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Bharadwaj, T.

Publications and source records attributed to Bharadwaj, T..

2 recordsLinked to original sources
bioRxiv2023

Japanese Encephalitis Virus: A pan-proteome analysis for aggregation propensities and in vitro validation with Capsid anchor and 2K peptide

Japanese encephalitis infection is a vector-borne disease caused by the flavivirus Japanese encephalitis virus (JEV). It is responsible of severe brain infection in humans worldwide. Given the ubiquitous nature of complications and tropism associated with Japanese encephalitis (JE) infection, a holistic understanding of its molecular mechanism is essential. The phenomenon of abnormal protein aggregation into pathogenic amyloids is now increasingly linked to multiple human diseases, also known as Amyloidosis. Most are neurodegenerative disorders but amyloidosis is not restricted to a specific organ or tissue type. The overlap of viral protein aggregation with human pathologies remains limited and it is gaining momentum, especially after the devastating Covid-19 pandemic. Therefore, in this study we have examined the likelihood of aggregation for the entire collection of proteins in JEV. Multiple independent web server tools were employed to scan for potential amyloid prone-regions (APRs), and it was followed by in vitro validation using two JEV transmembrane domains, Capsid anchor and 2K peptides. These synthetic viral peptides were introduced to artificial aggregation-inducing conditions and then analyzed using a different dye-based assays and microscopy methods confirming amyloid-like fibril structure formation. We found these aggregates cytotoxic to human neuronal cell line and membrane damaging to human blood derived RBCs. The aggregation kinetics of both peptides is enhanced in the presence of artificial membrane models and seeds of self and diabetes hallmark protein Amylin. Our findings thereby strongly suggest the possibility of JEV protein aggregation playing a vital role in its pathogenesis, opening up a broad scope of future study. Also, the interplay between JEV protein aggregation and initiation/progression of other proteopathies is possible and needs further exploration. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=116 SRC="FIGDIR/small/556571v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@2956d0org.highwire.dtl.DTLVardef@28128org.highwire.dtl.DTLVardef@6d7faaorg.highwire.dtl.DTLVardef@d89f40_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗
bioRxiv2022

Towards universal synthetic heterotrophy using a metabolic coordinator

Engineering the utilization of non-native substrates, or synthetic heterotrophy, in proven industrial microbes such as Saccharomyces cerevisiae represents an opportunity to valorize plentiful and renewable sources of carbon and energy as potential inputs to biotechnological processes. We previously demonstrated that activation of the galactose (GAL) regulon, a regulatory structure used by this yeast to coordinate substrate utilization with biomass formation during growth on galactose, during growth on the non-native substrate xylose results in a vastly altered gene expression profile and faster growth compared with constitutive overexpression of the same heterologous catabolic pathway. However, this effort involved the creation of a xylose-inducible variant of Gal3p (Gal3pS25144.1), the sensor protein of the GAL regulon, preventing this semi-synthetic regulon approach from being easily adapted to additional non-native substrates. Here, we report the construction of a variant Gal3pMC (metabolic coordinator) that exhibits robust GAL regulon activation in the presence of structurally diverse substrates and recapitulates the dynamics of the native system. Multiple molecular modeling studies confirm that Gal3pMC occupies conformational states corresponding to galactose-bound Gal3p in an inducer-independent manner. Using Gal3pMC to test a regulon approach to the assimilation of the non-native lignocellulosic sugars xylose, arabinose, and cellobiose yields higher growth rates and final cell densities when compared with a constitutive overexpression of the same set of catabolic genes. The subsequent demonstration of rapid and complete co-utilization of all three non-native substrates suggests that Gal3pMC-mediated dynamic global gene expression changes by GAL regulon activation may be universally beneficial for engineering synthetic heterotrophy.

synthetic biology↗