -8 as well as structures of pro-MMP-3 and MMP-1 followed [33]. The most common structural features are (Figure5 and Table2)[1,2,8,9,13–19,23]:

1- A signal N-terminal peptide with variable length, that targets the peptide for secretion; 2- A pro-domain (with about 80 aa), which keeps MMP inactive and is removed when the enzyme is proteolytically activated; 3- A catalytic domain (with about 160 aa), with a zinc , that consists of five β-sheets, three α-helixes and three ; 4- A linker of variable length (14–69 aa), which links the catalytic domain to hemopexin-like domain—“hinge region”; 5- A hemopexin-like domain (with about 210 aa) that is characterized by four β-propeller and 6- An additional transmembrane domain with the small cytoplasmatic C-terminal domain, only present in MMPs-14, -15, -16 and -24. Cells 2020, 9, 1076 7 of 18 Cells 2020, 9, x FOR PEER REVIEW 7 of 19

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FigureFigure 5. Schematic5. Schematic representation representation of the the general general structure structure of MMP. of MMP. Table 2. Domain and presence in MMPs. Table 2. Domain and presence in MMPs.

DomainDomain Presence Presence FigureSignal 5. Schematic Peptide representation of the Allgeneral MMPs structure of MMP. All MMPs Pro-domain All MMPs Pro-domain All MMPs CatalyticTable 2. Domain and presenceAll MMPsin MMPs. CatalyticHemopexin-like All MMPs, except All in MMPsMMP-7, -23, and -26 Hemopexin-likeFibronectinDomain All MMPs, Only except PresenceMMP-2 in MMP-7, and -9 -23, and -26 FibronectinVitronectinSignal Peptide insert Only Only All MMP-2 MMPsMMP-21 and -9 VitronectinType I transmembranePro-domain insert Only MMP-14, OnlyAll MMPs -15, MMP-21 -16, and -24 Type I transmembraneCytoplasmicCatalytic Only Only MMP-14, All MMPs -15, -15, -16, -16, and and-24 -24 CytoplasmicHemopexin-likeGPI anchor All MMPs, Only Only MMP-14,exce MMP-17pt in MMP-7, -15, and -16, -23,-25 and -26 -24 Fibronectin Only MMP-2 and -9 CysteineGPI anchor Array Region Only Only MMP-17 MMP-23 and -25 Vitronectin insert Only MMP-21 IgG-like domain Only MMP-23 CysteineType Array I transmembrane Region Only MMP-14, Only -15, MMP-23 -16, and -24 IgG-like domain Only MMP-23 All MMPs are synthesizedCytoplasmic with an N-terminal Only MMP-14, signal -15, sequence,-16, and -24 which is removed in the GPI anchor Only MMP-17 and -25 endoplasmic reticulum, prCysteineoducing Array pro-enzymes Region [13]. Only MMP-23 All MMPsThe pro-domain are synthesized (FigureIgG-like 6) withcontains domain an threeN-terminal α-helixes Only signal[8,12,13,17,18]. MMP-23 sequence, In the which case of is MMP-1 removed and -2 in the endoplasmic[13], theAll first reticulum, MMPs loop hasare producing asynt cleavagehesized region—“bait-regio pro-enzymes with an N-terminal [13].n” signal[8,17]—which sequence, is proteasewhich is sensitiveremoved [12,13,18]in the The(EKRRNendoplasmic pro-domain for pro-MMP-1 reticulum, (Figure prand6oducing) contains SCN*LF pro-enzymes threefor pro-MMP-2)α -helixes[13]. [13]. [8,12 The,13, 17α(3)-helixes,18]. In the are case followed of MMP-1 by a and -2 [13],“ the firstThe switch” looppro-domain has [2,6,12,18], a cleavage (Figure 6)a region—“bait-region” containsvery conserved three α-helixes region [8,12,13,17,18]. [8 ,17(PRCGXPD)]—which In isthe[1,2,17–19,21], case of MMP-1 sensitive where and [ -212the ,13 ,18] (EKRRNsulfhydryl[13], for the pro-MMP-1 firstgroup loop (Cys has and residue) a cleavage SCN*LF coordinates region—“bait-regio for pro-MMP-2) the catalyticn” [13 [8,17]—which ].zinc The [1,2,6α(3)-helixes,8,17,18,21], is protease are forming sensitive followed a [12,13,18]tetrahedral by a “cysteine coordination sphere, thereby blocking enzyme [2,15,17,18,21]. switch”(EKRRN [2,6,12, 18for], pro-MMP-1 a very conserved and SCN*LF region for (PRCGXPD) pro-MMP-2) [13]. [1,2 ,17The–19 α(3)-helixes,21], where are the followed sulfhydryl by a group “cysteine switch” [2,6,12,18], a very conserved region (PRCGXPD) [1,2,17–19,21], where the (Cys residue) coordinates the catalytic zinc [1,2,6,8,17,18,21], forming a tetrahedral coordination sphere, sulfhydryl group (Cys residue) coordinates the catalytic zinc [1,2,6,8,17,18,21], forming a tetrahedral therebycoordination blocking enzyme sphere, [thereby2,15,17 bl,18ocking,21]. enzyme [2,15,17,18,21].

