Anjum Sohail

Assessment of gelatinases (MMP-2 and MMP-9) by gelatin zymography.

Gelatin zymography is a simple yet powerful method to detect proteolytic enzymes capable of degrading gelatin from various biological sources. It is particularly useful for the assessment of two key members of the matrix metalloproteinase family, MMP-2 (gelatinase A) and MMP-9 (gelatinase B), due to their potent gelatin-degrading activity. This polyacrylamide gel electrophoresis-based method can provide a reliable assessment of the type of gelatinase, relative amount, and activation status (latent, compared with active enzyme forms) in cultured cells, tissues, and biological fluids. The method can be used to investigate factors that regulate gelatinase expression and modulate zymogen activation in experimental systems. The system provides information on the pattern of gelatinase expression and activation in human cancer tissues and how this relates to cancer progression. Interpretation of the data obtained in gelatin zymography requires a thorough understanding of the principles and pitfalls of the technique; this is particularly important when evaluating enzyme levels and the presence of active gelatinase species. If properly used, gelatin zymography is an excellent tool for the study of gelatinases in biological systems.

MT4-(MMP17) and MT6-MMP (MMP25), A unique set of membrane-anchored matrix metalloproteinases: properties and expression in cancer

The process of cancer progression involves the action of multiple proteolytic systems, among which the family of matrix metalloproteinases (MMPs) play a pivotal role. The MMPs evolved to accomplish their proteolytic tasks in multiple cellular and tissue microenvironments including lipid rafts by incorporation and deletions of specific structural domains. The membrane type-MMPs (MT-MMPs) incorporated membrane anchoring domains that display these proteases at the cell surface, and thus they are optimal pericellular proteolytic machines. Two members of the MT-MMP subfamily, MMP-17 (MT4-MMP) and MMP-25 (MT6-MMP), are anchored to the plasma membrane via a glycosyl-phosphatidyl inositol (GPI) anchor, which confers these enzymes a unique set of regulatory and functional mechanisms that separates them from the rest of the MMP family. Discovered almost a decade ago, the body of work on GPI-MT-MMPs today is still surprisingly limited when compared to other MT-MMPs. However, new evidence shows that the GPI-MT-MMPs are highly expressed in human cancer, where they are associated with progression. Accumulating biochemical and functional evidence also highlights their distinct properties. In this review, we summarize the structural, biochemical, and biological properties of GPI-MT-MMPs and present an overview of their expression and role in cancer. We further discuss the potential implications of GPI-anchoring for enzyme function. Finally, we comment on the new scientific challenges that lie ahead to better understand the function and role in cancer of these intriguing but yet unique MMPs.

Discoidin domain receptors: unique receptor tyrosine kinases in collagen-mediated signaling

The discoidin domain receptors (DDRs) are receptor tyrosine kinases that recognize collagens as their ligands. DDRs display unique structural features and distinctive activation kinetics, which set them apart from other members of the kinase superfamily. DDRs regulate cell-collagen interactions in normal and pathological conditions and thus are emerging as major sensors of collagen matrices and potential novel therapeutic targets. New structural and biological information has shed light on the molecular mechanisms that regulate DDR signaling, turnover, and function. This minireview provides an overview of these areas of DDR research with the goal of fostering further investigation of these intriguing and unique receptors.

Mismatch Repair in Methylated DNA STRUCTURE AND ACTIVITY OF THE MISMATCH-SPECIFIC THYMINE GLYCOSYLASE DOMAIN OF METHYL-CpG-BINDING PROTEIN MBD4

MBD4 is a member of the methyl-CpG-binding protein family. It contains two DNA binding domains, an amino-proximal methyl-CpG binding domain (MBD) and a C-terminal mismatch-specific glycosylase domain. Limited in vitro proteolysis of mouse MBD4 yields two stable fragments: a 139-residue fragment including the MBD, and the other 155-residue fragment including the glycosylase domain. Here we show that the latter fragment is active as a glycosylase on a DNA duplex containing a G:T mismatch within a CpG sequence context. The crystal structure confirmed the C-terminal domain is a member of the helix-hairpin-helix DNA glycosylase superfamily. The MBD4 active site is situated in a cleft that likely orients and binds DNA. Modeling studies suggest the mismatched target nucleotide will be flipped out into the active site where candidate residues for catalysis and substrate specificity are present.

