Mutational and Structural Analyses of the Hinge Region of Membrane Type 1-Matrix Metalloproteinase and Enzyme Processing

Osenkowski, Pamela; Meroueh, Samy O.; Pavel, Dumitru; Mobashery, Shahriar; Fridman, Rafael

doi:10.1074/jbc.m414379200

Cited by 14 publications

(13 citation statements)

References 85 publications

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“…S7A). Interestingly, a similar cleavage mechanism was also observed in the hinge domain of MT1-MMP, which contains a highly exposed loop and is the prime target for proteolysis (32). The aforementioned result provides evidence that this conformational change induces the instability of MMP-2.…”

Section: Discussionsupporting

confidence: 54%

The Regulatory Mechanism of Extracellular Hsp90α on Matrix Metalloproteinase-2 Processing and Tumor Angiogenesis

Song

Wang

Zhuo

et al. 2010

Journal of Biological Chemistry

108

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Heat shock protein 90␣ (Hsp90␣) is a ubiquitously expressed molecular chaperone that is essential for eukaryotic homeostasis. Hsp90␣ can also be secreted extracellularly, where it has been shown to be involved in tumor metastasis. Extracellular Hsp90␣ interacts with and promotes the proteolytic activity of matrix metalloproteinase-2 (MMP-2). However, the regulatory mechanism of Hsp90␣ on MMP-2 activity is still unknown. Here we show that Hsp90␣ stabilizes MMP-2 and protects it from degradation in tumor cells. Further investigation reveals that this stabilization effect is isoform-specific, ATP-independent, and mediated by the interaction between the Hsp90␣ middle domain and the MMP-2 C-terminal hemopexin domain. Moreover, this mechanism also applies to endothelial cells that secrete more Hsp90␣ in their proliferating status. Furthermore, endothelial cell transmigration, Matrigel plug, and tumor angiogenesis assays demonstrate that extracellular Hsp90␣ promotes angiogenesis in an MMP-2-dependent manner. In sum, this study provides new insights into the molecular mechanism of how Hsp90␣ regulates its extracellular client proteins and also reveals for the first time the function of extracellular Hsp90␣ in promoting tumor angiogenesis.Heat shock protein 90 (Hsp90) 2 is an ATP-dependent molecular chaperone that is ubiquitously expressed and essential for cell viability (1). Unlike other types of chaperones, Hsp90 is not required for the biogenesis of most polypeptides but instead functions in the maintenance of the active state of several conformationally labile signaling proteins (2-4). Many of the Hsp90 client proteins are mutated, chimeric, or overexpressed oncogenic proteins (3). Therefore, the chaperoning function of Hsp90 is subverted to a biochemical buffer for genetic lesions in tumor cells, facilitating the malignant transformations of the cells (3). Hsp90 has emerged as a promising target for cancer therapy (5).There are two isoforms of Hsp90 in the cytosol, referred to as Hsp90␣ and Hsp90␤ (6). Intriguingly, the Hsp90␣ isoform also exists extracellularly (7). Recent studies indicate that extracellular Hsp90␣ is significantly correlated with tumor invasiveness and metastasis (8), and the antibody or impermeable inhibitor of Hsp90␣ can suppress tumor metastasis efficiently in mouse models (9 -11). Furthermore, Hsp90␣ can be detected in the blood of cancer patients, and the level of Hsp90␣ is positively associated with tumor malignancy (9). In addition to tumor cells, extracellular Hsp90␣ has also been identified in neuron cells, dermal fibroblasts, keratinocyte, macrophages, and epithelial cells and participates in neuronal cell migration, wound healing, and viral and bacteria infections (7).Accumulating evidence indicates that extracellular Hsp90␣ plays important roles in both physiological and pathological processes, especially in tumor progression (7). However, the molecular mechanism of how extracellular Hsp90␣ functions is still largely unknown (12). Eustace et al. (8) reported that extracellular Hsp90...

