Membrane type 1 matrix metalloproteinase (MT1-MMP) is a collagenolytic enzyme that has been implicated in normal development and in pathological processes such as cancer metastasis. The activity of MT1-MMP is regulated extensively at the post-translational level, and the current data support the hypothesis that MT1-MMP activity is modulated by glycosylation. Enzymatic deglycosylation, site-directed mutagenesis, and lectin precipitation assays were used to demonstrate that MT1-MMP contains O-linked complex carbohydrates on the Thr 291 , Thr 299 , Thr 300 , and/or Ser 301 residues in the proline-rich linker region. MT1-MMP glycoforms were detected in human cancer cell lines, suggesting that MT1-MMP activity may be regulated by differential glycosylation in vivo. Although the autolytic processing and interstitial collagenase activity of MT1-MMP were not impaired in glycosylation-deficient mutants, cell surface MT1-MMP-catalyzed activation of pro-matrix metalloproteinase-2 (proMMP-2) required proper glycosylation of MT1-MMP. The inability of carbohydrate-free MT1-MMP to activate proMMP-2 was not a result of defective MT1-MMP zymogen activation, aberrant protein stability, or inability of the mature enzyme to oligomerize. Rather, our data support a mechanism whereby glycosylation affects the recruitment of tissue inhibitor of metalloproteinases-2 (TIMP-2) to the cell surface, resulting in defective formation of the MT1-MMP/TIMP-2/proMMP-2 trimeric activation complex. These data provide evidence for an additional mechanism for post-translational control of MT1-MMP activity and suggest that glycosylation of MT1-MMP may regulate its substrate targeting. Matrix metalloproteinases (MMPs)1 are a family of zinc-dependent proteinases (1-3) with activity against a variety of extracellular matrix proteins including collagens, laminins, and fibronectin (4). Recent research has identified a variety of non-traditional MMP activities including shedding of growth factors, cytokines and their receptors (5-8), disruption of cellmatrix and cell-cell junctional protein complexes (9 -11), and initiation of proteinase cascades that impact much broader substrates (12, 13). As a result, MMPs play a central role in many physiological and pathological processes, including development, wound healing, tissue resorption, angiogenesis, and tumor invasion (3,14,15). Most MMPs are secreted proteins and share a similar modular domain structure including propeptide, catalytic domain, linker region, and hemopexinlike domain (2). However, a subgroup of MMPs, designated membrane type (MT)-MMPs, contains either an additional transmembrane and cytoplasmic domain (16 -19) or a glycosylphosphatidylinositol anchor (20, 21), suggesting unique substrate specificity and distinct regulatory mechanisms as a consequence of cell surface localization (22).Among MT-MMPs, MT1-MMP is the most extensively studied and the best characterized. A primary function of MT1-MMP is pericelluar collagenolysis (reviewed in Ref. 23). This is supported by both in vitro experiments ...

The Respiratory Syncytial Virus (RSV) fusogenic glycoprotein F(1) was characterized using biochemical and biophysical techniques. Two heptad-repeat (HR) regions within F(1) were shown to interact. Proteinase-K digestion experiments highlight the HR1 region (located proximal to the fusion peptide sequence) of the F(1) protein to which an HR2-derived (located proximal to the membrane-spanning domain) peptide binds, thus protecting both the protein and peptide from digestion. Solution-phase analysis of HR1-derived peptides shows that these peptides adopt helical secondary structure as measured by circular dichroism. Sedimentation equilibrium studies indicate that these HR1 peptides self-associate in a monomer/trimer equilibrium with an association constant of 5.2 x 10(8) M(-2). In contrast, HR2-derived peptides form random monomers in solution. CD analysis of mixtures containing peptides from the two regions demonstrate their propensity to interact and form a very stable (T(m) = 87 degrees C), helical (86% helicity) complex comprised of three HR1 and three HR2 members.

Squamous cell carcinoma of the oral cavity is characterized by persistent, disorganized expression of integrin ␣3␤1 and enhanced production of urinary-type plasminogen activator (uPA) and its receptor (uPAR) relative to normal oral mucosa. Because multivalent aggregation of ␣3␤1 integrin up-regulates uPA and induces a dramatic co-clustering of uPAR, we explored the hypothesis that lateral ligation of ␣3␤1 integrin by uPAR contributes to uPA regulation in oral mucosal cells. To investigate mechanisms by which uPAR/␣3␤1 binding enhances uPA expression, integrin-dependent signal activation was assessed. Both Src and ERK1/2 were phosphorylated in response to integrin aggregation, and blocking Src kinase activity completely abrogated ERK1/2 activation and uPA induction, whereas inhibition of epidermal growth factor receptor tyrosine kinase activity did not alter uPA expression. Proteinase up-regulation occurred at the transcriptional level and mutation of the AP1 (؊1967) site in the uPA promoter blocked the uPAR/integrinmediated transcriptional activation. Because uPAR is redistributed to clustered ␣3␤1 integrins, the requirement for uPAR/␣3␤1 interaction in uPA regulation was assessed. Clustering of ␣3␤1 in the presence of a peptide (␣325) that disrupts uPAR/␣3␤1 integrin binding prevented uPA induction. Depletion of cell surface uPAR using small interfering RNA also blocked uPA induction following integrin ␣3␤1 clustering. These results were confirmed using a genetic strategy in which ␣3 null epithelial cells reconstituted with wild type ␣3 integrin, but not a mutant ␣3 unable to bind uPAR, induced uPA expression upon integrin clustering, confirming the critical role of uPAR in integrin-regulated proteinase expression. Disruption of uPAR/␣3␤1 binding using peptide ␣325 or small interfering RNA blocked filopodia formation and matrix invasion, indicating that this interaction stimulates invasive behavior. Together these data support a model wherein matrix-induced clustering of ␣3␤1 integrin promotes uPAR/␣3␤1 interaction, thereby potentiating cellular signal transduction pathways culminating in activation of uPA expression and enhanced uPA-dependent invasive behavior.

An ecofriendly solid catalyst has been synthesized by anchoring palladium(II) into post synthetically modified metal organic framework IRMOF-3. The pore of IRMOF-3 was first modified with pyridine-2-aldehyde. The amine group of IRMOF-3 upon condensation with pyridine-2-aldehyde afforded a bidentate Schiff base moiety in the porous matrix. The Schiff base moieties were used to anchor palladium(II) ions. The prepared catalyst has been characterized by UV-vis, IR spectroscopy, X-ray powder diffraction, and nitrogen sorption measurements. Framework structure of the catalyst is not being destroyed in the multistep synthesis procedure as evidenced in X-ray powder diffraction studies. The catalyst has shown high activity toward the Suzuki and Stille cross-coupling reaction in 20% H2O/EtOH and EtOH medium, respectively, at 80 °C. The immobilized complex did not leach or decompose during the catalytic reactions, showing practical advantages over the homogeneous catalysis.