MMP25 (MT6-MMP) Is Highly Expressed in Human Colon Cancer, Promotes Tumor Growth, and Exhibits Unique Biochemical Properties

MMP25 (MT6-MMP) is one of the two glycosylphosphatidylinositol-anchored matrix metalloproteinases (MMPs) that have been suggested to play a role in pericellular proteolysis. However, its role in cancer is unknown, and its biochemical properties are not well established. Here we found a marked increase in MT6-MMP expression within in situ dysplasia and invasive cancer in 61 samples of human colon cancer. Expression of MT6-MMP in HCT-116 human colon cancer cells promoted tumori-genesis in nude mice. Histologically, the MT6-MMP-expressing tumors demonstrated an infiltrative leading edge in contrast to a rounded leading edge in vector control tumors. Biochemical and biosynthesis analyses revealed that MT6-MMP displayed on the cell surface exists as a major form of 120 kDa that likely represents enzyme homodimers linked by disulfide bonds. Upon reduction, a single 57-kDa active MT6-MMP was detected. Interestingly, neither membrane-anchored nor phosphatidylinositol-specific phospholipase C-released MT6-MMPs were found to be associated with tissue inhibitor of metalloproteinases (TIMPs) and did not activate pro-gelatinases (pro-MMP-2 and pro-MMP-9) even in the presence of exogenous TIMP-2 or TIMP-1. A catalytic domain of MT6-MMP was inhibited preferentially by TIMP-1 (Ki = 0.2 nm) over TIMP-2 (Ki = 2.0 nm), because of a slower association rate. These results show that MT6-MMP may play a role in colon cancer and exhibit unique biochemical and structural properties that may regulate proteolytic function at the cell surface.

Shedding of Discoidin Domain Receptor 1 by Membrane-type Matrix Metalloproteinases

Background: DDR1 is a receptor tyrosine kinase that signals in response to collagen and regulates cell-collagen interactions. MT-MMPs are membrane-anchored proteases that accomplish pericellular collagenolysis. Results: MT-MMPs cleave DDR1 and regulate collagen-induced receptor phosphorylation. Conclusion: MT-MMPs negatively regulate DDR1 activation by promoting receptor ectodomain shedding. Significance: Cross-talk between membrane-anchored collagenases and RTKs integrates collagen-induced signaling and pericellular proteolysis. The discoidin domain receptors (DDRs) are receptor tyrosine kinases that upon binding to collagens undergo receptor phosphorylation, which in turn activates signal transduction pathways that regulate cell-collagen interactions. We report here that collagen-dependent DDR1 activation is partly regulated by the proteolytic activity of the membrane-anchored collagenases, MT1-, MT2-, and MT3-matrix metalloproteinase (MMP). These collagenases cleave DDR1 and attenuate collagen I- and IV-induced receptor phosphorylation. This effect is not due to ligand degradation, as it proceeds even when the receptor is stimulated with collagenase-resistant collagen I (r/r) or with a triple-helical peptide harboring the DDR recognition motif in collagens. Moreover, the secreted collagenases MMP-1 and MMP-13 and the glycosylphosphatidylinositol-anchored membrane-type MMPs (MT4- and MT6-MMP) have no effect on DDR1 cleavage or activation. N-terminal sequencing of the MT1-MMP-mediated cleaved products and mutational analyses show that cleavage of DDR1 takes place within the extracellular juxtamembrane region, generating a membrane-anchored C-terminal fragment. Metalloproteinase inhibitor studies show that constitutive shedding of endogenous DDR1 in breast cancer HCC1806 cells is partly mediated by MT1-MMP, which also regulates collagen-induced receptor activation. Taken together, these data suggest a role for the collagenase of membrane-type MMPs in regulation of DDR1 cleavage and activation at the cell-matrix interface.

Posttranslational Regulation of Membrane Type 1-Matrix Metalloproteinase (MT1-MMP) in Mouse PTEN Null Prostate Cancer Cells: Enhanced Surface Expression and Differential O-Glycosylation of MT1-MMP

Membrane type 1 (MT1)-matrix metalloproteinase (MT1-MMP) is a membrane-tethered MMP that has been shown to play a key role in promoting cancer cell invasion. MT1-MMP is highly expressed in bone metastasis of prostate cancer (PC) patients and promotes intraosseous tumor growth of PC cells in mice. The majority of metastatic prostate cancers harbor loss-of-function mutations or deletions of the tumor suppressor PTEN (phosphatase and tensin homologue deleted on chromosome ten). However, the role of PTEN inactivation in MT1-MMP expression in PC cells has not been examined. In this study, prostate epithelial cell lines derived from mice that are either heterozygous (PTEN(+/-)) or homozygous (PTEN(-/-)) for PTEN deletion or harboring a wild-type PTEN (PTEN(+/+)) were used to investigate the expression of MT1-MMP. We found that biallelic loss of PTEN is associated with posttranslational regulation of MT1-MMP protein in mouse PC cells. PTEN(-/-) PC cells display higher levels of MT1-MMP at the cell surface when compared to PTEN(+/+) and PTEN(+/-) cells and consequently exhibited enhanced migratory and collagen-invasive activities. MT1-MMP displayed by PTEN(-/-) cells is differentially O-glycosylated and exhibits a slow rate of turnover. MT1-MMP expression in PTEN(-/-) cells is under control of the PI3K/AKT signaling pathway, as determined using pharmacological inhibitors. Interestingly, rapamycin, an mTOR inhibitor, upregulates MT1-MMP expression in PTEN(+/+) cells via PI3K activity. Collectively, these data in a mouse prostate cell system uncover for the first time a novel and complex relationship between PTEN loss-mediated PI3K/AKT activation and posttranslational regulation of MT1-MMP, which may play a role in PC progression.

