ChaC1 upregulation reflects poor prognosis in a variety of cancers: analysis of the major missense SNPs of ChaC1 as an aid to refining prognosis.

The ChaC1 enzyme that catalyzes cytosolic glutathione degradation is highly upregulated in several cancers. In a systematic review of gene signature panels for cancer prognosis based on oxidative stress and ferroptosis genes, we observed that ChaC1 was found in panels in a wide variety of different cancers, with the upregulation correlating with poor prognosis. Since SNPs can have an impact on functionality and prognosis, ChaC1 SNPs from various databases were also investigated. Six frequently observed missense SNPs were chosen for reconstruction, and their functionality was evaluated. Three out of six SNPs resulted in either a partial or complete loss of ChaC1 function, and these SNPs had the changes R72Q, A156V, and G173S in their proteins. This study highlights the importance of ChaC1 in cancer prognosis across a wide variety of cancers. Additionally, the information on the SNPs of ChaC1 with altered enzymatic activities would improve the prognostic ability of these panels and facilitate treatment regimens.

The ChaC1 active site: Defining the residues and determining the role of ChaC1-exclusive residues in the structural and functional stability.

The glutathione degrading enzyme ChaC1 is highly upregulated in several cancers and viral infections making it a potential pharmacological target for cancer therapy. As an enzyme, however, ChaC1 has a relatively high Km (~2 mM) towards its natural substrate, and therefore finding its inhibitors becomes very difficult. Given this limitation, a careful mapping of the active site has become necessary. In the current study, the enzyme-substrate complex was generated by docking glutathione with the modeled hChaC1 structure. Using a combination of in silico and wet lab approaches, the active site residues forming direct interactions with the substrate glutathione were identified and validated. Furthermore, the role of residues exclusively conserved in the ChaC family and forming the surface of the active site were also explored for their putative role in active site stabilization. Mutants of these residues have been analysed for their structural stability and interaction with the substrate through MD simulations and MMGBSA binding energy calculations. These findings were experimentally validated by assessment of their function through in vivo assays in yeast. The experimental evidences along with the molecular modeling suggest that residues 38'YGSL'41, D68, R72, E115, and Y143 are responsible for high affinity binding of hChaC1 with the substrate/inhibitor, whereas the residues exclusive to the ChaC family are required for the structural stability of the enzyme and its active site. Such a characterization of essential active site and conserved residues is significant as a key step toward rational design of novel inhibitors of the ChaC1 enzyme.

Cysteine-phenylalanine-derived self-assembled nanoparticles as glutathione-responsive drug-delivery systems in yeast.

Despite the availability of different antifungal drugs in the market, their overall usefulness remains questionable due to the relatively high toxic profiles exerted by them in many cases. In addition, the emergence of drug resistance against these antifungal agents is a matter of concern. Thus, it becomes imperative to explore innovative drug-delivery vehicles to deliver these antifungal drugs for enhanced efficacy, mitigating unwanted side effects and tackling the surge in antifungal resistance. Considering this fact, in this piece of work, we have synthesized stimulus (glutathione)-responsive dipeptide-based self-assembled nanoparticles (NPs) to explore and establish the redox-responsive antifungal drug delivery of a relatively hydrophobic drug, terbinafine (Terb), in Saccharomyces cerevisiae (S. cerevisiae). The NPs were prepared using a relatively aqueous environment as opposed to other Terb formulations that are administered in mostly non-polar solvents and with limited biocompatibility. The NPs demonstrated an encapsulation efficiency of around 99% for Terb and resulted in complete inhibition of yeast-cell growth at a dose of 200 µg mL-1 of the drug-loaded formulation. Thus, these biocompatible and aqueous dipeptide-based redox-responsive NPs can offer a promising drug-delivery platform to provide enhanced antifungal drug delivery with heightened efficacy and biocompatibility.

Allosteric inhibition of MTHFR prevents futile SAM cycling and maintains nucleotide pools in one-carbon metabolism.