Figure 6. MMP-2 with pro-domain. (a) Surface of MMP-2, where the pro-domain is represented in orange. (b) Three-dimensional structure of MMP-2, where “bait-region” and “cysteine switch” are represented in blue. Cells 2020, 9, x FOR PEER REVIEW 8 of 19

Figure 6. MMP-2 with pro-domain. (a) Surface of MMP-2, where the pro-domain is represented in Cells 2020orange., 9, 1076 (b) Three-dimensional structure of MMP-2, where “bait-region” and “cysteine switch” are8 of 18 represented in blue.

Active site site (Figure (Figure 7)7) consists consists of of two two regions: regions: a cavity a cavity on the on surface the surface of the of protein the protein that houses that thehouses zinc theion in zinc the ioncenter in and the a center specific and site a S1 specific′ [16]. The site catalytic S10 [16 domains]. The catalyticof the known domains MMP of share the theknown same MMP structural share organization: the same structural Three α organization:-helixes, five β Three-sheetsα -helixes,(four parallel: five β β-sheets2–β1–β (four3–β5 and parallel: one anti-parallel:β2–β1–β3–β 5β and4), connected one anti-parallel: by eight βloops4), connected [8,9,12,13,16,17,19,21]. by eight loops They [8,9, 12are,13 sphere,16,17, 19shaped,,21]. They with are a diametersphere shaped, of ~40 withÅ [14]. a diameter Catalytic of domain ~40 Å [14is ].highly Catalytic conserved domain and is highlyin addition conserved to catalytic and in zinc addition ions 2+ containto catalytic another zinc ions Zn contain2+, that another has Zna structural, that has afunction structural [1,6,17,19,21], function [1,6, 17three,19,21 ],calcium three calcium ions [6,8,9,12,15,17,19,23]ions [6,8,9,12,15,17,19 and,23] andthree three histidine histidine residue residue [1,18,19,21] [1,18,19,21 that] that are are part part of of a a highly highly : VAAHEXGHXXGXXH VAAHEXGHXXGXXH [1,19,21].[1,19,21]. TheThe ΩΩ-loop-loop is isfound found between between α2-helixα2-helix and and α3-helix,α3-helix, its lengthits length and and amino amino acid acid composition composition vary vary signific significantlyantly among among MMPs MMPs [19]. [19]. This This loop loop is is the the least conserved fragmentfragment within within the the catalytic catalytic domain, domain, which which is most is likelymost responsible likely responsible for different for selectivitydifferent 2+ selectivityof MMPs [ 19of]. MMPs In the [19]. terminal In the zone terminal of the zone catalytic of the domain, catalytic located domain, 8 residues located down 8 residues from thedown Zn from[1], thethere Zn is2+ a [1], region there that is a forms region the that outer forms wall the of outer pocket wall S10 of[1 ,pocket19]—“met-turn” S1′ [1,19]—“met-turn” (XBMX) [1,13 (XBMX)]. The catalytic [1,13]. Thezinc catalytic ion and zinc the “met-turn”ion and the “met-turn” are conserved are andcons alsoerved are and present also are in present ADAM in and ADAM ADAMTS and ADAMTS families, families, andastacins bacterial and bacterial serralysins [8]. The [8]. second The second zinc ionzinc and ion theand three the three calcium calcium ions, ions, that that are arepresent present is all is MMPs,all MMPs, have have only only a structurala structural function function and and they they been been crucial crucial forfor maintainingmaintaining protein conformation [23]. [23]. Glutamic acid adjacent adjacent to to the the fi firstrst histidine residue residue is is essential for the catalytic process [[1,12,16,21].1,12,16,21].

Figure 7. MMP-1 catalytic domain. Figure 7. MMP-1 catalytic domain. Catalytic and hemopexin-like domains are linked by a proline-rich linker [9,13,17] of variable Catalytic and hemopexin-like domains are linked by a proline-rich linker [9,13,17] of variable length [18] that allows inter-domain flexibility [17], enzyme stability [13] and is involved in the length [18] that allows inter-domain flexibility [17], enzyme stability [13] and is involved in the hydrolysis of some structurally complex substrates [9,13,23] (Figure8). Mutations in the linker region hydrolysis of some structurally complex substrates [9,13,23] (Figure 8). Mutations in the linker region in MMP-1 [34] and MMP-8 [35] reduce collagenolytic activity, supporting the hypothesis that correct in MMP-1 [34] and MMP-8 [35] reduce collagenolytic activity, supporting the hypothesis that correct movement and rearrangement between the catalytic domain and the hemopexin-like domain is movement and rearrangement between the catalytic domain and the hemopexin-like domain is important for activity [17]. important for activity [17].

