Biomedical subjects

Alex Y Strongin

Publications and source records attributed to Alex Y Strongin.

At least 37 records · Page 2Linked to original sources

PubMed2005

Membrane type-1 matrix metalloproteinase (MT1-MMP) protects malignant cells from tumoricidal activity of re-engineered anthrax lethal toxin.

Protective antigen (PA) and lethal factor (LF) are the two components of anthrax lethal toxin. PA is responsible for interacting with cell receptors and for the subsequent translocation of LF inside the cell compartment. A re-engineered toxin comprised of PA and a fusion chimera LF/Pseudomonas exotoxin (FP59) is a promising choice for tumor cell surface targeting. We demonstrated, however, that in vitro in cell-free system and in cultured human colon carcinoma LoVo, fibrosarcoma HT1080 and glioma U251 cells membrane type-1 matrix metalloproteinase (MT1-MMP) cleaves both the PA83 precursor and the PA63 mature protein. Exhaustive MT1-MMP cleavage of PA83 in vitro generates several major degradation fragments with an N-terminus at Glu40, Leu48, and Gln512. In cultured cells, MT1-MMP-dependent cleavage releases the cell-bound PA83 and PA63 species from the cell surface. As a result, MT1-MMP expressing cells have less PA63 to internalize. In agreement, our observations demonstrate that MT1-MMP proteolysis of PA makes the MT1-MMP-expressing aggressive invasive cells resistant to the cytotoxic effect of a bipartite PA/FP59 toxin. We infer from our studies that synthetic inhibitors of MMPs are likely to increase the therapeutic anti-cancer effect of anthrax toxin. In addition, our study supports a unique role of furin in the activation of PA, thereby suggesting that furin inhibitors are the likely specific drugs for short-term therapy of anthrax infection.

Antigens, Bacterial↗

PubMed2004

Matrix metalloproteinase-26 is associated with estrogen-dependent malignancies and targets alpha1-antitrypsin serpin.

Proteases exert control over cell behavior and affect many biological processes by making proteolytic modification of regulatory proteins. The purpose of this paper is to describe novel, important functions of matrix metalloproteinase (MMP)-26. alpha1-Antitrypsin (AAT) is a serpin, the primary function of which is to regulate the activity of neutrophil/leukocyte elastase. Insufficient antiprotease activity because of AAT deficiency in the lungs is a contributing factor to early-onset emphysema. We recently discovered that AAT is efficiently cleaved by a novel metalloproteinase, MMP-26, which exhibits an unconventional PH(81)CGVPD Cys switch motif and is autocatalytically activated in cells and tissues. An elevated expression of MMP-26 in macrophages and polymorphonuclear leukocytes supports the functional role of MMP-26 in the AAT cleavage and inflammation. We have demonstrated a direct functional link of MMP-26 expression with an estrogen dependency and confirmed the presence of the estrogen-response element in the MMP-26 promoter. Immunostaining of tumor cell lines and biopsy specimen microarrays confirmed the existence of the inverse correlations of MMP-26 and AAT in cells/tissues. An expression of MMP-26 in the estrogen-dependent neoplasms is likely to contribute to the inactivation of AAT, to the follow-up liberation of the Ser protease activity, and because of these biochemical events, to promote matrix destruction and malignant progression. In summary, we hypothesize that MMP-26, by cleaving and inactivating the AAT serpin, operates as a unique functional link that regulates a coordinated interplay between Ser and metalloproteinases in estrogen-dependent neoplasms.

Cell Line, Tumor↗

PubMed2004

Cell-surface-associated tissue transglutaminase is a target of MMP-2 proteolysis.