Methylenetetrahydrofolate reductase (MTHFR) links the folate cycle to the methionine cycle in one-carbon metabolism. The enzyme is known to be allosterically inhibited by SAM for decades, but the importance of this regulatory control to one-carbon metabolism has never been adequately understood. To shed light on this issue, we exchanged selected amino acid residues in a highly conserved stretch within the regulatory region of yeast MTHFR to create a series of feedback-insensitive, deregulated mutants. These were exploited to investigate the impact of defective allosteric regulation on one-carbon metabolism. We observed a strong growth defect in the presence of methionine. Biochemical and metabolite analysis revealed that both the folate and methionine cycles were affected in these mutants, as was the transsulfuration pathway, leading also to a disruption in redox homeostasis. The major consequences, however, appeared to be in the depletion of nucleotides. 13C isotope labeling and metabolic studies revealed that the deregulated MTHFR cells undergo continuous transmethylation of homocysteine by methyltetrahydrofolate (CH3THF) to form methionine. This reaction also drives SAM formation and further depletes ATP reserves. SAM was then cycled back to methionine, leading to futile cycles of SAM synthesis and recycling and explaining the necessity for MTHFR to be regulated by SAM. The study has yielded valuable new insights into the regulation of one-carbon metabolism, and the mutants appear as powerful new tools to further dissect out the intersection of one-carbon metabolism with various pathways both in yeasts and in humans.

In silico screening of proteins targeting circulating miRNAs for improved diagnosis of multiple myeloma.

Multiple Myeloma (MM) is a B-cell malignancy, which is characterized by the expansion of clonal plasma cells in the bone marrow, leading to abnormal accumulation of monoclonal antibodies in circulation. Certain circulating miRNAs are deregulated in MM and their differential expression profiles in body fluids can be quantified and used to discriminate between the premalignant and malignant stages of MM. Our study identifies protein which would show affinity for a selected panel of circulating miRNAs deregulated in MM. Human RNA binding proteins were identified based on their unique RNA binding domains and their interacting probabilities with the panel of miRNAs deregulated in MM. miR-26 was used as a negative control for interaction studies. 3-D structure of candidate proteins were determined and molecular docking was performed to confirm the results. Five RNA binding proteins TROVE2, CUGBP2, DHX8, PUM2 and DKC1 were used for molecular docking studies. DKC1 showed significant hydrogen bonding as well as remarkable binding affinity values of -17.4 kcal/mol with miR-720 (2 H-bonds), -16 kcal/mol with miR-1246 (1 H-bond) and -16.9 kcal/mol with miR-1308 (3 H-bonds). Identified protein-miRNA interaction could be used to develop an economical and reliable ELISA based methodology for improved and sensitive diagnosis of MM patients.

Genetic and epigenetic markers in colorectal cancer screening: recent advances.

INTRODUCTION: Colorectal cancer (CRC) is a heterogenous disease which develops from benign intraepithelial lesions known as adenomas to malignant carcinomas. Acquired alterations in Wnt signaling, TGFß, MAPK pathway genes and clonal propagation of altered cells are responsible for this transformation. Detection of adenomas or early stage cancer in asymptomatic patients and better prognostic and predictive markers is important for improving the clinical management of CRC. Area covered: In this review, the authors have evaluated the potential of genetic and epigenetic alterations as markers for early detection, prognosis and therapeutic predictive potential in the context of CRC. We have discussed molecular heterogeneity present in CRC and its correlation to prognosis and response to therapy. Expert commentary: Molecular marker based CRC screening methods still fail to gain trust of clinicians. Invasive screening methods, molecular heterogeneity, chemoresistance and low quality test samples are some key challenges which need to be addressed in the present context. New sequencing technologies and integrated omics data analysis of individual or population cohort results in GWAS. MPE studies following a GWAS could be future line of research to establish accurate correlations between CRC and its risk factors. This strategy would identify most reliable biomarkers for CRC screening and management.