Sanjay Pal

Nuclear Magnetic Resonance Mapping and Functional Confirmation of the Collagen Binding Sites of Matrix Metalloproteinase-2 †

Interactions of matrix metalloproteinase-2 (MMP-2) with native and denatured forms of several types of collagen are mediated by the collagen binding domain (CBD). CBD positions substrates relative to the catalytic site and is essential for their cleavage. Our previous studies identified a CBD binding site on the alpha1(I) collagen chain. The corresponding synthetic collagen peptide P713 bound CBD with high affinity and was used in this study to identify specific collagen binding residues by NMR analysis of (15)N-labeled CBD complexed with P713. Results obtained showed that P713 caused chemical shift perturbations of several surface-exposed CBD backbone amide resonances in a concentration-dependent manner. The 10 residues that underwent the largest chemical shift perturbations (R(252) in module 1, R(296), F(297), Y(302), E(321), Y(323), and Y(329) in module 2, and R(368), W(374), and Y(381) in module 3) were investigated by site-specific substitution with alanine. The structural integrity of the CBD variants was also analyzed by one-dimensional (1)H NMR. Surface plasmon resonance and microwell protein binding assays of control and CBD variants showed that residues in all three CBD modules contributed to collagen binding. Single-residue substitutions altered the affinity for peptide P713, as well as native and denatured type I collagen, with the greatest effects observed for residues in modules 2 and 3. Additional alanine substitutions involving residues in two or three modules simultaneously further reduced the level of binding of CBD to native and denatured type I collagen and demonstrated that all three modules contribute to substrate binding. These results have localized and confirmed the key collagen binding site residues in the three fibronectin type II-like modules of MMP-2.

Fragmentation of fibronectin by inherent autolytic and matrix metalloproteinase activities.

Fibronectin (FN) purified by gelatin affinity chromatography is unstable and undergoes fragmentation. The cleavage has been ascribed to inherent autolytic protease activities as well as co-purified matrix metalloproteinases (MMP). Understanding the mechanism by which the proteolysis of FN occurs is important, because the FN fragments have biological activities that differ from those of intact FN. Having excluded contributions of other plasma-derived proteases, the present experiments demonstrated that cleavage of FN by MMP-2 to distinct fragments occurred in synergy with inherent FN activities. Limited heat treatment of FN at 56°C for 30 min inactivated the inherent protease activities sharply reducing autolysis of FN in a manner similar to that seen in the presence of serine proteinase inhibitors. Heat treatment did not alter cell attachment to FN, but significantly increased the susceptibility of FN to enzymatic cleavage by MMP-2. The carboxyl-terminal hemopexin-like domain (PEX) of MMP-2 was shown to possess critical exodomain properties required for the interactions of MMP-2 with FN, and FN was cleaved at a significantly reduced rate by an MMP-2 variant with deletion of PEX. Verifying the specificity of interactions, isolated PEX competed FN cleavage by MMP-2 in a concentration-dependent manner. These results have further elucidated the synergistic contributions of inherent autolytic serine protease-like activities and MMP-2 to fragmentation of FN and provide the rationale and basis for modified preparation and handling of FN used in biological research.

A positive feedback loop involving Erk5 and Akt turns on mesangial cell proliferation in response to PDGF

Platelet-derived growth factor BB and its receptor (PDGFRβ) play a pivotal role in the development of renal glomerular mesangial cells. Their roles in increased mesangial cell proliferation during mesangioproliferative glomerulonephritis have long been noted, but the operating logic of signaling mechanisms regulating these changes remains poorly understood. We examined the role of a recently identified MAPK, Erk5, in this process. PDGF increased the activating phosphorylation of Erk5 and tyrosine phosphorylation of proteins in a time-dependent manner. A pharmacologic inhibitor of Erk5, XMD8-92, abrogated PDGF-induced DNA synthesis and mesangial cell proliferation. Similarly, expression of dominant negative Erk5 or siRNAs against Erk5 blocked PDGF-stimulated DNA synthesis and proliferation. Inhibition of Erk5 attenuated expression of cyclin D1 mRNA and protein, resulting in suppression of CDK4-mediated phosphorylation of the tumor suppressor protein pRb. Expression of cyclin D1 or CDK4 prevented the dominant negative Erk5- or siErk5-mediated inhibition of DNA synthesis and mesangial cell proliferation induced by PDGF. We have previously shown that phosphatidylinositol 3-kinase (PI3-kinase) contributes to PDGF-induced proliferation of mesangial cells. Inhibition of PI3-kinase blocked PDGF-induced phosphorylation of Erk5. Since PI3-kinase acts through Akt, we determined the role of Erk5 on Akt phosphorylation. XMD8-92, dominant negative Erk5, and siErk5 inhibited phosphorylation of Akt by PDGF. Interestingly, we found inhibition of PDGF-induced Erk5 phosphorylation by a pharmacological inhibitor of Akt kinase and kinase dead Akt in mesangial cells. Thus our data unfold the presence of a positive feedback microcircuit between Erk5 and Akt downstream of PI3-kinase nodal point for PDGF-induced mesangial cell proliferation.

