Signal sequence insufficiency contributes to neurodegeneration caused by transmembrane prion protein

Protein translocation into the endoplasmic reticulum is mediated by signal sequences that vary widely in primary structure. In vitro studies suggest that such signal sequence variations may correspond to subtly different functional properties. Whether comparable functional differences exist in vivo and are of sufficient magnitude to impact organism physiology is unknown. Here, we investigate this issue by analyzing in transgenic mice the impact of signal sequence efficiency for mammalian prion protein (PrP). We find that replacement of the average efficiency signal sequence of PrP with more efficient signals rescues mice from neurodegeneration caused by otherwise pathogenic PrP mutants in a downstream hydrophobic domain (HD). This effect is explained by the demonstration that efficient signal sequence function precludes generation of a cytosolically exposed, disease-causing transmembrane form of PrP mediated by the HD mutants. Thus, signal sequences are functionally nonequivalent in vivo, with intrinsic inefficiency of the native PrP signal being required for pathogenesis of a subset of disease-causing PrP mutations.     

Insight towards the conserved water mediated recognition of catalytic and structural Zn+2 ions in human Matrix Metalloproteinase-8 enzyme: A study by MD-simulation methods

Human matrix metalloproteinase-8 (hMMP-8) plays a important role in the progression of colorectal cancer, metastasis, multiple sclerosis and rheumetoid arthritis. Extensive MD-simulation of the PDB and solvated structures of hMMP-8 has revealed the presence of few conserved water molecules around the catalytic and structural zinc (ZnC and ZnS) ions. The coordination of two conserved water molecules (W and WS) to ZnS and the H-bonding interaction of WS to S151 have indicated the plausible involvement of that metal ion in the catalytic process. Beside this the coupling of ZnC and ZnS metal ions (ZnC – W_H (W₁)…..W₂ ….H₁₆₂ - ZnS) through two conserved hydrophilic centers (occupied by water molecules) may also provide some rational on the recognition of two zinc ions which were separated by ~13 Å in their X-ray structures. This unique recognition of both the Zn⁺² ions in the enzyme through conserved water molecules may be implemented/ exploited for the design of antiproteolytic agent using water mimic drug design protocol.     

Combination of LC3 shRNA Plasmid Transfection and Genistein Treatment Inhibited Autophagy and Increased Apoptosis in Malignant Neuroblastoma in Cell Culture and Animal Models

Malignant neuroblastoma is an extracranial solid tumor that usually occurs in children. Autophagy, which is a survival mechanism in many solid tumors including malignant neuroblastoma, deters the efficacy of conventional chemotherapeutic agents. To mimic starvation, we used 200 nM rapamycin that induced autophagy in human malignant neuroblastoma SK-N-BE2 and IMR-32 cells in cell culture and animal models. Combination of microtubule associated protein light chain 3 short hairpin RNA (LC3 shRNA) plasmid transfection and genistein (GST) treatment was tested for inhibiting rapamycin-induced autophagy and promoting apoptosis. The best synergistic efficacy caused the highest decrease in cell viability due to combination of 50 nM LC3 shRNA plasmid transfection and 25 µM GST treatment in rapamycin-treated SK-N-BE2 cells while combination of 100 nM LC3 shRNA plasmid transfection and 25 µM GST treatment in rapamycin-treated IMR-32 cells. Quantitation of acidic vesicular organelles confirmed that combination of LC3 shRNA plasmid transfection and GST treatment prevented rapamycin-induced autophagy due to down regulation of autophagy promoting marker molecules (LC3 II, Beclin 1, TLR-4, and Myd88) and upregulation of autophagy inhibiting marker molecules (p62 and mTOR) in both cell lines. Apoptosis assays showed that combination therapy most effectively activated mitochondrial pathway of apoptosis in human malignant neuroblastoma in cell culture and animal models. Collectively, our current combination of LC3 shRNA plasmid transfection and GST treatment could serve as a promising therapeutic strategy for inhibiting autophagy and increasing apoptosis in human malignant neuroblastoma in cell culture and animal models.     

Ubiquitin-Like Protein from Human Placental Extract Exhibits Collagenase Activity

An aqueous extract of human placenta exhibits strong gelatinase/collagenase activity in zymography. 2-D gel electrophoresis of the extract with gelatin zymography in the second dimension displayed a single spot, identified as ubiquitin-like component upon MALDI/TOF MS/MS analysis. Immunoblot indicated presence of ubiquitin and absence of collagenase in the extract. Collagenase activity of the ubiquitin-like component was confirmed from the change in solubility of collagen in aqueous buffer, degradation of collagen by size-exclusion HPLC and atomic force microscopy. Quantification with DQ-gelatin showed that the extract contains 0.04 U/ml of collagenase activity that was inhibited up to 95% by ubiquitin antibody. Ubiquitin from bovine erythrocytes demonstrated mild collagenase activity. Bioinformatics studies suggest that placental ubiquitin and collagenase follow structurally divergent evolution. This thermostable intrinsic collagenase activity of placental extract might have wide physiological relevance in degrading and remodeling collagen as it is used as a drug for wound healing and pelvic inflammatory diseases.     

A profound computational study to prioritize the natural compound inhibitors against the P. falciparum orotidine-5-monophosphate decarboxylase enzyme

Abstract

In the current contribution, a multicomplex-based pharmacophore modeling approach was employed on the structural proteome of Plasmodium falciparum orotidine-5-monophosphate decarboxylase enzyme (PfOMPDC). Among the constructed pharmacophore models, the representative hypotheses were selected as the primary filter to screen the molecules with the complementary features responsible for showing inhibition. Thereafter, auxiliary evaluations were performed on the screened candidates via drug-likeness and molecular docking studies. Subsequently, the stability of the docked protein-ligand complexes was scrutinized by employing molecular dynamics simulations and molecular mechanics-Poisson Boltzmann surface area based free binding energy calculations. The stability the docked candidates was compared with the highly active crystallized inhibitor (3S9Y-FNU) to seek more potential candidates. All the docked molecules displayed stable dynamic behavior and high binding free energy in comparison to 3S9Y-FNU. The employed workflow resulted in the retrieval of five drug-like candidates with diverse scaffolds that may show inhibitory activity against PfOMPDC and could be further used as the novel scaffold to develop novel antimalarials.

Communicated by Ramaswamy H. Sarma     

Non-Healing Ulcer Due to Trichosporon loubieri in an Immunocompetent Host and Review of Published Reports

We report here a case of non-healing ulcer due to Trichosporon loubieri in an apparently immunocompetent female. The identity of isolate was confirmed by DNA sequencing of D1/D2 region of 26S rDNA. The minimum inhibitory concentrations of the isolate were amphotericin B—0.5 μg/ml; fluconazole—4 μg/ml; posaconazole—0.25 μg/ml; voriconazole—0.06 μg/ml. The patient was managed by extensive debridement and oral fluconazole 150 mg daily for 6 weeks. She responded to therapy. To the best of our knowledge, till date, this is the fourth report of human infection due to T. loubieri and the first of its kind in an immunocompetent host. A review of published literature on infections due to T. loubieri is also included.