Gelatin degradation assay reveals MMP-9 inhibitors and function of O-glycosylated domain

Vandooren, Jennifer; Geurts, Nathalie; Martens, Erik; Steen, Philippe E. Van den; Jonghe, Steven De; Herdewijn, Piet; Opdenakker, Ghislain

doi:10.4331/wjbc.v2.i1.14

Cited by 58 publications

(52 citation statements)

References 45 publications

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“…We first tested whether sarcoma cells embedded in hypoxic hydrogel remodel the matrix material. We examined the proteolytic degradation of the HI Gtn matrices using DQ Gtn (Invitrogen), which emits green fluorescence when degraded by a protease secreted by the cells (22,23). Interestingly, we observed higher fluorescence intensity in the cells cultured within the hypoxic microenvironments [480 relative fluorescent units (RFU)] compared with the nonhypoxic matrix (50 RFU) (Fig.…”

Section: Resultsmentioning

confidence: 98%

Intratumoral oxygen gradients mediate sarcoma cell invasion

Lewis

Park

Tang

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

104

114

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Hypoxia is a critical factor in the progression and metastasis of many cancers, including soft tissue sarcomas. Frequently, oxygen (O 2 ) gradients develop in tumors as they grow beyond their vascular supply, leading to heterogeneous areas of O 2 depletion. Here, we report the impact of hypoxic O 2 gradients on sarcoma cell invasion and migration. O 2 gradient measurements showed that large sarcoma mouse tumors (>300 mm 3 ) contain a severely hypoxic core [≤0.1% partial pressure of O 2 (pO 2 )] whereas smaller tumors possessed hypoxic gradients throughout the tumor mass (0.1-6% pO 2 ). To analyze tumor invasion, we used O 2 -controllable hydrogels to recreate the physiopathological O 2 levels in vitro. Small tumor grafts encapsulated in the hydrogels revealed increased invasion that was both faster and extended over a longer distance in the hypoxic hydrogels compared with nonhypoxic hydrogels. To model the effect of the O 2 gradient accurately, we examined individual sarcoma cells embedded in the O 2 -controllable hydrogel. We observed that hypoxic gradients guide sarcoma cell motility and matrix remodeling through hypoxia-inducible factor-1α (HIF-1α) activation. We further found that in the hypoxic gradient, individual cells migrate more quickly, across longer distances, and in the direction of increasing O 2 tension. Treatment with minoxidil, an inhibitor of hypoxia-induced sarcoma metastasis, abrogated cell migration and matrix remodeling in the hypoxic gradient. Overall, we show that O 2 acts as a 3D physicotactic agent during sarcoma tumor invasion and propose the O 2 -controllable hydrogels as a predictive system to study early stages of the metastatic process and therapeutic targets.hydrogel | sarcoma | hypoxia | gradients | migration S oft tissue sarcomas are a heterogeneous group of malignant cancers derived from transformed cells of mesenchymal origin (1, 2). Approximately 13,000 new cases per year are diagnosed in the United States alone, with 25-50% of patients developing recurrent and metastatic disease (3-5). Current clinical data suggest that undifferentiated pleomorphic sarcoma (UPS) is one of the most aggressive sarcoma subtypes, which frequently results in lethal pulmonary metastases that are insensitive to radio/chemotherapy. It has recently become apparent that sarcoma progression and metastasis are regulated by microenvironmental cues, such as extracellular matrix (ECM) remodeling, stiffness modulation, cellto-cell/matrix interactions, signaling factors, and spatial gradients (6-8). Of all these factors, low intratumoral oxygen (O 2 ; hypoxia) is most dramatically associated with pulmonary metastasis and poor clinical outcomes (9, 10).Intratumoral hypoxia occurs when the partial pressure of O 2 (pO 2 ) falls below 5%, and it is a commonly observed feature of many sarcomas. Regional hypoxia develops as rapidly growing tumors outstrip their blood supply and as a consequence of aberrant tumor angiogenesis. As a result, O 2 gradients develop throughout the growing tumor. Tumor hypoxia promotes c...