MT1-MMP, a prototypic member of a membrane-type metalloproteinase subfamily, is an invasion promoting protease and an activator of MMP-2. In addition, MT1-MMP proteolysis regulates the functionality of cell-surface adhesion/signaling receptors including tissue transglutaminase (tTG). tTG is known to serve as an adhesion coreceptor for beta1/beta3 integrins and as an enzyme that catalyzes the cross-linking of proteins and the conjugation of polyamines to proteins. Here, we report that MMP-2, functioning in concert with MT1-MMP, hydrolyzes cell-surface-associated tTG, thereby further promoting the effect initiated by the activator of MMP-2. tTG, in return, preferentially associates with the activation intermediate of MMP-2. This event decreases the rate of MMP-2 maturation and protects tTG against proteolysis by MMP-2. Our cell culture, in vitro experiments, and in silico modeling indicate that the catalytic domain of MMP-2 directly associates with the core enzymatic domain II of tTG (the K(d) = 380 nM). The follow-up cleavage of the domain II eliminates both the receptor and the enzymatic activity of tTG. Our data illuminate the coordinated interplay involving the MT1-MMP/MMP-2 protease tandem in the regulation of the cell receptors and explain the underlying biochemical mechanisms of the extensive tTG proteolysis that exists at the normal tissue/tumor boundary. Our findings also suggest that neoplasms, which express functionally active MT1-MMP and, therefore, activate soluble MMP-2, can contribute to the degradation of tTG expressed in neighboring host cells. The loss of adhesive and enzymatic activities of tTG at the interface between tumor and normal tissue will decrease cell-matrix interactions and inhibit matrix cross-linking, causing multiple pathological alterations in host cell adhesion and locomotion.

Catalytic Domain↗

PubMed2004

Non-proteolytic, receptor/ligand interactions associate cellular membrane type-1 matrix metalloproteinase with the complement component C1q.

Membrane type-1 matrix metalloproteinase (MT1-MMP), a prototypic member of the membrane-tethered MMP family, is an essential component of a cellular proteolysis apparatus. Recognition of protein cleavage targets followed by proteolysis is a main function of MT1-MMP. For the first time, however, we present evidence that MT1-MMP and other structurally related membrane MMPs bind C1q, the recognition unit of the first component of complement C1 that initiates activation of the classical pathway of complement. These interactions involve the catalytic domain of MT1-MMP and the C1q globular domain. In silico modeling followed by mutagenesis and the in vitro and cell-based binding studies showed that the His(171)-Glu-Lys-Gln-Ala-Asp(176) and Val(223)-Arg-Asn(224) peptide sequences of MT1-MMP are directly involved in the binding with C1q. These sequence regions are spatially distant from the active site of the protease. As a result, the catalytically active and the catalytically latent forms of cellular MT1-MMP are both efficient in binding with C1q. In agreement, despite the MT1-MMP/C1q interactions, C1q is totally resistant to MT1-MMP proteolysis. The discovery of the unconventional, receptor/ligand-like interactions of MT1-MMP with C1q, an essential component of immunity, is a significant step toward a more complete understanding of the role of this membrane-tethered protease in cancer.

Amino Acid Sequence↗

PubMed2004

Cellular membrane type-1 matrix metalloproteinase (MT1-MMP) cleaves C3b, an essential component of the complement system.

Neoplasms have developed numerous strategies to protect themselves against the host immune system. Membrane type-1 matrix metalloproteinase (MT1-MMP) is strongly associated with many cancer types and is up-regulated in the aggressive, metastatic neoplasms. During the past few years, there has been an increasing appreciation of the important, albeit incompletely understood, role of MT1-MMP in cancer. We have discovered, using cell-free and cell-based assays in vitro, that MT1-MMP proteolysis specifically targets C3b, an essential component of the complement propagation pathway. MT1-MMP proteolysis liberates the deposited C3 activation fragments from the cell surface. The shedding of these cell-deposited opsonins by MT1-MMP inhibits the complement cascade and protects breast carcinoma MCF7 cells from direct complement-mediated injury in the in vitro tests. The functional link associating MT1-MMP with the host immune system, heretofore unrecognized, may empower tumors with an escape mechanism that contributes to the protection against the host anti-tumor immunity as well as to the survival of invading and metastatic malignant cells in the bloodstream.

Blotting, Western↗

PubMed2004

Aberrant, persistent inclusion into lipid rafts limits the tumorigenic function of membrane type-1 matrix metalloproteinase in malignant cells.

