Stabilizing Exposure of Conserved Epitopes by Structure Guided Insertion of Disulfide Bond in HIV-1 Envelope Glycoprotein

Entry of HIV-1 into target cells requires binding of the viral envelope glycoprotein (Env) to cellular receptors and subsequent conformational changes that culminates in fusion of viral and target cell membranes. Recent structural information has revealed that these conformational transitions are regulated by three conserved but potentially flexible layers stacked between the receptor-binding domain (gp120) and the fusion arm (gp41) of Env. We hypothesized that artificial insertion of a covalent bond will ‘snap’ Env into a conformation that is less mobile and stably expose conserved sites. Therefore, we analyzed the interface between these gp120 layers (layers 1, 2 and 3) and identified residues that may form disulfide bonds when substituted with cysteines. We subsequently probed the structures of the resultant mutant gp120 proteins by assaying their binding to a variety of ligands using Surface Plasmon Resonance (SPR) assay. We found that a single disulfide bond strategically inserted between the highly conserved layers 1 and 2 (C65-C115) is able to ‘lock’ gp120 in a CD4 receptor bound conformation (in the absence of CD4), as indicated by the lower dissociation constant (Kd) for the CD4-induced (CD4i) epitope binding 17b antibody. When disulfide-stabilized monomeric (gp120) and trimeric (gp140) Envs were used to immunize rabbits, they were found to elicit a higher proportion of antibodies directed against both CD4i and CD4 binding site epitopes than the wild-type proteins. These results demonstrate that structure-guided stabilization of inter-layer interactions within HIV-1 Env can be used to expose conserved epitopes and potentially overcome the sequence diversity of these molecules.     

Isolation and Characterisation of a Recombinant Antibody Fragment That Binds NCAM1-Expressing Intervertebral Disc Cells

Degeneration of the intervertebral discs (IVD) is a leading cause of neck and low back pain. Degeneration begins in the central nucleus pulposus region, leading to loss of IVD osmotic properties. Regeneration approaches include administration of matrix-mimicking scaffolds, cells and/or therapeutic factors. Cell-targeting strategies are likely to improve delivery due to the low cell numbers in the IVD. Single-chain antibody fragments (scFvs) that bind IVD cells were isolated for potential delivery of therapeutics to degenerated IVD. The most cell-distal domain of neural cell adhesion molecule 1 (NCAM1) was cloned and expressed in Escherichia coli. Phage display technology was used to isolate a human scFv against the recombinant domain by panning a scFv library on the immobilised protein. The isolated scFv bound cultured rat astrocytes, as well as bovine nucleus pulposus and annulus fibrosus cells in immunocytochemical studies. The scFv also labelled cells in bovine spinal cord and six-month and two-year old bovine IVD sections by immunohistochemistry. Antibody fragments can provide cell-binding moieties at improved cost, time, yield and functionalisation potential over whole antibodies. The described scFv has potential application in delivery of therapeutics to NCAM1-expressing cells in degenerated IVD.     

Resveratrol depletes mitochondrial DNA and inhibition of autophagy enhances resveratrol-induced caspase activation

We recently demonstrated that resveratrol induces caspase-dependent apoptosis in multiple cancer cell types. Whether apoptosis is also regulated by other cell death mechanisms such as autophagy is not clearly defined. Here we show that inhibition of autophagy enhanced resveratrol-induced caspase activation and apoptosis. Resveratrol inhibited colony formation and cell proliferation in multiple cancer cell types. Resveratrol treatment induced accumulation of LC3-II, which is a key marker for autophagy. Pretreatment with 3-methyladenine (3-MA), an autophagy inhibitor, increased resveratrol-mediated caspase activation and cell death in breast and colon cancer cells. Inhibition of autophagy by silencing key autophagy regulators such as ATG5 and Beclin-1 enhanced resveratrol-induced caspase activation. Mechanistic analysis revealed that Beclin-1 did not interact with proapoptotic proteins Bax and Bak; however, Beclin-1 was found to interact with p53 in the cytosol and mitochondria upon resveratrol treatment. Importantly, resveratrol depleted ATPase 8 gene, and thus, reduced mitochondrial DNA (mtDNA) content, suggesting that resveratrol induces damage to mtDNA causing accumulation of dysfunctional mitochondria triggering autophagy induction. Together, our findings indicate that induction of autophagy during resveratrol-induced apoptosis is an adaptive response.     