Identification of collagen binding domain residues that govern catalytic activities of matrix metalloproteinase-2 (MMP-2).

An innovative approach to enhance the selectivity of matrix metalloproteinase (MMP) inhibitors comprises targeting these inhibitors to catalytically required substrate binding sites (exosites) that are located outside the catalytic cleft. In MMP-2, positioning of collagen substrate molecules occurs via a unique fibronectin-like domain (CBD) that contains three distinct modular collagen binding sites. To characterize the contributions of these exosites to gelatinolysis by MMP-2, seven MMP-2 variants were generated with single, or concurrent double and triple alanine substitutions in the three fibronectin type II modules of the CBD. Circular dichroism spectroscopy verified that recombinant MMP-2 wild-type (WT) and variants had the same fold. Moreover, the MMP-2 WT and variants had the same activity on a short FRET peptide substrate that is hydrolyzed independently of CBD binding. Among single-point variants, substitution in the module 3 binding site had greatest impact on the affinity of MMP-2 for gelatin. Simultaneous substitutions in two or three CBD modules further reduced gelatin binding. The rates of gelatinolysis of MMP-2 variants were reduced by 20-40% following single-point substitutions, by 60-75% after double-point modifications, and by >90% for triple-point variants. Intriguingly, the three CBD modules contributed differentially to cleavage of dissociated α-1(I) and α-2(I) collagen chains. Importantly, kinetic analyses (k(cat)/K(m)) revealed that catalysis of a triple-helical FRET peptide substrate by MMP-2 relied primarily on the module 3 binding site. Thus, we have identified three collagen binding site residues that are essential for gelatinolysis and constitute promising targets for selective inhibition of MMP-2.

Co-purified Gelatinases Alter the Stability and Biological Activities of Human Plasma Fibronectin Preparations

BACKGROUND AND OBJECTIVE Fibronectin (FN) is an important cell adhesion molecule that is used widely to characterize cell behavior. Preparations of FN purified from human plasma by gelatin-Sepharose affinity chromatography typically also contain gelatin-binding gelatinases that may cleave FN, reduce its stability and alter its biological activities. Available methods for separating gelatinases from FN are resource demanding. Therefore, our objective was to devise a time- and cost-efficient protocol for purification of gelatinase-free FN. MATERIAL AND METHODS Experiments tested the elution profiles for FN and gelatinases from gelatin-Sepharose using a concentration range (1-7%) of dimethyl sulfoxide (DMSO) and 4 m urea as eluants. Subsequently, we explored the sequential application of those eluants for differential elution of gelatinases and FN using a single affinity column. Finally, experiments characterized the stability of purified FN with or without contaminating gelatinases, as well as the effects of FN degradation on cell attachment and migration. RESULTS Assay optimization demonstrated that pre-elution with 3% DMSO efficiently eliminated gelatinases but not FN from gelatin-Sepharose, whereas subsequent elution with 4 m urea released FN. Sequential elutions with DMSO and urea produced gelatinase-free FN, which was more stable than FN eluted by urea only. Fibronectin degradation did not affect human gingival fibroblast attachment, but increased cell migration significantly. CONCLUSION The present experiments devised a time- and cost-efficient protocol for eliminating gelatinases during purification of human plasma FN. Gelatinase-free FN preparations had greater stability, which may be essential for experiments because FN fragments have altered biological activities compared with intact FN.

Peptide from the C-terminal domain of tissue inhibitor of matrix metalloproteinases-2 (TIMP-2) inhibits membrane activation of matrix metalloproteinase-2 (MMP-2)

Cellular activation of latent matrix metalloproteinase-2 (proMMP-2) requires formation of a cell membrane-associated activation complex that involves specific binding between the hemopexin domain of proMMP-2 (PEX) and the C-terminal domain of tissue inhibitor of matrix metalloproteinases-2 (C-TIMP-2). In this study, we tested the feasibility of inhibiting activation of proMMP-2 by exogenous inhibitors, which block the binding between PEX and TIMP-2. The recombinant C-TIMP-2 and synthetic peptides from C-TIMP-2 were used as inhibitors for proMMP-2 activation. Recombinant C-TIMP-2 bound specifically to both the catalytically inactive MMP-2(E404A) and the C-terminal domain of MMP-2 (PEX) in a concentration dependent manner with apparent K(d) of 3.9×10(-7)M and 1.7×10(-7)M, respectively. Moreover, C-TIMP-2 competed the binding between MMP-2(E404A) and full-length TIMP-2. Finally, activity assays showed that addition of C-TIMP-2 to HT-1080 fibrosarcoma cells inhibited proMMP-2 activation in a concentration-dependent manner. We then designed a synthetic peptide, P175L, consisting of 20 residues from the PEX-binding tail region of C-TIMP-2. P175L bound PEX and inhibited cell membrane-mediated activation of proMMP-2 in a concentration dependent manner. Deletion of the last 9 tail residues of C-TIMP-2 in P175L abrogated the inhibitory activities of the peptide showing that these residues were essential for function. Overall, these experiments have demonstrated that proMMP-2 activation can be inhibited by exogenous inhibitors which points to a potential strategy for MMP-2 specific inhibition.