Membrane type-1 matrix metalloproteinase (MT1-MMP) is a key enzyme in cell locomotion and tissue remodeling. Trafficking to the plasma membrane and internalization into the transient storage compartment both regulate the cell surface presentation of MT1-MMP. Our data indicate that mutant MT1-MMP lacking the cytoplasmic tail is recruited to the caveolae-enriched lipid raft membrane microdomains in breast carcinoma MCF7 cells. In contrast, the wild-type protease is not permanently associated with lipid rafts. Trafficking to lipid rafts correlated with poor internalization and the persistent presentation of MT1-MMP at the cell surface. The tail mutant efficiently functioned in inducing the activation of the latent proMMP-2 zymogen, matrix remodeling, and contraction of three-dimensional collagen lattices. Recruitment of the tail mutant to lipid raft antagonized, however, the cleavage of the plasma membrane-associated E-cadherin. These events limited the contribution of the tail mutant to cell locomotion and malignant growth. It is conceivable that the tail peptide sequence plays a crucial role in the translocations of MT1-MMP across the cell and contributes to coordinated cellular functions. It is tempting to hypothesize that the mechanisms involved in trafficking of MT1-MMP to caveolin-enriched lipid rafts may be targeted in a clinically advantageous manner.

Amino Acid Substitution↗

PubMed2004

Beta-catenin regulates the gene of MMP-26, a novel metalloproteinase expressed both in carcinomas and normal epithelial cells.

There are several unorthodox features, which distinguish the non-redundant and unique novel matrix metalloproteinase-26 (MMP-26) (an enzyme that has recently evolved and does not exist in rodents but is present in humans) from other members of the MMP superfamily. This report describes our recent efforts to gain a better understanding of the mechanisms which restrict expression of MMP-26 to certain cell/tissue types. We examined transcriptional regulation of the human MMP-26 gene in normal and malignant cells. The AP-1 and Tcf-4 sites of the MMP-26 promoter appear most potent in regulating the expression of the MMP-26-luciferase chimera in HEK293 embryonic kidney and MCF7 breast carcinoma cells. Key regulators of the Wnt pathway (beta-catenin and lymphoid enhancer-binding factor/T-cell factor with which beta-catenin associates) enhanced the transcriptional activity of MMP-26 suggesting that the MMP-26 gene is a likely target of the Wnt pathway. Immunostaining, gene arrays and reverse-transcriptase polymerase chain reaction (RT-PCR) confirm the presence of MMP-26 in normal cells, including the apical epithelial conjunctiva cells of the human eye, as well as in malignant cells of epithelial origin. MMP-26 predominantly accumulates in its proenzyme form in the intracellular milieu of the transfected breast carcinoma MCF7 cells. This study brings us a step forward towards a better understanding of the unconventional role, regulation and functions of epithelial cell MMP-26 in physiological conditions and in neoplasms.

Carcinoma↗

PubMed2004

Prointegrin maturation follows rapid trafficking and processing of MT1-MMP in Furin-Negative Colon Carcinoma LoVo Cells.

Understanding the function of invasion-promoting membrane type-1 matrix metalloproteinase (MT1-MMP) is of paramount importance for understanding cancer biology. MT1-MMP is synthesized in cells as a latent zymogen that requires the cleavage of its prodomain to exert the proteolytic activity. The mature alphav integrin subunit is also generated by endoproteolytic cleavage of the alphav subunit precursor (pro-alphav). Cleavage by furin is considered to be a principal event in the activation of both MT1-MMP and pro-alphav. To elucidate the alternative activation pathway of MT1-MMP and pro-alphav, we employed furin-negative LoVo cells, which co-express MT1-MMP with integrin alphavbeta3. In these cells the MT1-MMP proenzyme was rapidly trafficked to the plasma membrane via an unconventional Brefeldin A-resistant pathway and, then, autocatalytically processed on the cell surface. Next, the MT1-MMP activity converted the cell surface-associated pro-alphav into the mature alphav integrin, represented by the disulfide-bonded heavy and light chains, and promoted the formation of the functional integrin alphavbeta3 heterodimer. These events stimulated cell motility in vitro, and malignant invasion and tumor growth in vivo. Our data suggest that in furin-negative colon carcinoma cells MT1-MMP is autocatalytically processed and the active protease then operates as a prointegrin convertase. Our findings argue strongly that the processing by furin is not a prerequisite for the activation of MT1-MMP.