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Section: Discussionsupporting

confidence: 54%

The Regulatory Mechanism of Extracellular Hsp90α on Matrix Metalloproteinase-2 Processing and Tumor Angiogenesis

Song

Wang

Zhuo

et al. 2010

Journal of Biological Chemistry

108

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“…The fact that 44⌬H lacks the hinge region suggests that this domain may play a role in regulating the function of the 44-kDa processed form. Accumulating evidence suggests that the hinge of MT1-MMP plays an important role in enzyme activity and stability because of its high mobility and O-glycosylation, respectively (25,36,60,81). Indeed, 44-MT1 is O-glycosylated, whereas 44⌬H is not (data not shown).…”

Section: Discussionmentioning

confidence: 99%

“…Interestingly, a FLAGtagged 44-kDa species lacking the hinge, which inhibited pro-MMP-2 activation in HT1080 cells, was found to be processed to a smaller form (57), possibly because of the lack of O-glycosylation. Because the hinge is the main target of autocatalytic processing (81), its presence in the 44-kDa form may also contribute to the stabilization of active MT1-MMP by interfering with processing, as proposed previously (59). On the other hand, significant deletions within the hinge region and/or presence of N-terminal tags may cause conformational or structural changes in the 44-kDa protein, which in turn may disrupt active MT1-MMP-44-kDa interactions leading to inhibition of enzyme function.…”

Section: Discussionmentioning

confidence: 99%

The Inactive 44-kDa Processed Form of Membrane Type 1 Matrix Metalloproteinase (MT1-MMP) Enhances Proteolytic Activity via Regulation of Endocytosis of Active MT1-MMP

Cho

Osenkowski

Zhao

et al. 2008

Journal of Biological Chemistry

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Membrane type 1 (MT1) matrix metalloproteinase (MMP-14)is a membrane-tethered MMP considered to be a major mediator of pericellular proteolysis. MT1-MMP is regulated by a complex array of mechanisms, including processing and endocytosis that determine the pool of active proteases on the plasma membrane. Autocatalytic processing of active MT1-MMP generates an inactive membrane-tethered 44-kDa product (44-MT1) lacking the catalytic domain. This form preserves all other enzyme domains and is retained at the cell surface. Paradoxically, accumulation of the 44-kDa form has been associated with increased enzymatic activity. Here we report that expression of a recom- Membrane type 1 matrix metalloproteinase (MT1-MMP, 2 MMP-14) is a type I-transmembrane protease and a major mediator of pericellular proteolysis. MT1-MMP is responsible for the proteolytic cleavage of multiple pericellular and membrane-associated substrates, including collagens and other extracellular matrix proteins, growth factors, growth factor receptors, cell adhesion proteins and their receptors, cytokines, protease inhibitors, and proteases, just to mention a few (1-5). MT1-MMP is also the major physiological activator of pro-MMP-2 (pro-gelatinase A) on the cell surface (6, 7), a process that further contributes to pericellular proteolysis. As a multifunctional protease, MT1-MMP elicits profound effects on cell behavior and has been implicated in the pathogenesis of various human diseases, including cancer (8 -10), diabetes (11, 12), vascular (13, 14), and connective tissue diseases (2).The importance of MT1-MMP for pericellular proteolysis demands a tight control of its catalytic activity at the cell surface. This is partly achieved by the action of endogenous protease inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), which bind to the active site inhibiting catalysis (15). In addition, by virtue of being a membrane-anchored protease, MT1-MMP developed a unique set of regulatory mechanisms that control enzymatic activity independently of TIMPs. These processes include the targeting of active enzyme to specific plasma membrane locations, endocytosis, and autocatalytic processing (1, 16 -19). Together, these distinct processes determine the level of active enzyme on the cell surface. However, how these processes are integrated to control the pool of active MT1-MMP is not understood.The processing of MT1-MMP is a cell surface event in which the active enzyme is usually autocatalytically cleaved in trans to generate a major membrane-anchored product of ϳ44 kDa (also referred to as the 43-or 45-kDa species in some studies) and a soluble ϳ18-kDa inactive fragment of the catalytic domain (6, 20 -25). The 44-kDa product of MT1-MMP is detected in cultured cells expressing natural MT1-MMP (20, 26 -32) and has been found in platelets (33), human tumors extracts (34 -36), and extracts of arthritic synovial tissues (37). MT1-MMP processing is stimulated by a variety of factors known to stimulate MT1-MMP expression, trafficking, and/or endocytosi...