MicroRNA Profiling in Prostate Cancer - The Diagnostic Potential of Urinary miR-205 and miR-214

Prostate cancer (PCa) is the most common type of cancer in men in the United States, which disproportionately affects African American descents. While metastasis is the most common cause of death among PCa patients, no specific markers have been assigned to severity and ethnic biasness of the disease. MicroRNAs represent a promising new class of biomarkers owing to their inherent stability and resilience. In the present study, we investigated potential miRNAs that can be used as biomarkers and/or therapeutic targets and can provide insight into the severity and ethnic biasness of PCa. PCR array was performed in FFPE PCa tissues (5 Caucasian American and 5 African American) and selected differentially expressed miRNAs were validated by qRT-PCR, in 40 (15 CA and 25 AA) paired PCa and adjacent normal tissues. Significantly deregulated miRNAs were also analyzed in urine samples to explore their potential as non-invasive biomarker for PCa. Out of 8 miRNAs selected for validation from PCR array data, miR-205 (p<0.0001), mir-214 (p<0.0001), miR-221(p<0.001) and miR-99b (p<0.0001) were significantly downregulated in PCa tissues. ROC curve shows that all four miRNAs successfully discriminated between PCa and adjacent normal tissues. MiR-99b showed significant down regulation (p<0.01) in AA PCa tissues as compared to CA PCa tissues and might be related to the aggressiveness associated with AA population. In urine, miR-205 (p<0.05) and miR-214 (p<0.05) were significantly downregulated in PCa patients and can discriminate PCa patients from healthy individuals with 89% sensitivity and 80% specificity. In conclusion, present study showed that miR-205 and miR-214 are downregulated in PCa and may serve as potential non-invasive molecular biomarker for PCa.     

Monosaccharide-Responsive Phenylboronate-Polyol Cell Scaffolds for Cell Sheet and Tissue Engineering Applications

Analyte-responsive smart polymeric materials are of great interest and have been actively investigated in the field of regenerative medicine. Phenylboronate containing copolymers form gels with polyols under alkaline conditions. Monosaccharides, by virtue of their higher affinity towards boronate, can displace polyols and solubilize such gels. In the present study, we investigate the possibility of utilizing phenylboronate-polyol interactions at physiological pH in order to develop monosaccharide-responsive degradable scaffold materials for systems dealing with cells and tissues. Amine assisted phenylboronate-polyol interactions were employed to develop novel hydrogel and cryogel scaffolds at neutral pH. The scaffolds displayed monosaccharide inducible gel-sol phase transformability. In vitro cell culture studies demonstrated the ability of scaffolds to support cell adhesion, viability and proliferation. Fructose induced gel degradation is used to recover cells cultured on the hydrogels. The cryogels displayed open macroporous structure and superior mechanical properties. These novel phase transformable phenylboronate-polyol based scaffolds displayed a great potential for various cell sheet and tissue engineering applications. Their monosaccharide responsiveness at physiological pH is very useful and can be utilized in the fields of cell immobilization, spheroid culture, saccharide recognition and analyte-responsive drug delivery.     

Computationally guided identification of Akt-3, a serine/threonine kinase inhibitors: Insights from homology modelling,structure-based screening,molecular dynamics and quantum mechanical calculations

Abstract

Chemical entities targeting kinase signalling pathways serve as a potential strategy to combat malignancies. Protein Kinase B or Akt is a validated target for various malignancies and Akt3 remains the least explored isoform among all its isoforms. Initially, homology modelling technique was used for generating protein structure and further validation was performed using molecular dynamics simulation and Ramachandran plot. The validated protein structure was then subjected for active site analysis which led to identification of active site residues based on metrics provided by site score. The important residues in binding site were identified as Thr81, Asp271 and Asp289 for binding energetics and inhibition. Subsequently, virtual screening methodologies were used for identification of novel hits for inhibition of Protein Kinase B or Akt3. This led to the identification of two hits, i.e. thiophene derivative and thieno-pyridine derivative which were selected on the basis of their binding affinity and drug likeliness. These identified hits were subjected for molecular dynamics simulations, quantum mechanical and synthetic accessibility studies. The role of crucial residues in binding site stood validated as suggested by molecular dynamics simulations studies.