Animals↗

PubMed2003

Membrane type-1 matrix metalloproteinase stimulates tumour cell-induced platelet aggregation: role of receptor glycoproteins.

1. Matrix metalloproteinase-2 (MMP-2) plays a role in agonist- and tumour cell-induced platelet aggregation (TCIPA). 2. MMP-2 is synthesized as a proenzyme and is activated at the cell surface by membrane type-1 matrix metalloproteinase (MT1-MMP, MMP-14). 3. The significance of tumour cell-associated MT1-MMP for TCIPA was investigated using human breast carcinoma MCF7 cells stably coexpressing the integrin alphavbeta3 with MT1-MMP, cells expressing alphavbeta3 alone and mock-transfected cells. 4. Western blot and zymography confirmed that alphavbeta3/MT1-MMP cells expressed MT1-MMP and efficiently processed proMMP-2 to MMP-2. 5. Aggregometry, phase-contrast and transmission electron microscopy and flow cytometry were used to characterize TCIPA induced by MCF7 cell lines. 6. The aggregating potency of cells was: alphavbeta3/MT1-MMP >alphavbeta3=mock cells, as shown by aggregometry and phase-contrast microscopy. 7. Electron microscopy revealed close, membrane-membrane interactions between activated platelets and alphavbeta3/MT1-MMP cells during TCIPA. 8. Inhibition of MMP-2 with the neutralizing anti-MMP-2 antibody (5 microg ml(-1)) and o-phenanthroline (100 microm) reduced aggregation induced by alphavbeta3/MT1-MMP cells. 9. TCIPA induced by alphavbeta3/MT1-MMP cells was also reduced by inhibiting the generation and actions of ADP with apyrase (250 microg ml(-1)) and 2-methylthio-AMP (2-MeSAMP) (30 microm), but not N(6)-methyl-2'-deoxyadenosine-3',5'-bisphosphate (MRS2179) (30 microm). 10. Flow cytometry demonstrated that TCIPA enhanced expression of glycoprotein (GP) Ib and IIb/IIIa receptors not only on platelets but also on breast cancer cells. 11. Thus, (a) human breast carcinoma cell surface-associated MT1-MMP, via activating proMMP-2, stimulates TCIPA; (b) ADP amplifies the effects of MMPs via stimulation of P2Y(12) receptors and (c) both tumour- and platelet-derived GPIb and GPIIb/IIIa are involved in the aggregatory effects of MT1-MMP.

Breast Neoplasms↗

PubMed2003

The low density lipoprotein receptor-related protein LRP is regulated by membrane type-1 matrix metalloproteinase (MT1-MMP) proteolysis in malignant cells.

We demonstrate that the presentation of LRP and the subsequent uptake of its ligands by malignant cells are both strongly regulated by MT1-MMP. Because LRP is essential for the clearance of multiple ligands, these findings have important implications for many pathophysiological processes including the pericellular proteolysis in neoplastic cells as well as the fate of the soluble matrix-degrading proteases such as MMP-2. MT1-MMP is a key protease in cell invasion and a physiological activator of MMP-2. Cellular LRP consists of a non-covalently associated 515-kDa extracellular alpha-chain (LRP-515) and an 85-kDa membrane-spanning beta-chain, and plays a dual role as a multifunctional endocytic receptor and a signaling molecule. Through the capture and uptake of several soluble proteases, LRP is involved in the regulation of matrix proteolysis. LRP-515 associates with the MT1-MMP catalytic domain and is highly susceptible to MT1-MMP proteolysis in vitro. Similar to MT1-MMP, the metalloproteinases MT2-MMP, MT3-MMP and MT4-MMP also degrade LRP. The N-terminal and C-terminal parts of the LRP-515 subunit are resistant and susceptible, respectively, to MT1-MMP proteolysis. In cells co-expressing LRP and MT1-MMP, the proteolytically competent protease decreases the levels of cellular LRP and releases its N-terminal portion in the extracellular milieu while the catalytically inert protease co-precipitates with LRP. These events implicate MT1-MMP, not only in the activation of MMP-2, but also in the mechanisms that control the subsequent fate of MMP-2 in cells and tissues.