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Section: Resultsmentioning

confidence: 98%

Intratumoral oxygen gradients mediate sarcoma cell invasion

Lewis

Park

Tang

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

104

114

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“…We tested the enzymatic activity of APMA-or trypsin-activated MMP9 using DQ gelatin as a substrate (22). DQ gelatin is a highly quenched, fluorescein-labeled gelatin that increases in fluorescence (excitation, 485 nm; emission, 520 nm) upon cleavage by MMP9.…”

Section: Characterization Of Activated Mmp9mentioning

confidence: 99%

Biochemical characterization and structure determination of a potent, selective antibody inhibitor of human MMP9

Appleby

Greenstein

Hung

et al. 2017

Journal of Biological Chemistry

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Matrix metalloproteinase 9 (MMP9) is a member of a large family of proteases that are secreted as inactive zymogens. It is a key regulator of the extracellular matrix, involved in the degradation of various extracellular matrix proteins. MMP9 plays a pathological role in a variety of inflammatory and oncology disorders and has long been considered an attractive therapeutic target. GS-5745, a potent, highly selective humanized monoclonal antibody inhibitor of MMP9, has shown promise in treating ulcerative colitis and gastric cancer. Here we describe the crystal structure of GS-5745⅐MMP9 complex and biochemical studies to elucidate the mechanism of inhibition of MMP9 by GS-5745. GS-5745 binds MMP9 distal to the active site, near the junction between the prodomain and catalytic domain, and inhibits MMP9 by two mechanisms. Binding to pro-MMP9 prevents MMP9 activation, whereas binding to active MMP9 allosterically inhibits activity.Human matrix metalloproteinase 9 (MMP9), 3 also referred to as gelatinase B, is a member of the MMP family, which are secreted from cells as inactive zymogens. The MMP family consists of ϳ25 members that share a common domain structure. The proenzyme (pro-MMP9) contains an N-terminal propeptide domain, a zinc-containing catalytic domain with an insertion of three fibronectin type II repeats, and a C-terminal hemopexin-like domain, which is connected to the catalytic domain by an O-glycosylated linker (1). The propeptide domain contains a conserved cysteine residue that coordinates the active site zinc, preventing substrate binding and catalysis. Stepwise proteolytic cleavage of the propeptide is required to generate catalytically competent MMP9 (2, 3). A related protease, MMP3, has been shown to activate MMP9 in vitro by cleaving between residues, Glu-59/Met-60 and Arg-106/Phe-107, releasing the prodomain (4, 5). Once active, MMP9 is capable of cleaving numerous substrates (6).In vivo, MMP9 activity is critical in remodeling components of the extracellular matrix, including collagen IV and laminin in the basement membrane (7). In addition to its role in extracellular matrix remodeling, MMP9 regulates other cellular processes, and its expression and secretion are up-regulated in pathological states such as cancer and chronic inflammation (7-13). MMP9 has also been shown to activate latent cytokines and growth factors and alter both trafficking and cell surface protein expression of both myeloid and lymphoid cells (14 -17).Because of its association with disease and correlation with poor prognosis in ulcerative colitis and colorectal cancer patients, MMP9 has long been considered a potential target for therapeutic intervention. Efforts to discover and develop safe and effective drugs selectively targeting MMP9 have been difficult due to the high structural conservation of the protease active site among MMP family members. A clinical stage, broad-spectrum peptidomimetic MMP inhibitor, marimastat, failed to meet efficacy end points and led to musculoskeletal syndrome in some patients (12,18).A...