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“…As shown in Figure 3A, Western blot analysis of surface-biotinylated proteins confirms MT1/MMP-1 CAT expression at the cell surface. Unlike wt MT1-MMP, and consistent with a change in enzymic properties, the chimeric construct does not undergo autocatalytic processing (i.e., the generation of the 43 kDa form of wt MT1-MMP; Figure 3A) (Osenkowski et al, 2005). Nonetheless, the MT1/MMP-1 CAT chimera maintains significant type I collagenolytic activity ( Figure 3B), and of note, confers the transfected COS cells with collagen-invasive activity in vitro (Figure 3, C and D).…”

Section: Mt1-mmp Drives Invasion By Functioning As a Pericellular Colmentioning

confidence: 99%

Molecular Dissection of the Structural Machinery Underlying the Tissue-invasive Activity of Membrane Type-1 Matrix Metalloproteinase

Ota

Yana

et al. 2008

MBoC

104

135

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Membrane type-1 matrix metalloproteinase (MT1-MMP) drives cell invasion through three-dimensional (3-D) extracellular matrix (ECM) barriers dominated by type I collagen or fibrin. Based largely on analyses of its impact on cell function under two-dimensional culture conditions, MT1-MMP is categorized as a multifunctional molecule with 1) a structurally distinct, N-terminal catalytic domain; 2) a C-terminal hemopexin domain that regulates substrate recognition as well as conformation; and 3) a type I transmembrane domain whose cytosolic tail controls protease trafficking and signaling cascades. The MT1-MMP domains that subserve cell trafficking through 3-D ECM barriers in vitro or in vivo, however, remain largely undefined. Herein, we demonstrate that collagen-invasive activity is not confined strictly to the catalytic, hemopexin, transmembrane, or cytosolic domain sequences of MT1-MMP. Indeed, even a secreted collagenase supports invasion when tethered to the cell surface in the absence of the MT1-MMP hemopexin, transmembrane, and cytosolic tail domains. By contrast, the ability of MT1-MMP to support fibrin-invasive activity diverges from collagenolytic potential, and alternatively, it requires the specific participation of MT-MMP catalytic and hemopexin domains. Hence, the tissue-invasive properties of MT1-MMP are unexpectedly embedded within distinct, but parsimonious, sequences that serve to tether the requisite matrix-degradative activity to the surface of migrating cells. INTRODUCTIONNormal as well as neoplastic cells traverse interstitial tissues by mobilizing proteolytic enzymes that dissolve intervening structural barriers that are dominated by cross-linked networks of either type I collagen or fibrin (Hiraoka et al., 1998;Chun et al., 2004;Sabeh et al., 2004;Hotary et al., 2006;. Although multiple proteinases have been implicated in tissue-invasive processes, recent studies suggest that the membrane type-1 matrix metalloproteinase (MT1-MMP) plays a critical role in conferring cells with the ability to remodel and penetrate extracellular matrix (ECM) barriers in vivo (Chun et al., 2004;Sabeh et al., 2004;Filippov et al., 2005;Hotary et al., 2006;. Nonetheless, the critical structural and functional properties that imbue MT1-MMP with proinvasive activity and that distinguish it from the bulk of the Ͼ500 proteinases encoded in the mammalian genome remain largely undefined (Puente et al., 2003).Similar in its overall domain structure to the larger family of secreted MMPs, the MT1-MMP zymogen is composed of a propeptide domain, a Zn-containing catalytic domain, a flexible linker peptide, and a hemopexin-like domain near its carboxy terminus . In contrast to the secreted MMPs, however, the MT1-MMP hemopexin domain is extended to include a glutamic acid-rich stem region that connects the extracellular face of the proteinase to a single-pass transmembrane segment that terminates in a short, 20-amino acid cytosolic tail . In its membrane-tethered configuration, studies to date have largely focused on the ability...

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Mutational and Structural Analyses of the Hinge Region of Membrane Type 1-Matrix Metalloproteinase and Enzyme Processing

Cited by 14 publications

References 85 publications

The Regulatory Mechanism of Extracellular Hsp90α on Matrix Metalloproteinase-2 Processing and Tumor Angiogenesis

The Regulatory Mechanism of Extracellular Hsp90α on Matrix Metalloproteinase-2 Processing and Tumor Angiogenesis

The Inactive 44-kDa Processed Form of Membrane Type 1 Matrix Metalloproteinase (MT1-MMP) Enhances Proteolytic Activity via Regulation of Endocytosis of Active MT1-MMP

Molecular Dissection of the Structural Machinery Underlying the Tissue-invasive Activity of Membrane Type-1 Matrix Metalloproteinase

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