Biology subjects

Nair, S. K.

Publications and source records attributed to Nair, S. K..

11 recordsLinked to original sources

bioRxiv2026

Discovery of the Phosphonate Flavophos Produced by Burkholderia

Phosphonate natural products have proven value to society as antibiotics and herbicides. They inhibit a range of enzyme targets usually by mimicking the enzyme substrates. In this study, we investigate a family of phosphonate biosynthetic gene clusters (BGCs) found in Burkholderia. Heterologous expression in Escherichia coli resulted in production of an antimicrobial compound. Spectroscopic characterization and chemical synthesis assigned its structure as 2,4-dioxopentylphosphonic acid. One of the biosynthetic enzymes is a member of the domain of unknown function (DUF) 849 family with homology to {beta}-keto acid cleavage enzymes (BKACEs). In vitro characterization shows that this enzyme catalyzes chemistry that is divergent from previously characterized BKACEs. The observed catalytic activity is explained by a series of co-crystal structures with substrates and intermediates. The BGC also contains a gene encoding lumazine synthase (LS), an essential enzyme in flavin biosynthesis. Biochemical experiments revealed that 2,4-dioxopentylphosphonic acid inhibits LS. In addition, expression of the LS encoded in the BGC, or LS orthologs from a range of organisms, in E. coli conferred resistance to the new phosphonate, which we therefore name flavophos.

biochemistry↗

bioRxiv2025

Enhancing enzymatic bioconjugation efficiency via installation of a substrate recruitment domain

Enzyme mediated bioconjugation provides a method for easy and rapid formation of protein-protein and protein-small molecule conjugates under mild conditions. Promiscuous enzymes are of particular interest because they can catalyze conjugation reactions on a broad set of substrates. However, this promiscuity carries the risk of undesirable off-target modifications. To mitigate this effect, we used computational design to install a substrate recruitment domain (SRD) onto the promiscuous enzyme, tyrosinase. The redesigned tyrosinase, called D42, preferentially modifies tyrosine residues within the peptide core (core) linked to a 6-amino acid recognition motif/sequence (RS) specific for the SRD. Incorporation of the recognition sequence along with a neighboring tyrosine in peptides or proteins allows for rapid D42-mediated conversion of the tyrosine to an orthoquinone, which can be selectively modified with a variety of nucleophiles. We demonstrate the utility of our design system by rapidly installing cytotoxic molecules on a monoclonal antibody. For Table of Contents Only O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=72 SRC="FIGDIR/small/684804v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@1a3b274org.highwire.dtl.DTLVardef@18eeedorg.highwire.dtl.DTLVardef@ee0aaeorg.highwire.dtl.DTLVardef@1ff3fa1_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2023

Detection of circular RNAs and their potential as biomarkers predictive of drug response

The introduction of high-throughput sequencing technologies has allowed for comprehensive RNA species detection, both coding and non-coding, which opened new avenues for the discovery of predictive and prognostic biomarkers. However the consistency of the detection of different RNA species depends on the RNA selection protocol used for RNA-sequencing. While preliminary reports indicated that non-coding RNAs, in particular circular RNAs, constitute a rich source of biomarkers predictive of drug response, the reproducibility of this novel class of biomarkers has not been rigorously investigated. To address this issue, we assessed the inter- lab consistency of circular RNA expression in cell lines profiled in large pharmacogenomic datasets. We found that circular RNA expression quantified from rRNA-depleted RNA-seq data is stable and yields robust prognostic markers in cancer. On the other hand, quantification of the expression of circular RNA from poly(A)-selected RNA-seq data yields highly inconsistent results, calling into question results from previous studies reporting their potential as predictive biomarkers in cancer. We have also identified median expression of transcripts and transcript length as potential factors influencing the consistency of RNA detection. Our study provides a framework to quantitatively assess the stability of coding and non-coding RNA expression through the analysis of biological replicates within and across independent studies.

bioinformatics↗

bioRxiv2022

Tail-tape-fused virion and non-virion RNA polymerases of a thermophilic virus with an extremely long tail

Thermus thermophilus bacteriophage P23-45 encodes a giant 5,002-residue tail tape measure protein (TMP)1 that defines the length of its extraordinarily long 800 nm tail2,3. We found that the N-terminal portion of P23-45 TMP is an unusual RNA polymerase (RNAP) homologous to cellular and viral two-barrel RNAPs. The TMP-fused virion RNAP transcribes pre-early phage genes, including a gene that encodes another, non-virion RNAP, that transcribes early and some middle phage genes. We determined the crystal structures of both P23-45 RNAPs. The non-virion RNAP has a crab claw-like architecture similar to previously reported two-barrel RNAPs. The virion RNAP adopts a unique flat structure without a clamp, which likely reflects the requirement for its extrusion through the narrow channel in the phage tail for delivery into the cell. Structure and sequence comparisons of the P23-45 RNAPs with other phage and cellular RNAPs suggest that, despite the extensive functional differences, the two P23-45 RNAPs originate from an ancient gene duplication in an ancestral phage. Our findings demonstrate remarkable adaptability of two-barrel RNAPs that can be attained within a single virus species.