Communicated by Ramaswamy H. Sarma     

A disintegrin and metalloproteinase-12 (ADAM12): Function,roles in disease progression,and clinical implications

Background

A disintegrin and metalloproteinase-12 (ADAM12) is a member of the greater ADAM family of enzymes: these are multifunctional, generally membrane-bound, zinc proteases for which there are forty genes known (21 of these appearing in humans). ADAM12 has been implicated in the pathogenesis of various cancers, liver fibrogenesis, hypertension, and asthma, and its elevation or decrease in human serum has been linked to these and other physiological/pathological conditions.

Scope

In this review, we begin with a brief overview of the ADAM family of enzymes and protein structure. We then discuss the role of ADAM12 in the progression and/or diagnosis of various disease conditions, and we will conclude with an exploration of currently known natural and synthetic inhibitors.

Major conclusion

ADAM12 has potential to emerge as a successful drug target, although targeting the metalloproteinase domain with any specificity will be difficult to achieve due to structural similarity between the members of the ADAM and MMP family of enzymes. Overall, more research is required to establish ADAM12 being as a highly desirable biomarker and drug target of different diseases, and their selective inhibitors as potential therapeutic agents.

General significance

Given the appearance of elevated levels of ADAM12 in various diseases, particularly breast cancer, our understanding of this enzyme both as a biomarker and a potential drug target could help make significant inroads into both early diagnosis and treatment of disease.     

Genetic and physiological relatedness of antagonistic Trichoderma isolates against soil borne plant pathogenic fungi

In this study, the in vitro potential of 42 Trichoderma spp. were evaluated against four isolates of soil borne phytopathogenic fungi viz., Rhizoctonia solani, Macrophomina sp., Sclerotium rolfsii and Pythium aphanidermatum in dual culture techniques and through production of volatile and non-volatile inhibitors. In vitro screening results showed that the proportion of isolates with antagonistic activities was highest for the S. rolfsii followed by R. solani, Macrophomina sp. and P. aphanidermatum, respectively. The isolates TNT1, TNP2 and TWP1 showed consistent results in volatile and non-volatile activity in vitro against any of the two pathogens tested. Based on genomic finger prints, potential isolates showed no particular correlation between the origin of the isolates and the Random Amplified Polymorphic DNA (RAPD) groups could not be established. However, the polymorphism shown by the isolates did not correlate to their level of antagonism. Whereas, in physiology studies using BIOLOG (microbial identification system), three groups were formed, one group consists with 14 different Trichoderma species and two groups with two isolates each comprised of only T. koningii and T. viride.     

Effects of interleukin-18 on natural killer cells: costimulation of activation through Fc receptors for immunoglobulin

The antitumor activity of monoclonal antibodies is mediated by effector cells, such as natural killer (NK) cells, that express Fc receptors for immunoglobulin. Efficacy of monoclonal antibodies, including the CD20 antibody rituximab, could be improved by agents that augment the function of NK cells. Interleukin (IL)-18 is an immunostimulatory cytokine that has antitumor activity in preclinical models. The effects of IL-18 on NK cell function mediated through Fcγ receptors were examined. Human NK cells stimulated with immobilized IgG in vitro secreted IFN-γ as expected; such IFN-γ production was partially inhibited by blocking CD16 with monoclonal antibodies. IL-18 augmented IFN-γ production by NK cells stimulated with immobilized IgG or CD16 antibodies. NK cell IFN-γ production in response to immobilized IgG and/or IL-18 was inhibited by chemical inhibitors of Syk and several other kinases involved in CD16 signaling pathways. IL-18 augmented antibody-dependent cellular cytotoxicity (ADCC) of human NK cells against rituximab-coated Raji cells in vitro. IL-18 and rituximab acted synergistically to promote regression of human lymphoma xenografts in SCID mice. Inasmuch as IL-18 costimulates IFN-γ production and ADCC of NK cells activated through Fc receptors in vitro and augments antitumor activity of rituximab in vivo, it is an attractive cytokine to combine with monoclonal antibodies for treatment of human cancer.