Biology subjects

Dibyachintan, S.

Publications and source records attributed to Dibyachintan, S..

6 recordsLinked to original sources

bioRxiv2026

The hydrophobicity of peptides limits their availability to bind to interacting domains

A challenge in synthetic biology is to transfer the knowledge acquired in vitro to in vivo applications. For instance, binding affinity between peptide binding domains (PBDs) and their binding peptides is usually measured in simple buffers and in the absence of the cell machinery responsible for synthesis and proteostasis. Consequently, there is often a discrepancy between interaction quantified in vitro and in vivo. To identify strongly binding PBD-peptide pairs for in vivo applications, we aim to better understand the determinants of synthetic interaction in living yeast cells. We designed libraries of peptides expected to bind to PBDs based on data collected by phage-display. Using an in vivo protein interaction assay, we assessed the interaction strength of >6,500 PBD-peptide variants in yeast. We found that most substitutions disrupt binding, but that 3.4% increased binding strength to levels higher than expected from the phage-display data. We asked if, by using our mutational data, it was possible to optimize peptide sequences to gain even more binding strength and show additivity effect up to four substitutions. We found that PBD specificity is consistent between in vitro and in vivo assays. We hypothesized that in vivo peptide abundance could contribute to the discrepancy between the PCA and the phage-display results. We performed assays to detect in vivo peptide availability, reporting on peptide abundance, and we found that peptides with a stronger binding also show a higher availability in cells. We show that peptide availability, which seems mainly defined by peptide hydrophobicity, is key for the design of strong synthetic interactions.

molecular biology↗

bioRxiv2026

Epistasis limits but does not prevent the transfer of mutation-drug resistance mapping across 600 million years of fungal evolution

Whether different pathogens acquire resistance to antimicrobials through the same mutations is a major question in evolution and microbiology. Most antifungal drugs are used to treat infections caused by multiple fungal species, many of which have diverged for millions of years. If the evolution of resistance was to converge onto the same set of mutations across species, knowing the mechanism of resistance in one would allow us to predict and track it in others. The extent of this convergence remains unknown due to the lack of systematic data on resistance mutations. Here, we quantify the conservation of resistance mutations in the cytosine deaminase (CD), a protein responsible for resistance to flucytosine, one of the oldest antifungal drugs. By comparing the crystal structures of this enzyme through 600 My of evolution, we show that the CD structure is highly conserved. We compared the full CD mutational resistance spectrum of resistance from an ascomycete and a basidiomycete. We found that resistance mutations in one ortholog can be used to predict resistance in the other at a high level of accuracy. However, because of epistasis, around 10% of mutations have distinct effects in the two orthologs, which imposes an upper limit to the transferability of the knowledge of resistance mutations from one species to another. Using biochemical assays and by structural characterization of several mutants, we identify distinct mechanisms of epistasis, one important being that the local physiochemical environment of some position has evolved in a way that makes the same substitutions destabilizing or entirely inactivating in an ortholog-specific manner. Our results show that resistance mutations can be conserved in fungi across hundreds of millions of years of evolution but that epistasis eventually limits the accuracy of these predictions.

microbiology↗

bioRxiv2024

Cryptic genetic variation shapes the fate of gene duplicates in a protein interaction network

Paralogous genes are often redundant for long periods of time before they diverge in function. While their functions are preserved, paralogous proteins can accumulate mutations that, through epistasis, could impact their fate in the future. By quantifying the impact of all single-amino acid substitutions on the binding of two myosin proteins to their interaction partners, we find that the future evolution of these proteins is highly contingent on their regulatory divergence and the mutations that have silently accumulated in their protein binding domains. Differences in the promoter strength of the two paralogs amplify the impact of mutations on binding in the lowly expressed one. While some mutations would be sufficient to non-functionalize one paralog, they would have minimal impact on the other. Our results reveal how functionally equivalent protein domains could be destined to specific fates by regulatory and cryptic coding sequence changes that currently have little to no functional impact.

evolutionary biology↗

bioRxiv2023

Deep mutational scanning of Pneumocystis jirovecii dihydrofolate reductase reveals allosteric mechanism of resistance to an antifolate

