Krishnakumar N. Menon

A simple and efficient method for processing of cell lysates for two-dimensional gel electrophoresis

Sample preparation is one of the major issues in 2‐DE for the separation of proteins. Although a 100% representation of cellular proteins onto a 2‐DE is virtually impossible, maximum representation of cellular proteins compared with the original cell lysate is important in the subsequent analysis. We demonstrate that lysis of cells in urea/thiourea solution with subsequent sonication to disrupt the nucleic acids and concentration of the lysate using centri‐con led to enrichment of proteins. The procedure resulted in minimal nucleic acid contamination with better resolution of spots. 2‐DE spot patterns of proteins prepared using urea–thiourea solubilization/centri‐con method to other protein enrichment methods such as phenol/chloroform/isoamyl alcohol extraction, methanol/ammonium acetate precipitation, acetone precipitation and ethanol precipitation were compared. Urea–thiourea solubilization combined with centri‐con method of protein enrichment represented higher number/unique spots particularly in the 50–250 kDa Mr compared with others. Lysis of cells in urea/thiourea from the beginning of lysate preparation preserves the proteins from protease activity due to denaturation of proteases. Thus, we demonstrate that the centri‐con methodology is simple and effective for the preparation of high‐quality sample that can be used for a qualitative representation of cellular proteins on a 2‐DE for proteomic analysis.

Exploitation of Detergent Thermodynamics in the Direct Solubilization of Myelin Membrane Proteins for Two-dimensional Gel Electrophoresis for Proteomic Analysis

Performing 2‐DE of lipid‐rich multilamellar membranes like myelin is a cumbersome task. However, for understanding its molecular organization and changes during diseases, identification of proteins of myelin is essential. Although the 2‐D‐proteomic approach of myelin has been employed to understand the myelin proteome, representation of myelin proteins in its entirety is still a challenge. 2‐DE profiling of myelin proteins is very important for the detection of immuno‐reactivity to myelin proteins from various biological fluids following Western blotting in diseases like multiple sclerosis. Here we developed a novel approach by exploiting the thermodynamic principles behind detergent‐mediated solubilization of myelin membranes without any conventional processing of myelin involving precipitation of myelin proteins. We show that the addition of myelin to ASB‐14‐4 resulted in significant increase in protein representation of myelin in 2‐DE compared with the addition of ASB‐14‐4 to myelin. Moreover, the number and resolution of spots are significantly higher in myelin to ASB‐14‐4 strategy than other strategies of myelin sample processing such as ASB‐14‐4 to myelin or ethanol or acetone or methanol–ammonium acetate precipitation of myelin proteins. In addition, the step involves no precipitation that selective removal of any proteins as a result of precipitation is nil and a qualitative representation of myelin proteins in a 2‐D gel is achieved.

Development and molecular characterization of polymeric micro-nanofibrous scaffold of a defined 3-D niche for in vitro chemosensitivity analysis against acute myeloid leukemia cells

Standard in vitro drug testing employs 2-D tissue culture plate systems to test anti-leukemic drugs against cell adhesion-mediated drug-resistant leukemic cells that harbor in 3-D bone marrow microenvironments. This drawback necessitates the fabrication of 3-D scaffolds that have cell adhesion-mediated drug-resistant properties similar to in vivo niches. We therefore aimed at exploiting the known property of polyurethane (PU)/poly-l-lactic acid (PLLA) in forming a micro-nanofibrous structure to fabricate unique, not presented before, as far as we are aware, 3-D micro-nanofibrous scaffold composites using a thermally induced phase separation technique. Among the different combinations of PU/PLLA composites generated, the unique PU/PLLA 60:40 composite displayed micro-nanofibrous morphology similar to decellularized bone marrow with increased protein and fibronectin adsorption. Culturing of acute myeloid leukemia (AML) KG1a cells in FN-coated PU/PLLA 60:40 shows increased cell adhesion and cell adhesion-mediated drug resistance to the drugs cytarabine and daunorubicin without changing the original CD34+/CD38−/CD33− phenotype for 168 hours compared to fibronectin tissue culture plate systems. Molecularly, as seen in vivo, increased chemoresistance is associated with the upregulation of anti-apoptotic Bcl2 and the cell cycle regulatory protein p27Kip1 leading to cell growth arrest. Abrogation of Bcl2 activity by the Bcl2-specific inhibitor ABT 737 led to cell death in the presence of both cytarabine and daunorubicin, demonstrating that the cell adhesion-mediated drug resistance induced by Bcl2 and p27Kip1 in the scaffold was similar to that seen in vivo. These results thus show the utility of a platform technology, wherein drug testing can be performed before administering to patients without the necessity for stromal cells.