Binding Sites↗

PubMed2003

Membrane type-1 matrix metalloproteinase (MT1-MMP) processing of pro-alphav integrin regulates cross-talk between alphavbeta3 and alpha2beta1 integrins in breast carcinoma cells.

We have recently demonstrated that in breast carcinoma MCF7 cells MT1-MMP processes the alphav, alpha3, and alpha5 integrin precursors generating the respective mature S-S-linked heavy and light alpha-chains. The precursor of alpha2 integrin subunit was found resistant to MT1-MMP proteolysis. The processing of the alphav subunit by MT1-MMP facilitated alphavbeta3-dependent adhesion, activation of FAK signaling pathway, and migration of MCF7 cells on vitronectin. To elucidate further the effects of MT1-MMP on cellular integrins, we examined the functional activity of alpha5beta1 and alpha2beta1 integrins in MCF7 cells expressing MT1-MMP. Either expression of MT1-MMP alone or its coexpression with alphavbeta3 failed to affect the functionality of alpha5beta1 integrin, and adhesion of cells to fibronectin. MT1-MMP, however, profoundly affected the cross-talk involving alphavbeta3 and alpha2beta1 integrins. In MT1-MMP-deficient cells, integrin alphavbeta3 suppressed the functional activity of the collagen-binding alpha2beta1 integrin receptor and diminished cell adhesion to type I collagen. Coexpression of MT1-MMP with integrin alphavbeta3 restored the functionality of alpha2beta1 integrin and, consequently, the ability of MCF7 cells to adhere efficiently to collagen. We conclude that the MT1-MMP-controlled cross-talk between alphavbeta3 and alpha2beta1 integrins supports binding of aggressive, MT1-MMP-, and alphavbeta3 integrin-expressing malignant cells on type I collagen, the most common substratum of the extracellular matrix.

Breast Neoplasms↗

PubMed2003

The structure and regulation of the human and mouse matrix metalloproteinase-21 gene and protein.

Matrix metalloproteinases (MMPs) play key roles in tissue remodelling under normal development and, especially, in diseases ranging from malignancies to stroke. We cloned and thoroughly characterized the novel human and mouse MMP gene encoding MMP-21. MMP-21 is the last uncharacterized MMP coded by the human genome. Human and mouse MMP-21 is the orthologue of Xenopus laevis X-MMP. The latent proenzyme of MMP-21 (569 amino acid residues) consists of the prodomain, the catalytic domain and the haemopexin-like domain, and is potentially capable of being activated in its secretory pathway to the extracellular milieu by furin-like proprotein convertases. Human MMP-21 is the probable target gene of the Wnt pathway. In addition, the expression of MMP-21 is controlled uniquely by Pax and Notch transcription factors known to be critical for organogenesis. MMP-21 is expressed transiently in mouse embryogenesis and increased in embryonic neuronal tissues. Our observations clearly indicate that there is an important specific function for MMP-21 in embryogenesis, especially in neuronal cells.

Amino Acid Sequence↗

PubMed2003

Prinomastat, a hydroxamate inhibitor of matrix metalloproteinases, has a complex effect on migration of breast carcinoma cells.

Membrane type-1 matrix metalloproteinase (MT1-MMP) and alphavbeta3 integrin have been directly implicated in tumor cell dissemination and metastasis. We have demonstrated that in the case of breast carcinoma MCF7 cells co-expressing MT1-MMP and alphavbeta3 integrin, the proteinase processes the pro-alphav integrin subunit, thus facilitating alphavbeta3 integrin maturation and cell migration on vitronectin. Our findings show that cell surface MT1-MMP is a short-lived protein with a life span in the range of several hours. In contrast, turnover of alphavbeta3 integrin is much slower. The half-life of alphavbeta3 heterodimer is about 24 hr. This large difference in life span allowed us to distinguish between the effects of MT1-MMP on cell migration brought by matrix proteolysis from those imposed through alphavbeta3 integrin maturation. We then modulated the enzyme's activity by a potent hydroxamate MMP inhibitor, Prinomastat (AG3340), to analyze the divergent effects of MT1-MMP on cell migration. Although Prinomastat immediately blocked MT1-MMP-mediated matrix degradation, the pool of MT1-MMP-modified alphavbeta3 integrin molecules was still capable of mediating cell-matrix interactions. To our considerable surprise, inhibition of MT1-MMP-dependent vitronectin proteolysis by Prinomastat allowed a several-fold increase in migration of MCF7 cells co-expressing MT1-MMP and alphavbeta3 integrin. In contrast, long-term Prinomastat inhibition of MT1-MMP-dependent pro-alphav cleavage and thus alphavbeta3 integrin maturation strongly inhibited cell motility. Our studies suggest that MT1-MMP could actually promote cell migration via modification of the cell surface receptors, including alphavbeta3 integrin, rather than facilitate cell migration through direct cleavage of the matrix proteins.