Pneumocystis jirovecii is a fungal pathogen that causes pneumocystis pneumonia, a disease that mainly affects immunocompromised individuals. This fungus has historically been hard to study because of our inability to grow it in vitro. One of the main drug targets in P. jirovecii is its dihydrofolate reductase (PjDHFR). Here, by using functional complementation of the bakers yeast ortholog, we show that PjDHFR can be inhibited by the antifolate methotrexate in a dose-dependent manner. Using deep mutational scanning of PjDHFR, we identify mutations conferring resistance to methotrexate. Thirty-one sites spanning the protein have at least one mutation that leads to resistance, for a total of 355 high-confidence resistance mutations. Most resistance-inducing mutations are found inside the active site, and many are structurally equivalent to mutations known to lead to resistance to different antifolates in other organisms. Some sites show specific resistance mutations, where only a single substitution confers resistance, whereas others are more permissive, as several substitutions at these sites confer resistance. Surprisingly, one of the permissive sites (F199) is without direct contact to either ligand or cofactor, suggesting that it acts through an allosteric mechanism. Modeling changes in binding energy between F199 mutants and drug shows that most mutations destabilize interactions between the protein and the drug. This evidence points towards a more important role of this position in resistance than previously estimated and highlights potential unknown allosteric mechanisms of resistance to antifolate in DHFRs. Our results offer unprecedented resources for the interpretation of mutation effects in the main drug target of an uncultivable fungal pathogen. Author summaryThe study of uncultivable microorganisms has always been a challenge. Such is the case of the human-specific pathogen Pneumocystis jirovecii, the causative agent of pneumocystis pneumonia. P. jirovecii is insensitive to classical antifungal drugs, making options for treatment and prophylaxis limited. In recent years, more and more cases of P. jirovecii infections have become resistant to treatment, highlighting the need to study and understand this pathogens mechanisms of resistance. Here, we use a yeast strain expressing P. jiroveciis DHFR as a reporter for resistance to an antifolate, one of the drug families used to treat infections. We observed that this DHFR was sensitive to methotrexate, a powerful antifolate, in a quantitative manner. Then, by using a large-scale mutational assay, we identified virtually all single mutations that confer this protein resistance to methotrexate. While any of them have also been reported in other eukaryotes, we find new mutations at positions of the protein not previously known to confer resistance or to be in contact with this competitive inhibitor. Overall, our results are a comprehensive portrait of this DHFRs resistance to methotrexate.

biochemistry↗

bioRxiv2021

A novel innate pathogen sensing strategy involving ubiquitination of bacterial surface proteins

Sensing of pathogens by ubiquitination is critical for maintaining cytosolic sanctity. However, universal ubiquitination targets on bacteria, especially of proteinaceous origin, remain unidentified. Here, we unveil a novel strategy, involving recognition of degron-like motifs for identification of first protein-based ubiquitination substrates on phylogenetically distinct bacteria. Such motifs can form a new class of intra-cytosolic pathogen associated molecular patterns (PAMPs) as their incorporation enables identification of non-ubiquitin targets by host Ub-ligases. We find SCFFBW7 E3-ligase, supported by the regulatory kinase, GSK3{beta}, is crucial for effective pathogen detection and clearance. This may explain the enhanced risk of infections in Chronic Lymphocytic Leukaemia patients bearing FBXW7 mutations. We conclude that exploitation of such ubiquitous pathogen sensing strategy allows conservation of cellular resources and boost anti-microbial immunity. One Sentence SummaryUbiquitination of bacterial surface proteins fosters sensing and clearance of diverse pathogens

microbiology↗

bioRxiv2020

The specificity of SRC Homology 3 (SH3) domain interactions is modulated by their protein context in vivo

Protein-protein interactions (PPIs) between modular binding domains and their target peptide motifs are thought to largely depend on the intrinsic binding specificities of the domains. By combining deletion, mutation, swapping and shuffling of SRC Homology 3 (SH3) domains and measuring their impact on protein interactions, we find that most SH3s do not autonomously dictate PPI specificity in vivo. The identity of the host protein and the position of the SH3 domains within their host are both critical for PPI specificity, for cellular functions and for key biophysical processes such as phase separation. Our work demonstrates the importance of the interplay between a modular PPI domain such as SH3 and its host protein in establishing specificity to wire PPI networks.Competing Interest StatementThe authors have declared no competing interest.View Full Text

systems biology↗