molecular biology↗

bioRxiv2022

Meta-analysis of preclinical pharmacogenomic studies to discover robust and translatable biomarkers of drug response

Preclinical pharmacogenomic studies provide an opportunity to discover novel biomarkers for drug response. However, pharamcogenomic studies linking gene expression profiles to drug response do not always agree on the significance or strength of biomarkers. We apply a statistical meta-analysis approach to 7 large independent pharmacogenomic studies, testing for tissue-specific gene expression markers predictive of response among cancer cell lines. We found 4,338 statistically-significant biomarkers across 8 tissue types and 34 drugs. Significant biomarkers were found to be closer than random to drug targets in a gene network built on pathway co-membership (average distance of 2 vs 2.9). However, functional relationships with the drug target did not predict reproducibility across studies. To validate these biomarkers, we utilized 10 clinical datasets, allowing 42/4338 biomarkers to be assessed for clinical translation. Of the 42 candidate biomarkers, the expression of ODC1 was found to be significantly predictive of Paclitaxel response as a neoadjuvant treatment of breast carcinoma across 2 independent clinical studies of >200 patients each. We expect that as more clinical transcriptomics data matched with response are available, our results can be used to prioritize which genes to evaluate as clinical biomarkers of drug response.

cancer biology↗

bioRxiv2022

Intratumoral Recall of Childhood Vaccine-Specific CD4+ T cells Coordinates Type I and II Antitumor Immunity

BackgroundCD4+ T cells are key contributors to cancer immune surveillance. However, means to effectively harness CD4+ T cell help for cancer immunotherapy are lacking, and antitumor mechanisms of CD4+ T cells remain crudely defined. MethodsThe impact of polio immunization on polio virotherapy was tested in syngeneic murine melanoma and breast cancer models. Antitumor effects of polio and tetanus toxoid antigens were assessed in polio and tetanus immunized mice. T and B cell knockout mice, CD4+ T cell adoptive transfer, and eosinophil depletion demonstrated cell-type specific contributions to the antitumor efficacy of polio recall. Phenotyping of adoptively transferred OT-I (OVA-specific) T cells in B16-OVA tumor bearing mice, as well as adoptive transfer of T cells to naive tumor-bearing recipients, measured the impact of intratumor polio/tetanus recall on antitumor T cell immunity. Pan-cancer human transcriptome data sets were queried to test associations between eosinophils and Tregs; cytokine profiles of polio and tetanus recall were defined in human peripheral blood of healthy donors and cancer patients; CD40L blockade was used to determine dependency of recall antigen therapy on CD40:CD40L signaling. ResultsPrior vaccination against poliovirus substantially bolstered the antitumor efficacy of polio virotherapy in mice, and intratumor recall of poliovirus or tetanus immunity delayed tumor growth in a manner complemented by pattern recognition receptor agonist therapy and PD1 blockade. Intratumor recall antigens augmented antitumor T cell function, and caused marked tumor infiltration of type 2 innate lymphoid cells (ILC2s) and eosinophils, coinciding with decreased proportions of intratumor Tregs. Antitumor effects of recall antigens were mediated by CD4+ T cells, independent of CD40L signaling, and were dependent on both eosinophils and CD8+ T cells. Human PBMCs mounted diverse cytokine/chemokine responses, which were not impaired in patients with advanced cancer, and an inverse relationship between eosinophil and Treg signatures was observed across TCGA cancer types. ConclusionThis work defines cancer immunotherapy potential of childhood vaccines, reveals their utility to engage CD4+ T cell help for antitumor CD8+ T cells, and implicates eosinophils as antitumor effectors of CD4+ T cells.

immunology↗

bioRxiv2022

Prevalence and mechanisms of evolutionary contingency in human influenza H3N2 neuraminidase

Neuraminidase (NA) of human influenza H3N2 virus has evolved rapidly and been accumulating mutations for more than half-century. However, biophysical constraints that govern the evolutionary trajectories of NA remain largely elusive. Here, we show that among 70 natural mutations that are present in the NA of a recent human H3N2 strain, >10% are deleterious for an ancestral strain. By mapping the permissive mutations using combinatorial mutagenesis and next-generation sequencing, an extensive epistatic network is revealed. Biophysical and structural analyses further demonstrate that certain epistatic interactions can be explained by non-additive stability effect, which in turn modulates membrane trafficking and enzymatic activity of NA. Additionally, our results suggest that other biophysical mechanisms also contribute to epistasis in NA evolution. Overall, these findings not only provide mechanistic insights into the evolution of human influenza NA and elucidate its sequence-structure-function relationship, but also have important implications for the development of next-generation influenza vaccines.

microbiology↗

bioRxiv2021

Nutrient limitation magnifies fitness costs of antimalarial drug resistance mutations