Integration of common feature pharmacophore modeling and in vitro study to identify potent AChE inhibitors

Alzheimer’s disease is a progressive neurodegenerative disorder arising due to genetic and non-genetic causes. One of the major therapies adapted for symptomatic Alzheimer’s disease is by targeting acetylcholinesterase enzyme based on the cholinergic hypothesis. Acetylcholinesterase is a substrate-specific enzyme that degrades the neuro-transmitter acetylcholine. An optimum level of acetylcholine should be maintained in the brain for its proper function. In order to identify potent and selective acetylcholinesterase inhibitors we adopted an integrated in silico and bioassay methodologies. In silico approach involves creating chemical features based 3D-pharmacophore models using AChE specific inhibitors. This model was then used for sequential virtual screening from the small molecule databases. Finally, five molecules were selected on the basis of the best docking scores and pharmacokinetics properties. These molecules were subjected to docking analysis with the recently solved crystal structure of human acetylcholinesterase enzyme, in order to reveal its binding mode and interactions at the dual binding sites of the enzyme. The acetylcholinesterase enzyme inhibitory activity of these five lead molecules was further assessed by in-vitro analysis.

Expression and Purification of Quinine Dihydro Pteridine Reductase from astrocytes and its significance in the astrocyte pathology

Quinine dihydropteridinereductase (QDPR) is involved in the synthesis of tetradihydrobiopteridine (BH4) that serve as cofactor for many aromatic hydroxylases including induced nitric oxide synthase (NOS) leading to NO production. Increased activity of QDPR has been associated with decrease levels of TGF-β, a cytokine that regulates the immune response and that elevated levels of NO has been associated with neurodegenerative diseases. Thus, expression of QDPR in astrocytes is essential to study the pathological changes observed in many neurodegenerative disorders. We have expressed QDPR in astrocytes and generated stably expressing clones that overexpresses QDPR. We further verified the specificity of QDPR expression using immunofluorescence and immunoblotting. To further confirm, we purified QDPR using Ni-NTA column and subjected the purified fraction to immunoblotting using anti-QDPR antibody and identified two major protein products of QDPR resolving at 25 and 17 kDa as reported in the literature. In order to further assess the significance of QDPR expression, we verified the expression of iNOS in QDPR over expressing cells. We show for the first time statistically significant up regulation of iNOS in QDPR overexpressing astrocytes. Increased expression of iNOS associated with astrocyte pathology seen in many neurodegenerative disorders may have implications in autoimmune neurodegenerative disorders.

Identification of prolargin expression in articular cartilage and its significance in rheumatoid arthritis pathology

Qualitative 2D gel-electrophoresis (2DE) protein profiling for osteoarthritis (OA) and rheumatoid arthritis (RA) is challenging because of selective protein loss due to discrepancies in protein precipitation methodologies. Thus, we aimed at developing qualitative protein representation from OA/RA articular cartilage without protein precipitation towards identification of clinically relevant proteins. Chondroitinase digested human articular cartilages from RA patients were subjected to protein extraction using guanidinium hydrochloride (GuHCl) or 8 M urea with 10 or 2% ASB-14-4 or 0.45 M urea with 2% ASB-14-4 with cetylpyridinium chloride (CPC). The GuHCl extract is further protein precipitated with acetone or ammonium acetate-methanol or centricon-fractionated using 100 kDa cut filters and protein precipitated using ethanol. Processed extracts were subjected to 2DE to identify protein profiles. Poor proteins representations were observed in 2D gels with protein precipitated samples compared to qualitative protein representations seen in 2D gels of 0.45 M urea and 2%ASB-14-4 extraction procedure reproducibly. The strategy circumventing protein precipitation generated qualitative 2D gels. RA vs OA gel comparison showed elevated prolargin levels in RA with biglycan levels remaining unaltered. Up regulation of prolargin in RA suggests the likelihood of an adaptive mechanism to control the increased osteoclastogenesis in RA and may have therapeutic value in controlling the disease.

Exploring the role of defective fibronectin matrix assembly in the VHL-associated CNS hemangioblastoma.

