Biology subjects

Agarwal, P. K.

Publications and source records attributed to Agarwal, P. K..

6 recordsLinked to original sources

bioRxiv2025

A novel non-catalytic function of PA2803-encoded PcrP contributes to polymyxin B resistance in P. aeruginosa and redefines the functional role of the PA2803 subfamily.

The opportunistic human pathogen Pseudomonas aeruginosa (Pa), a leading cause of severe infections, becomes increasingly resistant to antibiotics, including the last resort antibiotic, polymyxin-B (PMB). Previous studies have shown that calcium (Ca2+) at the levels encountered during infections increases Pa resistance to PMB. However, the mechanisms of this Ca2+ regulation are not known. Here, we identified three novel genes (PA2803, PA3237 and PA5317) that contribute to the Ca2+-dependent PMB resistance in Pa. PA2803, the focus of this work, encodes a putative phosphonatase and is a founding member of the PA2803 subfamily from the Haloacid Dehalogenase Superfamily. Since the transcription of this gene is regulated by both Ca2+ and inorganic phosphate (Pi), we named it "Pi and Ca2+ regulated protein, PcrP". Congruent with sequence-based predictions, we showed that PcrP lacks catalytic activity and instead binds protein partners, revealing a novel non-catalytic function for PA2803 subfamily proteins. By using pull-down assays and bacterial two-hybrid system, we identified and validated two protein partners of PcrP: Acp3 and PA3518. We show that PcrP is involved in oxidative stress responses in Pa, which are likely mediated by its interactions with Acp3, and may support its role in PMB resistance. In addition, PcrP imparts a Ca2+-dependent growth advantage to Pa during Pi starvation and plays a role in polyphosphate accumulation in a Ca2+-dependent manner. Overall, this study identified a novel protein-binding function for the PA2803 subfamily representative that mediates Pa responses to elevated Ca2+ and Pi starvation and enhances PMB resistance. IMPORTANCEPseudomonas aeruginosa (Pa) is a critical human pathogen that presents significant clinical challenges, underscoring the urgent need for understanding its resistance mechanisms. Previous studies have shown that calcium (Ca2+) at the levels detected during infections increase Pa resistance to the last resort antibiotic polymyxin-B (PMB). For the first time, we identified three novel genes, whose products are required for the Ca2+-dependent PMB resistance in Pa. One of them, PA2803, regulated by Ca2+ and phosphate, was named phosphate and Ca2+ regulated protein, PcrP. This study discovered a novel protein-binding function of PcrP and identified two protein partners. The protein-binding function is likely shared by the entire PA2803 subfamily of proteins, which redefines their previous functional assignment as enzymes.

microbiology↗

bioRxiv2025

APE1 active site residue Asn174 stabilizes the AP-site and is essential for catalysis

Apurinic/Apyrimidinic (AP)-sites are common and highly mutagenic DNA lesions that can arise spontaneously or as intermediates during Base Excision Repair (BER). The enzyme apurinic/apyrimidinic endonuclease 1 (APE1) initiates repair of AP-sites by cleaving the DNA backbone at the AP-site via its endonuclease activity. Here, we investigated the functional role of the APE1 active site residue N174 that contacts the AP-site during catalysis. We analyzed the effects of three rationally designed APE1 mutations that alter the hydrogen bonding potential, size, and charge of N174: N174A, N174D, and N174Q. We found impaired catalysis of the APE1N174A and APE1N174D mutants due to disruption of hydrogen bonding and electrostatic interactions between residue 174 and the AP-site. In comparison, the APE1N174Q mutant was less impaired due to retaining similar hydrogen bonding and electrostatic characteristics as N174 in wild-type APE1. Structures and computational simulations further revealed that the AP-site was destabilized within the active sites of the APE1N174A and APE1N174D mutants due to loss of hydrogen bonding between residue 174 and the AP-site. Cumulatively, we show that N174 stabilizes the AP-site within the APE1 active site through hydrogen bonding and electrostatic interactions to enable effective catalysis. These findings highlight the importance of N174 in APE1s function and provide new insights into the molecular mechanism by which APE1 processes AP-sites during DNA repair.

biochemistry↗

bioRxiv2024

Dynamic Differences in Clinically relevant Pen β-Lactamases from Burkholderia spp.

Antimicrobial resistance (AMR) is a global threat, with Burkholderia species contributing significantly to difficult-to-treat infections. Ambler Class A Pen {beta}-lactamases are produced by all Burkholderia spp., and their mutation or overproduction leads to the resistance of {beta}-lactam antibiotics. This study investigates the dynamic differences among four Pen {beta}-lactamases (PenA, PenI, PenL and PenP) using machine learning driven enhanced sampling molecular dynamics simulations, Markov State Models (MSMs), convolutional variational autoencoder-based deep learning (CVAE) and the BindSiteS-CNN model. Although sharing the same catalytic mechanisms, these enzymes exhibit distinct dynamic features due low sequence identity, resulting in different substrate profiles and catalytic turnover. The BindSiteS-CNN model further reveals local active site dynamics, offering insights into the Pen {beta}-lactamase evolutionary adaptation. Our findings reported here identify critical mutations and proposes new hotspots affecting Pen {beta}-lactamase flexibility and function, which can be used to fight emerging resistance in these enzymes.