Antineoplastic Agents↗

PubMed2003

Compensation mechanism in tumor cell migration: mesenchymal-amoeboid transition after blocking of pericellular proteolysis.

Invasive tumor dissemination in vitro and in vivo involves the proteolytic degradation of ECM barriers. This process, however, is only incompletely attenuated by protease inhibitor-based treatment, suggesting the existence of migratory compensation strategies. In three-dimensional collagen matrices, spindle-shaped proteolytically potent HT-1080 fibrosarcoma and MDA-MB-231 carcinoma cells exhibited a constitutive mesenchymal-type movement including the coclustering of beta 1 integrins and MT1-matrix metalloproteinase (MMP) at fiber bindings sites and the generation of tube-like proteolytic degradation tracks. Near-total inhibition of MMPs, serine proteases, cathepsins, and other proteases, however, induced a conversion toward spherical morphology at near undiminished migration rates. Sustained protease-independent migration resulted from a flexible amoeba-like shape change, i.e., propulsive squeezing through preexisting matrix gaps and formation of constriction rings in the absence of matrix degradation, concomitant loss of clustered beta 1 integrins and MT1-MMP from fiber binding sites, and a diffuse cortical distribution of the actin cytoskeleton. Acquisition of protease-independent amoeboid dissemination was confirmed for HT-1080 cells injected into the mouse dermis monitored by intravital multiphoton microscopy. In conclusion, the transition from proteolytic mesenchymal toward nonproteolytic amoeboid movement highlights a supramolecular plasticity mechanism in cell migration and further represents a putative escape mechanism in tumor cell dissemination after abrogation of pericellular proteolysis.

Actins↗

PubMed2003

Membrane type-1 matrix metalloproteinase functions as a proprotein self-convertase. Expression of the latent zymogen in Pichia pastoris, autolytic activation, and the peptide sequence of the cleavage forms.

An understanding of the regulatory mechanisms that control the activity of membrane type-1 matrix metalloproteinase (MT1-MMP), a key proteinase in tumor cell invasion, is essential for the design of potent and safe anti-cancer therapies. A unique proteolytic pathway regulates MT1-MMP at cancer cell surfaces. The abundance of proteolytic enzymes in cancer cells makes it difficult to identify the autocatalytic events in this pathway. To identify these events, a soluble form of MT1-MMP, lacking the C-terminal transmembrane and cytoplasmic domains, was expressed in Pichia pastoris. Following secretion, the latent zymogen and active enzyme were each purified from media by fast protein liquid chromatography. Trace amounts of active MT1-MMP induced activation of the zymogen and its self-proteolysis. This autocatalytic processing generated six main forms of MT1-MMP, each of which was subjected to the N-terminal microsequencing to identify the cleavage sites. Our data indicate that MT1-MMP functions as a self-convertase and is capable of cleaving its own prodomain at the furin cleavage motif RRKR downward arrow Y(112), thus autocatalytically generating the mature MT1-MMP enzyme with an N terminus starting at Tyr(112). The mature enzyme undergoes further autocatalysis to the two distinct intermediates (N terminus at Trp(119) and at Asn(130)) and, next, to the three inactive ectodomain forms (N terminus at Thr(222), at Gly(284), and at Thr(299)). These findings provide, for the first time, a structural basis for understanding the unconventional mechanisms of MT1-MMP activation and regulation. Finally, our data strongly imply that MT1-MMP is a likely substitute for the general proprotein convertase activity of furin-like proteinases, especially in furin-deficient cancer cells.