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Figure 8. MMP-1 catalytic domain, hemopexin-like domain and linker. FigureFigure 8. 8. MMP-1MMP-1 catalytic catalytic domain, domain, hemopexin-like hemopexin-like domain domain and and linker. linker. The hemopexin-like domain has four β-propeller structural elements [8,12,13,17]. This domain is TheThe hemopexin-likehemopexin-like domaindomain hashas fourfour ββ-propeller-propeller structuralstructural elementselements [8,12,13,17].[8,12,13,17]. ThisThis domaindomain necessary for collagen triple helix degradation [8,11,17] and is important for substrate specificity [11–13,21] isis necessarynecessary forfor collagencollagen tripletriple helixhelix degradationdegradation [8,11,17][8,11,17] andand isis importantimportant forfor substratesubstrate specificityspecificity (Figure9). It also may be essential for the recognition and subsequent catalytic degradation of fibrillar [11–13,21][11–13,21] (Figure (Figure 9). 9). It It also also may may be be essential essential for for the the recognition recognition and and subsequent subsequent catalytic catalytic degradation degradation collagen, whereas the catalytic domain is sufficient for the degradation of non-collagen substrates [1]. ofof fibrillarfibrillar collagen,collagen, whereaswhereas thethe catalyticcatalytic domaindomain isis sufficientsufficient forfor thethe degradationdegradation ofof non-collagennon-collagen The only MMPs that don’t have the hemopexin-like domain are MMP-7, -23, and -26 [8,15,17]. In the substratessubstrates [1].[1]. TheThe onlyonly MMPsMMPs thatthat don’tdon’t havehave thethe hemopexin-likehemopexin-like domaindomain areare MMP-7,MMP-7, -23,-23, andand -26-26 case MMP-23, this domain is substituted by an immunoglobulin-like domain and a cysteine-rich [8,15,17].[8,15,17]. InIn thethe casecase MMP-23,MMP-23, thisthis domaindomain isis substitutedsubstituted byby anan immunoglobulin-likeimmunoglobulin-like domaindomain andand aa domain [8,15,17], located immediately after the C-terminal of the catalytic domain [17]. cysteine-richcysteine-rich domain domain [8,15,17], [8,15,17], located located immediately immediately after after the the C C-terminal-terminal of of the the catalytic catalytic domain domain [17]. [17].

Figure 9. (a) MMP-1 hemopexin-like domain, composed to 4 β-propeller (4 β- sheets and 1 α-helix). Figure 9. (a) MMP-1 hemopexin-like domain, composed to 4 β-propeller (4 β- sheets and 1 α-helix). Figure(b) Surface 9. (a) of MMP-1 pro-MMP-2-TIMP-2 hemopexin-like complex domain, (pro-domain, composed catalyticto 4 β-propeller domain and(4 β- hemopexin-like sheets and 1 α domain-helix). (b) Surface of pro-MMP-2-TIMP-2 complex (pro-domain, catalytic domain and hemopexin-like (areb) representedSurface of pro-MMP-2-TIMP-2 in green; TIMP-2 is representedcomplex (pro-d in blue).omain, catalytic domain and hemopexin-like domain are represented in green; TIMP-2 is represented in blue). domain are represented in green; TIMP-2 is represented in blue). S10 Pocket Selectivity 5.1. S1′ Pocket Selectivity 5.1. S1′ Pocket Selectivity MMPs possess six pockets (S1,S2,S3,S10,S20, and S30)[19] and the fragments of the substances MMPs possess six pockets (S1, S2, S3, S1′, S2′, and S3′) [19] and the fragments of the substances and and inhibitorsMMPs possess are consequently six pockets (S named1, S2, S3 after, S1′, theS2′, and pocket S3′) that [19]they and interactthe fragments with (P of1,P the2,P substances3,P10,P20, and and inhibitors are consequently named after the pocket that they interact with (P1, P2, P3, P1′, P2′, and P3′) inhibitors are consequently named after the pocket that they interact with (P1, P2, P3, P1′, P2′, and P3′) [2,6]. The S1, S2, and S3 pockets are unprimed pockets, which are localized on the right side of zinc ion [2,6]. The S1, S2, and S3 pockets are unprimed pockets, which are localized on the right side of zinc ion