biophysics↗

bioRxiv2023

Pandemic preparedness through genomic surveillance: Overview of mutations in SARS-CoV-2 over the course of COVID-19 outbreak

Genomic surveillance is a vital strategy for preparedness against the spread of infectious diseases and to aid in development of new treatments. In an unprecedented effort, millions of samples from COVID-19 patients have been sequenced worldwide for SARS-CoV-2. Using more than 8 million sequences that are currently available in GenBanks SARS-CoV-2 database, we report a comprehensive overview of mutations in all 26 proteins and open reading frames (ORFs) from the virus. The results indicate that the spike protein, NSP6, nucleocapsid protein, envelope protein and ORF7b have shown the highest mutational propensities so far (in that order). In particular, the spike protein has shown rapid acceleration in mutations in the post-vaccination period. Monitoring the rate of non-synonymous mutations (Ka) provides a fairly reliable signal for genomic surveillance, successfully predicting surges in 2022. Further, the external proteins (spike, membrane, envelope, and nucleocapsid proteins) show a significant number of mutations compared to the NSPs. Interestingly, these four proteins showed significant changes in Ka typically 2 to 4 weeks before the increase in number of human infections ("surges"). Therefore, our analysis provides real time surveillance of mutations of SARS-CoV-2, accessible through the project website http://pandemics.okstate.edu/covid19/. Based on ongoing mutation trends of the virus, predictions of what proteins are likely to mutate next are also made possible by our approach. The proposed framework is general and is thus applicable to other pathogens. The approach is fully automated and provides the needed genomic surveillance to address a fast-moving pandemic such as COVID-19.

genomics↗

bioRxiv2021

Allosteric communication in Class A β-lactamases occurs via Cooperative Coupling of Loop Dynamics

Allosteric effects control protein (e.g. enzyme) activity in ways that are not fully understood. Better understanding of allosteric effects, and tools to identify them, would offer promising alternative strategies to inhibitor development. Through a combination of equilibrium and nonequilibrium molecular dynamics simulations, we identify allosteric effects and communication pathways from two distant ligand binding sites to important active site structural elements that control enzymatic activity in two prototypical class A {beta}-lactamases, TEM-1 and KPC-2. Both of these enzymes are important determinants of antibiotic resistance in widespread bacterial pathogens. The simulations show that the allosteric sites are connected to the active site in both enzymes, (e.g. affecting the conformation of the {Omega}-loop) highlighting how allosteric inhibitors may exert their effects. Nonequilibrium simulations reveal pathways of communication operating over distances of 30 [A] or more. In these identified signaling pathways, the propagation of the signal occurs through cooperative coupling of loop dynamics. Notably, 50% or more clinically relevant amino acid substitutions in each enzyme map onto the identified signal transduction pathways. This suggests that clinically important variation may affect, or be driven by, differences in allosteric behavior, providing a mechanism by which amino acid substitutions may affect the relationship between spectrum of activity, catalytic turnover and potential allosteric behavior in this clinically important enzyme family. Simulations of the type presented here will help in identifying and analyzing such differences.

biophysics↗

bioRxiv2020

Autocrine Signaling by Receptor Tyrosine Kinases in Urothelial Carcinoma of the Bladder

Comprehensive characterizations of bladder cancer (BCa) have established molecular phenotype classes with distinct alterations and survival trends. Extending these studies within the tyrosine kinase (TK) family to identify disease drivers could improve our use of TK inhibitors to treat specific patient groups or individuals. We examined the expression distribution of TKs as a class (n = 89) in The Cancer Genome Atlas (TCGA) muscle invasive BCa data set (n >400). Patient profiles of potentially oncogenic alterations (overexpression and/or amplification) clustered TKs into 3 groups; alterations of group 1 and 3 TKs were associated with significantly worse patient survival relative to those without alterations. Many TK pathways induce epithelial-to-mesenchymal transition (EMT), which promotes tumor invasiveness and metastasis. Overexpression and/or amplification among 9 EMT transcriptional activators occurred in 43% of TCGA cases. Co-occurring alterations of TKs and EMT transcriptional activators involved most group 1 TKs; 24% of these events were associated with significantly worse patient survival. Co-occurring alterations of receptor TKs and their cognate ligands occurred in 16% of TCGA cases and several BCa-derived cell lines. Suppression of GAS6, MST1 or CSF1, or their respective receptors (AXL, MST1R and CSF1R), in BCa cell lines was associated with decreased receptor activation, cell migration, cell proliferation and anchorage independent cell growth. These studies reveal the patterns and prevalence of potentially oncogenic TK pathway-related alterations in BCa and identify specific alterations associated with reduced BCa patient survival. Detection of these features in BCa patients could better inform TK inhibitor use and improve clinical outcomes.

cancer biology↗