Abstract Background: Central nervous system (CNS) hemangioblastoma (HB) is the most common tumor in the von Hippel Lindau (VHL) disorder, the hereditary tumor syndrome caused by the biallelic mutations of the VHL gene. The disrupted VHL and Elongin protein interaction on hypoxia-inducible factor-1α (HIF-1α) induces a set of hypoxia-inducible genes, resulting in an unchecked endothelial cell proliferation that then leads to hemangioblastoma formation. However, recent studies have shown that disruptive germline mutations of VHL need not result in hemangioblastoma, though it can cause other manifestations of the VHL syndrome. Similarly, sporadic hemangioblastoma can occur rarely without a somatic biallelic VHL mutation. The VHL protein was earlier found to be associated with the deposition of matrix fibronectin (FN) protein in the renal extracellular matrix. Methods: The present study was designed to investigate the deposition of the matrix FN protein in VHL-associated hemangioblastoma. Results: Seven HB tumor samples from the VHL syndrome had lower expressions of tissue FN compared to the control cerebellum samples or the control blood vessel sample. On comparing the VHL and FN protein expressions in a timed endothelial tube assay, the VHL protein expression was absent during the initial phase of tube formation but started expressing after 6 h. The levels of matrix form of FN gradually increased along with the VHL expression during the maturation of tube formation. Tube formation was found to be enhanced with extraneously added soluble FN and inhibited by matrix FN. Similarly, tube formation was inhibited by a modified tripeptide (RGD) inhibitor of integrin (-αVβ3), namely, Cyclo-Ala-Arg-Gly-Asp-3-aminomethylbenzoyl. Conclusions: Our study implicates that the extracellular deposition and matrix formation of FN is important for vascular endothelial proliferation, and that its absence has roles in the development of hemangioblastoma in the VHL syndrome.

Significance of elevated Prohibitin 1 levels in Multiple Sclerosis patients lymphocytes towards the assessment of subclinical disease activity and its role in the central nervous system pathology of disease

Multiple Sclerosis (MS) is an autoimmune-neurodegenerative disorder managed therapeutically by modulating lymphocytes activity which has potential in disease management. Prohibitin 1(PHB) that controls the reactive oxygen species (ROS) and present on the activated lymphocytes have significance in the therapy of MS as esters of fumaric acid that regulates ROS is in phase II/III clinical trials. Thus, we evaluated the expression levels of PHB1 in experimental autoimmune encephalomyelitis (EAE), the animal model of MS and on MS patients lymphocytes. PHB levels in brain tissue of EAE animals were determined by immunoblotting and on blood lymphocytes from MS relapse, Remission, Optic Neuritis, Neurological controls and Healthy volunteers by FACS using anti-PHB and anti-CD45 antibodies. We observed significant elevation of PHB in EAE brains (91.0 ± 17.59%) vs controls (29.8 ± 12.9%) (p = 0.01) and on lymphocytes of MS patients in acute (73.5 ± 11.20%) or relapsing (69.3 ± 17.33%) phase compared to remission (45.9 ± 8.08%) [p = 0.034 acute vs remission; p = 0.004 relapse vs remission]. Up regulation of PHB in relapsing vs remission MS patients imply the potential use of PHB to clinically evaluate subclinical disease status towards prognosis of an oncoming relapse. Elevated PHB levels in EAE brains signify the role of PHB in regulating ROS and implies PHBs role in oxidative stress.

Recombinant expression of extracellular domain of mutant Epidermal Growth Factor Receptor in prokaryotic and baculovirus expression systems

Epidermal Growth Factor Receptor variant III (EGFRvIII) is a tumor specific antigen detected in various tumors including gliomas, breast cancer, lung cancer, head and neck squamous cell carcinoma (HNSCC). Screening of EGFRvIII targeting drug molecules can be accelerated by developing drug screening platforms using recombinantly expressed protein. Choice of expression system is one of the major factors deciding the success of recombinant expression of a protein. In our study, we have tried to express and purify the extracellular domain (ECD) of this highly unstable protein using bacterial and baculovirus expression systems to select the expression system suited for our purpose. Even though the protein was successfully expressed in prokaryotic system, purification could be done only under denaturing conditions. But in the baculovirus expression system, the protein was expressed in soluble form and could be purified under native conditions, with single step of purification. Based on our results, we conclude that insect cells are better choice over E. coli cells for expressing EGFRvIII ECD in soluble form. This study provides insights for other researchers involved in expression of similar unstable membrane proteins, on selecting the best expression system and challenges involved.