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Cells 2020, 9, x FOR PEER REVIEW 10 of 19 P30)[2,6]. The S1,S2, and S3 pockets are unprimed pockets, which are localized on the right side of zinc ion[19]. [19 The]. The S1′, S12′0,,S and20, andS3′ pockets S30 pockets are the are primed the primed pockets, pockets, which which are localized are localized on the on left the side left sideof zinc of zincion [2,19]. ion [2, 19It ].has It hasbeen been demonstrated demonstrated that that the the S1 S′ 1is0 isthe the most most variable variable pocket pocket in in MMPs MMPs [[1,6,8,15,16],1,6,8,15,16], followedfollowed byby SS22,S, S330′,,S S1,, and S3 pockets with an equivalentequivalent degreedegree ofof variance,variance, whilewhile thethe SS220′ hashas thethe lowestlowest variability.variability. TheThe S S220′ and SS33′0 pockets are shallower than S1′0 andand are are more more exposed to solvent [[1].1]. TheThe SS33 pocketpocket maymay alsoalso contributecontribute toto substratesubstrate specificityspecificity [[1].1]. SS110′ pocket is the most important since it is a determining factor for substrate specificityspecificity [[1,2,8,16],1,2,8,16], ItsIts cavitycavity [[1,19],1,19], formedformed byby thethe ΩΩ-loop-loop [[19]19] isis aa highlyhighly hydrophobic.hydrophobic. ByBy analyzinganalyzing thethe depthdepth ofof thethe didifferentfferent SS110′ subsites, MMPs can be divided into three didifferentfferent subgroupssubgroups [[1,2,17,19,36]1,2,17,19,36] (Figure(Figure 1010):): thethe shallow,shallow, the the intermediumintermedium and and the the deepdeep pockets.pockets. ThisThis didifferentiationfferentiation isis relatedrelated toto thethe sizesize ofof aminoamino acids:acids: MMP-1 and -7-7 havehave anan ArgArg214214 andand TyrTyr214214 residue, respectively [[13,36],13,36], insteadinstead ofof aa leucineleucine presentpresent inin MMP-2,MMP-2, -3,-3, -8,-8, -9,-9, -10,-10, -12,-12, -13,-13, andand -14-14 [[16,36],16,36], givinggiving originorigin to to shallow shallow S S110′ pocketpocket [[15].15].

FigureFigure 10.10. SchematicSchematic representationrepresentation ofof didifferentfferent subgroups subgroups of of S S110′ pocket.pocket. 6. MMP Activity Regulation 6. MMP Activity Regulation MMP expression is closely controlled for transcription, secretion, activation, and inhibition of the MMP expression is closely controlled for transcription, secretion, activation, and inhibition of activated enzyme [1,2,9,14,18]. Some MMPs are not constitutively expressed by cells, in vivo, instead the activated enzyme [1,2,9,14,18]. Some MMPs are not constitutively expressed by cells, in vivo, their expression is induced by exogenous signals such as cytokines [1,14,16], growth factors [1,14,16], instead their expression is induced by exogenous signals such as cytokines [1,14,16], growth factors hormones [1,16], and changes in cell–matrix and cell–cell interactions [8,9,15]. [1,14,16], hormones [1,16], and changes in cell–matrix and cell–cell interactions [8,9,15]. MMPs are produced by various tissues and cells [1]. MMPs are secreted by connective tissue, MMPs are produced by various tissues and cells [1]. MMPs are secreted by connective tissue, pro-inflammatory and uteroplacental cells, such as fibroblast, endothelial cells, osteoblasts, vascular pro-inflammatory and uteroplacental cells, such as , endothelial cells, osteoblasts, vascular smooth muscle, macrophages, lymphocytes, cytotrophoblasts, and [1]. MMPs are smooth muscle, macrophages, lymphocytes, cytotrophoblasts, and neutrophils [1]. MMPs are synthesized as pre-proMMPs, from which the signal peptide is removed during translation to generate synthesized as pre-proMMPs, from which the signal peptide is removed during translation to proMMPs or zymogens [1]. MMPs are secreted in the form of latent precursors- “zymogens”, which generate proMMPs or zymogens [1]. MMPs are secreted in the form of latent precursors- are proteolytically activated in extracellular medium [1,2,6,13,14,16–19,24]. The important activation “zymogens”, which are proteolytically activated in extracellular medium [1,2,6,13,14,16–19,24]. The mechanism is the proteolytic removal of the pro-domain by endopeptidases, such as, other MMPs, important activation mechanism is the proteolytic removal of the pro-domain by endopeptidases, proteases, or furin [1,11–16,21]. The first proteolytic attack occurs in the “bait region” [8,19,24] such as, other MMPs, serine proteases, plasmin or furin [1,11–16,21]. The first proteolytic attack and the specificity of cleavage is dictated by a sequence found in each MMP [9,12,18]. This leads occurs in the “bait region” [8,19,24] and the specificity of cleavage is dictated by a sequence found in to the removal of part of the pro-domain, destabilizing the rest of the pro-domain [13], including each MMP [9,12,18]. This leads to the removal of part of the pro-domain, destabilizing the rest of the cysteine switch-zinc interaction [17,24], which allows intramolecular processing [8,9,12,18]—“stepwise pro-domain [13], including cysteine switch-zinc interaction [17,24], which allows intramolecular activation” [8,18] (Figure 11). One question that remains unanswered is how a simple bait-region processing [8,9,12,18]—“stepwise activation” [8,18] (Figure 11). One question that remains cleavage can result in destabilization of the pro-domain and cause conformational changes in the zone unanswered is how a simple bait-region cleavage can result in destabilization of the pro-domain and between the pro-domain and the catalytic domain [13]. cause conformational changes in the zone between the pro-domain and the catalytic domain [13].