Identification and Role of the Homodimerization Interface of the Glycosylphosphatidylinositol-anchored Membrane Type 6 Matrix Metalloproteinase (MMP25)

The membrane type (MT) 6 matrix metalloproteinase (MMP) (MMP25) is a glycosylphosphatidylinositol-anchored matrix metalloproteinase (MMP) that is highly expressed in leukocytes and in some cancer tissues. We previously showed that natural MT6-MMP is expressed on the cell surface as a major reduction-sensitive form of Mr 120, likely representing enzyme homodimers held by disulfide bridges. Among the membrane type-MMPs, the stem region of MT6-MMP contains three cysteine residues at positions 530, 532, and 534 which may contribute to dimerization. A systematic site-directed mutagenesis study of the Cys residues in the stem region shows that Cys532 is involved in MT6-MMP dimerization by forming an intermolecular disulfide bond. The mutagenesis data also suggest that Cys530 and Cys534 form an intramolecular disulfide bond. The experimental observations on cysteines were also investigated by computational studies of the stem peptide, which validate these proposals. Dimerization is not essential for transport of MT6-MMP to the cell surface, partitioning into lipid rafts or cleavage of α-1-proteinase inhibitor. However, monomeric forms of MT6-MMP exhibited enhanced autolysis and metalloprotease-dependent degradation. Collectively, these studies establish the stem region of MT6-MMP as the dimerization interface, an event whose outcome imparts protease stability to the protein.

Characterization of the Dimerization Interface of Membrane Type 4 (MT4)-Matrix Metalloproteinase

MT4-MMP (MMP17) belongs to a unique subset of membrane type-matrix metalloproteinases that are anchored to the cell surface via a glycosylphosphatidylinositol moiety. However, little is known about its biochemical properties. Here, we report that MT4-MMP is displayed on the cell surface as a mixed population of monomeric, dimeric, and oligomeric forms. Sucrose gradient fractionation demonstrated that these forms of MT4-MMP are all present in lipid rafts. Mutational and computational analyses revealed that Cys564, which is present within the stem region, mediates MT4-MMP homodimerization by forming a disulfide bond. Substitution of Cys564 results in a more rapid MT4-MMP turnover, when compared with the wild-type enzyme, consistent with a role for dimerization in protein stability. Expression of MT4-MMP in Madin-Darby canine kidney cells enhanced cell migration and invasion of Matrigel, a process that requires catalytic activity. However, a serine substitution at Cys564 did not reduce MT4-MMP-stimulated cell invasion of Matrigel suggesting that homodimerization is not required for this process. Deglycosylation studies showed that MT4-MMP is modified by N-glycosylation. Moreover, inhibition of N-glycosylation by tunicamycin diminished the extent of MT4-MMP dimerization suggesting that N-glycans may confer stability to the dimeric form. Taken together, the data presented here provide a new insight into the characteristics of MT4-MMP and highlight the common and distinct properties of the glycosylphosphatidylinositol-anchored membrane type-matrix metalloproteinases.

Dissociation kinetics of an enzyme-inhibitor system using single-molecule force measurements.

We report on an improved method to interpret single molecule dissociation measurements using atomic force microscopy. We describe an easy to use methodology to reject nonspecific binding events, as well as estimating the number of multiple binding events. The method takes nonlinearities in the force profiles into account that result from the deformation of the used polymeric linkers. This new method is applied to a relevant enzyme-inhibitor system, latent matrix metalloprotease 9 (ProMMP-9, a gelatinase), and its inhibitor, tissue inhibitor of metalloproteases 1 (TIMP 1), which are important players in cancer metastasis. Our method provides a measured kinetic off-rate of 0.010 ± 0.003 s(-1) for the dissociation of ProMMP9 and TIMP1, which is consistent with values measured by ensemble methods.