Biology subjects

Asthana, P.

Publications and source records attributed to Asthana, P..

4 recordsLinked to original sources

bioRxiv2025

Metabolic modulation of intratumoral cholesterol with gut microbiota for the treatment of colorectal cancer

Excess cholesterol is positively correlated with colorectal cancer (CRC). Current therapeutic strategies for modulating cholesterol levels in CRC are limited and often come with complications. Here, we demonstrated that microbiome shunting of intestinal cholesterol to anticancer metabolites is a safe and applicable therapeutic approach for CRC. By screening major microbial metabolic products of cholesterol, we found that 4-cholesten-3-one (4-C-3) was selectively depleted in fecal samples and tumor tissues of patients with CRC. 4-C-3 exhibits strong antitumor effects on human CRC cell lines, patient-derived organoids, and patient-derived xenograft (PDX) models. Mechanistically, 4-C-3 suppresses CRC tumorigenesis by dually targeting the epidermal growth factor receptor (EGFR) and Kirsten rat sarcoma viral oncogene homologue (KRAS). 4-C-3 directly binds to EGFR, blocking its signal transduction by inhibiting the binding of EGFR ligands. Additionally, 4-C-3 inhibits oncogenic KRAS variants, such as KRASG12D, by suppressing nucleotide exchange activity and effector engagement. By targeting both EGFR and KRAS, 4-C-3 reduces primary resistance to anti-EGFR therapies caused by KRAS mutations in CRC. As a proof-of-concept study, we showed that delivery of 4-C-3 by Oscillibacter ruminantium, a 4-C-3- producing commensal bacterium that is reduced in CRC patients, or a nonpathogenic Escherichia coli strain engineered to specifically convert intestinal cholesterol into 4-C-3, removed intratumoral cholesterol and led to rapid tumor regression in multiple models of CRC. These results suggest that microbial therapies for restoring intestinal cholesterol homeostasis represent a new therapeutic avenue for CRC.

cancer biology↗

bioRxiv2025

Distal mutations enhance catalysis in designed enzymes by facilitating substrate binding and product release

The role of amino-acid residues distant from an enzymes active site in facilitating the complete catalytic cycle--including substrate binding, chemical transformation, and product release-- remains poorly understood. Here, we investigate how distal mutations promote the catalytic cycle by engineering mutants of three de novo Kemp eliminases containing either active-site or distal mutations identified through directed evolution. Kinetic analyses, X-ray crystallography, and molecular dynamics simulations reveal that while active-site mutations create preorganized catalytic sites for efficient chemical transformation, distal mutations enhance catalysis by facilitating substrate binding and product release through tuning structural dynamics to widen the active-site entrance and reorganize surface loops. These distinct contributions work synergistically to improve overall activity, demonstrating that a well-organized active site, though necessary, is not sufficient for optimal catalysis. Our findings reveal critical roles that distal residues play in shaping the catalytic cycle to enhance efficiency, yielding valuable insights for enzyme design.

biochemistry↗

bioRxiv2023

Structural characterization of ligand binding and pH-specific enzymatic activity of mouse Acidic Mammalian Chitinase

Chitin is an abundant biopolymer and pathogen-associated molecular pattern that stimulates a host innate immune response. Mammals express chitin-binding and chitin-degrading proteins to remove chitin from the body. One of these proteins, Acidic Mammalian Chitinase (AMCase), is an enzyme known for its ability to function under acidic conditions in the stomach but is also active in tissues with more neutral pHs, such as the lung. Here, we used a combination of biochemical, structural, and computational modeling approaches to examine how the mouse homolog (mAMCase) can act in both acidic and neutral environments. We measured kinetic properties of mAMCase activity across a broad pH range, quantifying its unusual dual activity optima at pH 2 and 7. We also solved high resolution crystal structures of mAMCase in complex with oligomeric GlcNAcn, the building block of chitin, where we identified extensive conformational ligand heterogeneity. Leveraging these data, we conducted molecular dynamics simulations that suggest how a key catalytic residue could be protonated via distinct mechanisms in each of the two environmental pH ranges. These results integrate structural, biochemical, and computational approaches to deliver a more complete understanding of the catalytic mechanism governing mAMCase activity at different pH. Engineering proteins with tunable pH optima may provide new opportunities to develop improved enzyme variants, including AMCase, for therapeutic purposes in chitin degradation.

biochemistry↗

bioRxiv2020

Structural insights on the substrate-binding proteins of the Mycobacterium tuberculosis mammalian-cell-entry (Mce) 1 and 4 complexes

Tuberculosis (Tb), caused by Mycobacterium tuberculosis (Mtb), is responsible for more than a million deaths annually. In the latent phase of infection, Mtb uses lipids as the source of carbon and energy for its survival. The lipid molecules are transported across the cell wall via multiple transport systems. One such set of widely present and less-studied transporters is the Mammalian-cell-entry (Mce) complexes. Here, we report the properties of the substrate-binding proteins (SBPs; MceA-F) of the Mce1 and Mce4 complexes from Mtb which are responsible for the import of mycolic acid/fatty acids, and cholesterol respectively. MceA-F are composed of four domains namely, transmembrane, MCE, helical and tail domains. Our studies show that MceA-F are predominantly monomeric when purified individually and do not form homohexamers unlike the reported homologs (MlaD, PqiB and LetB) from other prokaryotes. The crystal structure of MCE domain of Mtb Mce4A (MtMce4A39-140) determined at 2.9 [A] shows the formation of an unexpected domain-swapped dimer in the crystals. Further, the purification and small-angle X-ray scattering (SAXS) analysis on MtMce1A, MtMce4A and their domains suggest that the helical domain requires hydrophobic interactions with the detergent molecules for its stability. Combining all the experimental data, we propose a heterohexameric arrangement of MtMceA-F SBPs, where the soluble MCE domain of the SBPs would remain in the periplasm with the helical domain extending to the lipid layer forming a hollow channel for the transport of lipids across the membranes. The tail domain would reach the cell surface assisting in lipid recognition and binding.

biochemistry↗