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Figure 11. Schematic representation of MMP activation. Figure 11. Schematic representation of MMP activation. Pro-MMP-2 forms a complex with TIMP-2 through interactions of the hemopexin-like domain with Pro-MMP-2 forms a complex with TIMP-2 through interactions of the hemopexin-like domain the non-inhibitory C-terminal domain of TIMP-2 [1,2,8,17,18,25]. Formation of this complex is essential with the non-inhibitory C-terminal domain of TIMP-2 [1,2,8,17,18,25]. Formation of this complex is for pro-MMP-2 activation by MT1-MMP [8,17,18]. The complex then reaches the cell surface where it essential for pro-MMP-2 activation by MT1-MMP [8,17,18]. The complex then reaches the cell surface binds to the active site of MT1-MMP [1], via the free inhibitory N-terminal of TIMP-2 [2], orienting where it binds to the active site of MT1-MMP [1], via the free inhibitory N-terminal of TIMP-2 [2], the pro-MMP-2 pro-domain adjacent to MT1-MMP [8,17,18]. With the aid of a second MT1-MMP [2], orienting the pro-MMP-2 pro-domain adjacent to MT1-MMP [8,17,18]. With the aid of a second MT1- interactions between the MT1-MMP occur through their hemopexin-like domains, forming a quaternary MMP [2], interactions between the MT1-MMP occur through their hemopexin-like domains, forming tetrameric complex [1,17]: an MT1-MMP acts as a for the pro-MMP-2-TIMP-2 complex and a quaternary tetrameric complex [1,17]: an MT1-MMP acts as a receptor for the pro-MMP-2-TIMP-2 the other as a pro-MMP-2 activator [2,8,18]. Excess of TIMP-2 prevents this activation process by complex and the other as a pro-MMP-2 activator [2,8,18]. Excess of TIMP-2 prevents this activation inhibiting the second MT1-MMP [2,8]. process by inhibiting the second MT1-MMP [2,8]. The MMP activation can also occur by physiochemical agents, such as heat, low pH, The MMP activation can also occur by physiochemical agents, such as heat, low pH, chaotropic chaotropic agents and -modifying agents (4-aminophenylmercurin acetate, mercury chloride and agents and thiol-modifying agents (4-aminophenylmercurin acetate, mercury chloride and N- N-ethylmaleimide) [1], which cause disruption of the cysteine-Zn2+ coordination [1]. ethylmaleimide) [1], which cause disruption of the cysteine-Zn2+ coordination [1]. Pro-enzymes and active forms of MMP are controlled by the specific stereochemical binding of Pro-enzymes and active forms of MMP are controlled by the specific stereochemical binding of -specific inhibitors (TIMPs: -1, -2, -3, and -4) [1,2,6,9,13,14,16,18,21,25]and by non-specific metalloproteinase-specific inhibitors (TIMPs: -1, -2, -3, and -4) [1,2,6,9,13,14,16,18,21,25] and by non- proteinase inhibitors such as the α1-proteinase inhibitor and α2-macroglobulin [8,9,13,14,16–18,21,25]. specific proteinase inhibitors such as the α1-proteinase inhibitor and α2-macroglobulin [8,9,13,14,16– 7.18,21,25]. Catalytic Mechanism

7. CatalyticOver the Mechanism past 30 years, the detailed structural characterization of different MMPs allowed disentangling of the individual catalytic steps that occur at the active site during [23]. TheOver catalytic the past activity 30 years, requires the catalyticdetailed zincstructur ion andal characterization a molecule of flanked different by MMPs three conserved allowed histidinedisentangling and aof conserved the individual glutamate, catalytic with steps a conserved that occur methionineat the active acting site during as a hydrophobic proteolysis [23]. base to supportThe thecatalytic structure activity surrounding requires catalytic the catalytic zinc io zincn and ion a [water1,2,14 ]molecule (Figure 12flanked). In the by initialthree conserved transition stateshistidine of theand MMP-substrate a conserved glutamate, interaction, with Zn2 a+ conservedis penta-coordinated methionine with acting a substrate’s as a hydrophobic carbonyl oxygenbase to atom,support one the oxygen structure atom surrounding from the glutamate-bound the catalytic zinc water ion and [1,2,14] the three (Figure conserved 12). In histidinethe initial residues transition [1]. 2+ Hydrolysisstates of the ofMMP-substrate the interaction, begins Zn with is the penta-coordinated nucleophilic attack with of a thesubstrate’s water coordinated carbonyl oxygen with zincatom, to one the oxygen carbonyl atom carbon from of the the gl substrateutamate-bound [13,14,17 water,18], subsequentlyand the three conserved proton transfer histidine to the residues amine nitrogen[1]. Hydrolysis occurs of through the peptide the bond glutamic begins acid with residue the nucleophilic [2,8,12,13, 15atta,16ck,18 of,21 the], water promoting coordinated a gem-diol with reactionzinc to the intermediate carbonyl carbon [9,11, 23of] the with substrate a tetrahedral [13,14,17,18], geometry subsequently [1,2,13]. This proton results transfer in the to breakdown the amine ofnitrogen the substrate occurs andthrough the releasethe glutamic of a water acid moleculeresidue [2,8,12,13,15,16,18,21], [1]. The peptide is stabilizedpromoting at a thegem-diol active reaction intermediate [9,11,23] with a tetrahedral geometry [1,2,13]. This results in the breakdown of site, by interaction between N-terminal residues and S10 pocket [1,12], and by new hydrogen bonds formedthe substrate between andN the-terminal, release of glutamate a water molecule and water [1]. [2 ,The9,11 ,peptide13,15,16 ,is18 stabilized,23]. The twoat the key active steps site, in theby catalyticinteraction process between involve N-terminal a structural residues rearrangement and S1′ pocket of the [1,12], active and site by and new the hydrogen fate of the bonds two obtained formed peptidesbetween [N23-terminal,]. Particularly glutamate relevant and to water the catalytic [2,9,11,13,15,16,18,23]. mechanism is the The flexibility two key of steps the loopin the that catalytic forms process involve a structural rearrangement of the active site and the fate of the two obtained peptides the exterior of the S10 pocket [23]. The internal flexibility of the catalytic domain plays an important role[23]. forParticularly enzymatic relevant activity, butto the it is catalytic also the causemechanism of drawbacks is the flexibility related to of inhibitor the loop selectivity. that forms the exterior of the S1′ pocket [23]. The internal flexibility of the catalytic domain plays an important role for enzymatic activity, but it is also the cause of drawbacks related to inhibitor selectivity.