Amino Acid Sequence↗

PubMed2002

The cytoplasmic tail peptide sequence of membrane type-1 matrix metalloproteinase (MT1-MMP) directly binds to gC1qR, a compartment-specific chaperone-like regulatory protein.

Membrane type-1 matrix metalloproteinase (MT1-MMP), a key enzyme in cell locomotion, is known to be primarily recruited to the leading edge of migrating cells. This raises a possibility that the C-terminal cytoplasmic tail of MT1-MMP interacts with intracellular regulatory proteins, which modulate translocations of the protease across the cell. Here, we demonstrated that MT1-MMP via its cytoplasmic tail directly associates with a chaperone-like compartment-specific regulator gC1qR. Although a direct functional link between these two proteins remains uncertain, our observations suggest that the transient associations of gC1qR with the cytoplasmic tail of MT1-MMP are likely to be involved in the mechanisms regulating presentation of the protease at the tumor cell surface.

Amino Acid Sequence↗

PubMed2002

A unique substrate binding mode discriminates membrane type-1 matrix metalloproteinase from other matrix metalloproteinases.

In our study, we characterized the substrate recognition properties of membrane type-1 matrix metalloproteinase (MT1-MMP; also known as MMP-14), a key enzyme in tumor cell invasion and metastasis. A panel of optimal peptide substrates for MT1-MMP was identified using substrate phage display. The substrates can be segregated into four groups based on their degree of selectivity for MT1-MMP. Substrates with poor selectivity for MT1-MMP are comprised predominately of the Pro-X-X- downward arrow-X(Hy) motif that is recognized by a number of MMPs. Highly selective substrates lack the characteristic Pro at the P(3) position; instead they contain an Arg at the P(4) position. This P(4) Arg is essential for efficient hydrolysis and for selectivity for MT1-MMP. Molecular modeling indicates that the selective substrates adopt a linear conformation that extends along the entire catalytic pocket of MT1-MMP, whereas non-selective substrates are kinked at the conserved P(3) Pro residue. Importantly, the selective substrates can be made non-selective by insertion of a proline kink at P(3), without significantly reducing overall k(cat)/K(m) values. Altogether the study provides a structural basis for selective and non-selective substrate recognition by MT1-MMP. The findings in this report are likely to explain several aspects of MT1-MMP biology.

Catalytic Domain↗

PubMed2002

Promoter characterization of the novel human matrix metalloproteinase-26 gene: regulation by the T-cell factor-4 implies specific expression of the gene in cancer cells of epithelial origin.

A novel matrix metalloproteinase-26 (MMP-26) is known to be specifically expressed in epithelial carcinomas. To facilitate studies of MMP-26 transcriptional regulation, we have cloned and characterized a 1 kb 5'-flanking region of the human MMP-26 gene. Altogether, our findings indicate that the MMP-26 promoter has distinctive structural and functional features among MMP genes. An unusual polyadenylation site proximal to the transcription-factor-binding sites protects transcription of the MMP-26 gene from the upstream promoters and represents a part of the stringent transcriptional regulation of the gene. The MMP-26 gene has a consensus TATA-box and one transcriptional start site located 60 and 35 nucleotides upstream of the translational start site, respectively. The MMP-26 promoter was able to drive luciferase expression in human A549 lung carcinoma, HT1080 fibrosarcoma and HEK293 embryonic kidney cells. The basal transcription efficiency of the MMP-26 promoter is relatively low, thereby explaining the minute expression of the gene in most cells and tissues. When compared with other MMP genes, the MMP-26 promoter contains binding sites for a few transcription factors. Sequential deletion and mutation analysis, and electrophoretic mobility-shift assay have identified the T-cell factor-4 (Tcf-4) motif and the activator protein-1 site as the major regulatory elements of the MMP-26 promoter. Since previous studies have established that the Tcf-4 transcription factor is subjected exclusively to regulation through the beta-catenin/E(epithelial)-cadherin pathway, this implies the specific expression of MMP-26 in cancer cells of epithelial origin.

Amino Acid Sequence↗