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“…1E). The inhibitory function of PEX9 was also demonstrated with a dye-quenched gelatin (DQ-gelatin) degradation assay (21). As shown in Fig.…”

Section: Resultsmentioning

confidence: 82%

“…DQ-Gelatin or Fluorigenic Peptide Degradation Assay-A DQ-gelatin degradation assay was performed as previously described (21). Briefly, to measure the degradation in fluorescence units, 0.1 nM of active MMP-9 was added to a solution of 2.5 g/ml of DQ-gelatin in a black 96-well plate.…”

Section: Mec-1 Cells and Transfectants-mentioning

confidence: 99%

Inhibition of MMP-9-dependent Degradation of Gelatin, but Not Other MMP-9 Substrates, by the MMP-9 Hemopexin Domain Blades 1 and 4

Ugarte-Berzal

Vandooren

Bailón

et al. 2016

Journal of Biological Chemistry

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Degradation and remodeling of the extracellular matrix by matrix metalloproteinases (MMPs) plays important roles in normal development, inflammation, and cancer. MMP-9 efficiently degrades the extracellular matrix component gelatin, and the hemopexin domain of MMP-9 (PEX9) inhibits this degradation. To study the molecular basis of this inhibition, we generated GST fusion proteins containing PEX9 or truncated forms corresponding to specific structural blades (B1-B4) of PEX9. GST-PEX9 inhibited MMP-9-driven gelatin proteolysis, measured by gelatin zymography, FITC-gelatin conversion, and DQ-gelatin degradation assays. However, GST-PEX9 did not prevent the degradation of other MMP-9 substrates, such as a fluorogenic peptide, ␣B crystalline, or nonmuscular actin. Therefore, PEX9 may inhibit gelatin degradation by shielding gelatin and specifically preventing its binding to MMP-9. Accordingly, GST-PEX9 also abolished the degradation of gelatin by MMP-2, confirming that PEX9 is not an MMP-9 antagonist. Moreover, GST-B4 and, to a lesser extent, GST-B1 also inhibited gelatin degradation by MMP-9, indicating that these regions are responsible for the inhibitory activity of PEX9. Accordingly, ELISAs demonstrated that GST-B4 and GST-B1 specifically bound to gelatin. Our results establish new functions of PEX9 attributed to blades B4 and B1 and should help in designing specific inhibitors of gelatin degradation. The matrix metalloprotease (MMP)5 family comprises more than 25 Zn 2ϩ -dependent proteases that mainly degrade extracellular matrix components but also signaling molecules, membrane receptors, and intracellular and nuclear proteins (1-6). MMPs play roles in normal developmental processes (embryogenesis, wound healing, and cell mobilization), as well as in many pathological conditions, such as cancer and inflammatory reactions (7,8). Indeed, the catalytic regions of MMPs have been tested as targets for cancer drugs, but most clinical trials failed (9). This was probably due to the high homology of these regions across MMPs, with the consequent low selectivity of the inhibitors and the induction of secondary effects of the drugs.MMP-9, also known as gelatinase B, is one of the most complex members of the MMP family. In contrast to gelatinase A (MMP-2), which is constitutively expressed, MMP-9 is highly regulated by numerous agonists/antagonists (10). Like other MMPs, MMP-9 is a multidomain enzyme composed of a prodomain, a catalytic domain, and a carboxyl-terminal hemopexin domain. Both MMP-2 and MMP-9 possess a domain with three fibronectin type II homology repeats that yield high affinity binding to gelatins. Only MMP-9 contains a serine-, threonine-, and proline-rich O-glycosylated domain, which confers high flexibility to the enzyme (10).The hemopexin domain of MMP-9 (PEX9) and other MMPs consists of four blade ␤-propeller structures (blades 1, 2, 3, and 4) (11). PEX9 is responsible for the interactions of MMP-9 with many molecules, including substrates, cell receptors, such as integrins and CD44, and tissue inhib...

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Gelatin degradation assay reveals MMP-9 inhibitors and function of O-glycosylated domain

Abstract: The DQ™-gelatin assay is useful in high-throughput drug screening and exosite targeting. We demonstrate that flexibility between the catalytic and hemopexin domain is functionally critical for gelatinolysis.

Cited by 58 publications

References 45 publications

Intratumoral oxygen gradients mediate sarcoma cell invasion

Intratumoral oxygen gradients mediate sarcoma cell invasion

Biochemical characterization and structure determination of a potent, selective antibody inhibitor of human MMP9

Inhibition of MMP-9-dependent Degradation of Gelatin, but Not Other MMP-9 Substrates, by the MMP-9 Hemopexin Domain Blades 1 and 4

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