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FigureFigure 12.12. Catalytic mechanism. 8. Conclusions 8. Conclusions MMPs are the most important enzymes for ECM maintenance. They are also involved in several MMPs are the most important enzymes for ECM maintenance. They are also involved in several biological and pathological diseases. However, MMPs remain a challenge for science, due to their high biological and pathological diseases. However, MMPs remain a challenge for science, due to their complexity, both in terms of regulation and activity. To that end, understanding of their role in the high complexity, both in terms of regulation and activity. To that end, understanding of their role in development of diseases and their mode of action require further studies. The development synthetic the development of diseases and their mode of action require further studies. The development inhibitorssynthetic ininhibitors particular, in particular, critically depend critically on depend the full on understanding the full understanding of the structural of the structural details about details S1 0 pocket in MMPs and its interaction with the substrate. about S1′ pocket in MMPs and its interaction with the substrate. Funding:Funding: ThisThis research was was funded funded by by A AMolecular Molecular View View of Dental of Dental Restoration, Restoration, grant grantnumber number PTDC/SAU- PTDC/ SAU-BMABMA/122444/2010/122444/2010 and andby Molecular by Molecular Design Design for Dental for Dental Restauration, Restauration, grant grantnumb numberer SAICT-POL/24288/2016. SAICT-POL/24288/ 2016.

ConflictsConflicts of of Interest: Interest:The The authors authors declare declareno no conflictconflict ofof interest.interest.

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Appendix A

Table A1. Collagenases type [1,9,11,24,36].

MMP Name Substrate Production Diseases Other Information Collagen type I, II, III, VII, VIII, Sensitive to oxidative stress. Rheumatoid arthritis, Identified in 1962. X and XI, gelatin, entactin, Cells: fibroblasts, , atherosclerosis, pulmonary Collagenase-1; Interstitial MMP-1 cleaves pro-MMP-2 and -9 into its active form. 1 tenascin, aggrecan, fibronectin, endothelial cells, macrophages, emphysema, fibrosis, or Fibroblast collagenase MMP-1 expression is increased by inflammatory vitronectin, myelin basic hepatocytes, , autoimmune disease, wound cytokines (TNF-α and IL-1). protein, ovostatin, casein platelets and osteoblasts. healing and cancer Identified in 1990 and it was discovered in cDNA Collagen type I, II and III, Cells: chondrocytes, endothelial Rheumatoid arthritis, asthma, library constructed from mRNA extracted from Collagenase-2; 8 fibronectin, aggrecan and cells, activated macrophages wound healing, periodontitis peripheral leukocytes of a patient with chronic collagenase ovostatin. and smooth muscle cells. and cancer. granulocytic leukemia. MMP-8 is secreted by pro-MMP-8 form and it is activated by MMP-3 and -10 Collagen type I, II, III, IV, IX, X and XIV, isoform, , lung diseases fibronectin, laminin, aggrecan Connective tissue (cartilage and (lung injury, viral infection and MMP-13 have gelatinolytic activity. 13 Collagenase-3 core protein, gelatin, developing bone) Cells: chronic obstructive pulmonary MMP-13 active the pro-MMP-2 and -9. plasminogen, , epithelial and neuronal cells. disease), liver fibroses, cancer casein, fibrillin-1 and serine and metastasis proteinases inhibitors Organs: mammary glands, Identified in 1990s, in sequence similarity studies. MMP-18 has not been directly , lung, pancreas, ovary, Show closest identity with MMPs-1, -3, -10 and -11. 18 Collagenase-4 Collagen and gelatin linked to a specific pathological intestine, spleen, thymus, MMP-18 differs from other MMPs in its amino acids condition. prostate, colon and heart sequence contains two cleavage sites for activation.