Drug resistance mutations tend to disrupt key physiological processes, and therefore carry a fitness cost. The size of these fitness costs is a central determinant of the rate of spread of these mutations in natural populations so are important to quantify. Head-to-head competition assays provide a standard approach to measuring differential fitness, and have been used extensively for malaria parasites. These assays typically use standardized culture media, containing RPMI 1640, which has a 1.4 to 5.5-fold (mean: 2.6-fold) higher concentration of amino acids than human blood. In this rich media we predict that fitness costs will be underestimated because resource competition is weak. We tested this prediction using an artemisinin sensitive parasite edited to contain kelch-C580Y or R561H mutations conferring resistance to artemisinin or synonymous control mutations. We examined the impact of these single amino acid mutations on fitness, using replicated head-to head competition experiments conducted in media containing (i) normal RPMI, (ii) modified RPMI with reduced amino acid concentration, (iii) RPMI containing only isoleucine, or (iv) 3-fold diluted RPMI. We found a significant 1.3 to 1.4-fold increase in fitness costs measured in modified and isoleucine-only media relative to normal media, while fitness costs were 2.5-fold higher in diluted media. We conclude that fitness costs are strongly affected by media composition and will be significantly underestimated in normal RPMI. Elevated fitness costs in nature will limit spread of ART-resistance but will also promote evolution of compensatory mutations that restore fitness, and can be exploited to maximize selection in laboratory experiments.

microbiology↗

bioRxiv2020

Bimodality of gene expression in cancer patient tumors as interpretable biomarkers for drug sensitivity

Identifying biomarkers predictive of cancer cells response to drug treatment constitutes one of the main challenges in precision oncology. Recent large-scale cancer pharmacogenomic studies have boosted the research for finding predictive biomarkers by profiling thousands of human cancer cell lines at the molecular level and screening them with hundreds of approved drugs and experimental chemical compounds. Many studies have leveraged these data to build predictive models of response using various statistical and machine learning methods. However, a common challenge in these methods is the lack of interpretability as to how they make the predictions and which features were the most associated with response, hindering the clinical translation of these models. To alleviate this issue, we develop a new machine learning pipeline based on the recent LOBICO approach that explores the space of bimodally expressed genes in multiple large in vitro pharmacogenomic studies and builds multivariate, nonlinear, yet interpretable logic-based models predictive of drug response. Using our method, we used a compendium of three of the largest pharmacogenomic data sets to build robust and interpretable models for 101 drugs that span 17 drug classes with high validation rate in independent datasets.

cancer biology↗

bioRxiv2020

Biochemical Basis for the Regulation of Biosynthesis of Antiparasitics by Bacterial Hormones

Diffusible small molecule microbial hormones drastically alter the expression profiles of antibiotics and other drugs in actinobacteria. For example, avenolide (a butenolide) regulates production of avermectin, derivatives of which are used in the treatment of river blindness and other parasitic diseases. Butenolides and {gamma}-butyrolactones control production of pharmaceutically important secondary metabolites by binding to TetR family transcriptional repressors. Here, we describe a concise, 22-step synthetic strategy for the production of avenolide. We present crystal structures of the butenolide receptor AvaR1 in isolation, and in complex with avenolide, as well as AvaR1 bound to an oligonucleotide derived from its operator. Biochemical studies guided by the co-crystal structures enable identification of 90 new actinobacteria that may be regulated by butenolides, two of which are experimentally verified. These studies provide a foundation for understanding regulation of microbial secondary metabolite production, which may be exploited for the discovery and production of novel medicines.

biochemistry↗

bioRxiv2019

Functional interactions between post-translationally modified amino acids of methyl-coenzyme M reductase in Methanosarcina acetivorans

Methyl-coenzyme M reductase (MCR) plays an important role in mediating global levels of methane by catalyzing a reversible reaction that leads to the production or consumption of this potent greenhouse gas in methanogenic and methanotrophic archaea. In methanogenic archaea, the alpha subunit of MCR (McrA) typically contains four to six post-translationally modified amino acids near the active site. Recent studies have identified genes that install two of these modifications (thioglycine and 5-(S)-methylarginine), yet little is known about the installation and function of the remaining post-translationally modified residues. Here, we provide in vivo evidence that a dedicated SAM-dependent methyltransferase encoded by a gene we designated mcmA is responsible for formation of S-methylcysteine in Methanosarcina acetivorans McrA. Phenotypic analysis of mutants incapable of cysteine methylation suggests that the S-methylcysteine residue plays an important role in adaptation to a mesophilic lifestyle. To examine the interactions between the S-methylcysteine residue and the previously characterized thioglycine, 5-(S)-methylarginine modifications, we generated M. acetivorans mutants lacking the three known modification genes in all possible combinations. Phenotypic analyses revealed complex, physiologically relevant interactions between the modified residues, which alter the thermal stability of MCR in a combinatorial fashion that is not readily predictable from the phenotypes of single mutants. Surprisingly, high-resolution crystal structures of the various unmodified MCRs were indistinguishable from the fully modified enzyme, suggesting that interactions between the post-translationally modified residues do not exert a major influence on the physical structure of the enzyme, but rather serve to fine-tune the activity and efficiency of MCR.

microbiology↗