Autolysin mediated adherence of Staphylococcus aureus with Fibronectin, Gelatin and Heparin

Major autolysin (Atl) of Staphylococcus aureusis a cell surface associated peptidoglycan hydrolase with amidase and glucosaminidase domains. Atl enzymes (amidase and glucosaminidase) are known to participate in biofilm formation and also can bind with host matrices. Earlier studies demonstrated the binding of Atlwithfibronectin, thrombospondin 1, vitronectin and heat shock cognate protein Hsc70. Here, we have shown, Atl mediates attachment of S.aureus to heparin and gelatine as well. The atl mutant strain demonstrated around 2.5 fold decreased adherence with fibronectin, gelatin and heparin coated microtiter plates. The microscopic studies confirmed the reduced binding of atl mutant with them compared to its parental wild type and complemented mutant strains. Amidase and glucosaminidase were expressed as N-terminal histidine tagged proteins from Escherichia coli, purified and refolded. We found refolded amidase bind with fibronectin, gelatin and heparin; whereas refolded glucosaminidase binds with only fibronectin and heparin but not gelatin. These results reemphasize Atl as one of the crucial proteins from Staphylococcus that facilitate their binding with multiple host cellular components during colonization and infection.

6-Shogaol induces caspase-independent paraptosis in cancer cells via proteasomal inhibition.

&NA; An &agr;, &bgr;‐unsaturated carbonyl compound of ginger, 6‐Shogaol (6S), induced extensive cytoplasmic vacuolation and cell death in breast cancer cell (MDA‐MB‐231) and non‐small lung cancer (A549) cells. In the presence of autophagic inhibitors the cells continued to exhibit cytoplasmic vacuolation and cell death clearly distinguishing it from the classic autophagic process. 6S induced death did not exhibit the characteristic apoptotic features like caspase cleavage, phosphatidyl serine exposure and DNA fragmentation. The immunofluorescence with the Endoplasmic Reticulum (ER) resident protein, calreticulin indicated that the vacuoles were of ER origin, typical of paraptosis. This was supported by the increase in level of microtubule associated protein light chain 3B (LC3 I and LC3 II) and polyubiquitin binding protein, p62. The level of ER stress markers like polyubiquitinated proteins, Bip and CHOP also consistently increased. We have found that 6S inhibits the 26S proteasome. The proteasomal inhibitory activity was elucidated by a) molecular docking of 6S onto the active site of &bgr;5 subunit and b) reduced fluorescence by the fluorogenic substrate of the chymotrypsin‐like subunit. In conclusion these studies demonstrate for the first time that proteasomal inhibition by 6S induces cell death via paraptosis. So 6‐shogaol may act as a template for anti‐cancer lead discovery against the apoptosis resistant cancer cells.

Polymer-Carbon Composites as Anti-corrosive Materials

The objective of this chapter is to focus on the recent developments of polymer-carbon composites in terms of utilizing various forms of carbon, i.e., graphene or graphene-like sheets, carbon nanotube, along with polymer binder for high-performance anti-corrosive paints. The attributes to final properties of dried film coating can be resolved into two major parts, i.e., attributes due to binder and additives. The role of binder and additives has been discussed separately in the subsequent sections. A general idea has also been discussed as a premise to give the right context before the objective of this chapter is discussed, i.e., Polymer-carbon composites as anti-corrosive material.

Studies on Interfacial Characteristics of Highly Dispersible Silica Reinforced Epoxidized Natural Rubber Compounds

ABSTRACT The present work examines the effect of two different specific surface areas of highly dispersible silica (HDS)-reinforced epoxidized natural rubber (ENR) composites. The influences of different blend ratio between ENRs consisting 25 and 50 mol% of epoxidation-based (ENR-25/ENR-50) composites was studied in detail. The primary objective is to investigate the interfacial area of HDS surface involved in filler-to-rubber interaction mechanisms for the better reinforcement. Notable improvement in overall properties of these green composites are corroborated with various meticulous characterization including cure characteristics, specific bound rubber content, physicomechanical, dynamic mechanical properties, etc. Increasing the specific surface area of HDS and their subsequent interface with ENR matrix invokes its superior dispersion. Small angle X-ray scattering (SAXS) has been used to analyze the particles network and clusters establishment in green composites. The present SAXS method provides a unique insight into the cluster formation according to the Beaucage model. However, SAXS results demonstrate that particles networks can be effectively suppressed by increasing specific surface area of HDS. GRAPHICAL ABSTRACT