Table A2. Gelatinases type [1,8,9,11,12,17,24,36].

MMP Name Substrate Production Diseases Other Information Collagen typeI, III, IV, V, VII and X, Cells: dermal fibroblasts, Promotion and inhibition MMP-2 expression is constitutive and gelatin, some of ECM, keratinocytes, endothelial cells, of inflammation, asthma, TNF-α and -β stimulate its 2 fibronectin, laminin, aggrecan, chondrocytes, osteoblasts, leukocytes, fibrosis, cardiovascular production, but IFN-τ suppresses its elastin, tenascin, myelin basic protein platelets and monocytes diseases and cancer production. and vitronectin Cells: neutrophils, macrophages, Collagen type IV, V, and XI, cytokines, polymorphonuclear leucocytes, Cardiovascular diseases, Identified such as neutrophil in 1974. elastin, aggrecan, , laminin, osteoblasts, epithelial cells, 9 inflammation and MMP-9 has a strongly entactin, myelin basic protein, casein, fibroblasts, dendritic cells, esophageal cancer O-glycosylated collagen type V insert , IL-8 and IL-1β granulocytes, T-cells and keratinocytes. Cells 2020, 9, 1076 14 of 18

Table A3. Stromelysin types [1,8,9,11,12,17,18,21,24,36].

MMP Name Substrate Production Diseases Other Information Collagen type I, II, III, IV, V, X Identified in 1985. MMP-3 is secreted as inactive and IX, fibronectin, gelatin, Cells: fibroblasts and platelets Arthritis, osteoarthritis, asthma, enzyme and actives pro-MMP-1, pro-MMP-13 laminin, aggrecan, vitronectin, MMP-3 has been detected in the aneurism, atherosclerosis, and gelatinases. MMP-3 retains protease 3 Stromelysin-1 entactin, tenascin, decorin, nucleus, and it may function as coronary artery diseases, capability regardless of metal center, but this myelin basic protein, ovostatin, a trans-regulator of connective periodontitis, wound healing, replace leads to sensitivity changes for different casein, osteonectin elastin and tissue growth factor. Alzheimer diseases and cancer. substrates. MMP-3 has a unique deep active site proteoglycans. that transverses the length of the enzyme. Collagen type III, IV, V, IX and Wound healing, arthritis, MMP-10 has 82% MMP-3 homologous sequence. X, proteoglycans, gelatin, Cells: keratinocytes, fibrosis, idiopathic pulmonary MMP-10 is secreted as pro-MMP-10. Actives 10 Stromelysin-2 fibronectin, laminin, elastin, macrophages and epithelium fibrosis, peripheral arterial others pro-MMPs, such as pro-collagenases. aggrecan, cassein and fibrilin-10 disease and cancer. MMP-10 plays a role in liver regeneration. No protein of major relevance to ECM can be degraded by Identified in 1990, in stromal cells surrounding MMP-11 but it degrades Cells: fibroblasts Organs: Would healing, progression of invasive breast carcinoma.MMP-11 is activated 11 - laminin receptor and serine , placenta and mammary epithelial malignancies and intracellularly by furin, since it has a furin proteinases inhibitors, glands cancer recognition sequence (RXRXKR) and secreted in α1-proteinases and active form. α1-antitrypsin inhibitors.

Table A4. Matrilysin types [8,9,11,12,17,24,36].

MMP Name Substrate Production Diseases Other Information Faz-, pro-TNF-α, E-cadherin, syndecan-1, fibronectin, laminin, elastin, MMP-7 was described such as putative uterine Cells: epithelia cells, Idiopathic pulmonary fibrosis, casein, gelatin type I, II, IV and metalloprotease- 1 in 1988. MMP-7 acts 7 - mammalian glands, liver, cancer, metastasis and V, collagen type I and IVm intracellularly in the intestine to process pancreas, prostate and skin inflammatory processes. vitronectin, entactin, tenascin, procryptidins to bactericidal forms. aggrecan, myelin, and proteoglycans MMP-26 contains a signal sequence for secretion (in vitro): collagen type IV, Carcinomas of the lung, and a prodomain with an unusual cysteine fibronectin, fibrinogen, gelatin, Matrilysin-2 or prostate and breast, switch for latency preservation. Active 26 vitronectin, α1-antipripsin, Cancer cells of epithelial origin endometase angiogenesis and tumor pro-MMP-9. MMP-26 is negatively regulated by β-casein, α2-macroglobulin and progression. TIMP-2 and -4, with TIMP-4 being more potent IGFBP-1 inhibitor. Cells 2020, 9, 1076 15 of 18

Table A5. Membrane-type metalloproteinases [1,8,9,11,12,17,24].

MMP Name Substrate Production Diseases Other Information Collagen type I, II and III; gelatin, fibronectin, laminin-1, vitronectin, cartilage Identified in 1994. MMP-14 actives the MMP-2, MT1-MMP Cells: fibroblasts, platelets and 14 proteoglycans, fibrilin-1, Cancer -8 and -13 latent forms. MMP-14 activates (Membrane-type) osteoblasts tenascin, entactin, aggrecan, pro-MMP-13 on the cell surface. α1-proteinase inhibitor and α2-macroglobulin. Laminin, fibronectin, entactin, MT2-MMP Organs: placenta, heart and Cancer (glioblastoma, ovarian Identified in 1995. MMP-15 can active MMP-2 15 aggrecan, gelatin, vibronectin (Membrane-type) brain and breast carcinoma) and -13 latent forms. and tenascin. Organs: , placenta, kidney, ovaries, intestine, prostate, MT3-MMP Gelatin, casein, collagen type Identified in 1997. MMP-16 can activate MMP-2 16 spleen, heart and skeletal Tumor invasion (Membrane-type) III, laminin and fibronectin. and -9. muscle Cells: cardiomyocytes progenitor cells In the mid-1990s, MMP-17 was cloned from a MT4-MMP Cells: leucocytes Organs: brain, Inflammatory processes, cancer human breast carcinoma cDNA library. 17 Gelatin, fibrinogen and fibrin (GPI-anchored) colon, ovaries and and tumor progression. ADAMTS-4 activator. MMP-17 cannot active pro-MMP-2. Identified in 1999 and cloned from a human brain cDNA library. MMP-24 can activate Brain tumor (astrocytomas and MT5-MMP Fibronectin, gelatin and Organs: brain, kidney, pancreas MMP-2 latent form. MMP-24 is neuro-specific 24 glioblastomas) and tumor (Membrane-type) proteoglycans and lung and contribute to neuronal circuit formation and progression and angiogenesis. plasticity. It has a role in the development of dermal neuro-immune synapses. The stem region contains three cysteine residues which may contribute to dimerization by inter- Cells: leucocytes and cancer MT6-MMP Collagen type IV, fibronectin, and intramolecular disulfide bond. MMP-25 25 tissue Organs: testicles, kidney Cancer (GPI-anchored) gelatin and proteoglycans. cannot degrade laminin-1. MMP-25 actives and skeletal muscle pro-MMP-2, but differently than the other MT-MMPS. Cells 2020, 9, 1076 16 of 18

Table A6. Other MMPs [9,12,17,21,24,36,37].

MMP Name Substrate Production Diseases Other Information Gelatin type I, elastin, fibronectin, laminin, vitronectin, Cells: chondrocytes, Chronic pulmonary disease, Macrophage proteoglycans, elastin, collagen macrophages and other stromal MMP-12 may affect the blood-brain barrier after 12 atherosclerosis, emphysema metalloelastase type I, IV and V, entactin, cells, osteoblasts. Organs: cerebral ischemia. and lung cancer. osteonectin, aggrecan, myelin, placenta fibrinogen and α1-antitripsin Collagen type I and IV, laminin Cells: leucocytes Organs: colon, and nidogen, tenascin-C intestine, ovary, testis, prostate, The MMP-19 can activate pro-MMP-9, but RASI-1 or Wound healing and arthritic 19 isoform, entactin, aggrecan, thymus, spleen, pancreas, kidney, cannot activate other latent forms (MMP-1, -2, -3, stromelysin-4 disease. fibronectin and gelatin type I, skeletal muscle, liver, lung, -13 and -14, in vitro) in vitro placenta, brain and heart MMP-20 is a tooth-specific MMP expressed in newly formed tooth enamel. MMP-20 contains a Ameloblasts, aggrecan, 20 Enamelysin Organs: dental tissue (enamel) Tooth development very basic hinge region compared to the hinge odontoblasts and amelogenin region of stromelysins (hydrophobic) or MMP-19 (acidic) Cells: leucocytes, macrophages, fibroblasts, basal and squamous Embryogenesis, pancreatic 21 Xenopus-MMP - cell Organs: ovary, kidney, lung, - cancer and tumor progression placenta, intestine, neuroectoderm, skin and brain. MMP-22 catalytic domain is closely related to 22 Chicken-MMP - - - stromelysin-3. MMP-23 lacks a signal sequence, it has a short pro-domain and the C-terminal domain is considerable shortened and shows no sequence Cysteine array Organs: ovary, testicles and similarity to hemopexin.MMP-23 is the only one 23 Gelatin - (CA)-MMP prostate that lacks the hemopexin domain, having a cysteine rich immunoglobulin-like domain.MMP-23 lacks the cysteine switch motif in propeptide. Ovarian or peritoneal Cells: B-lymphocytes Organs: endometriotic lesions, breast MMP-27 is classified as stromelysin and holds 27 - Gelatin testicles, intestine, lung and skin. cancer development and tumor 51.6% structural homology with MMP-10. progression. Cells: basal keratinocytes Organs: Tissue homeostasis and repair, epidermis. High levels- testis. 28 Epilysin Casein osteoarthritis and rheumatoid - Low levels-lungs heart, intestine, arthritis. colon, placenta and brain. Cells 2020, 9, 1076 17 of 18

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