Patel, Trushar
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- ItemStructural dynamics of dengue virus UTRs and their cyclization(Cell Press, 2025) Robinson, Zachary E.; Pereira, Higor Sette; D'Souza, Michael H.; Patel, Trushar R.The dengue virus (DENV) poses a significant threat to human health, accounting for approximately 400 million infections each year. Its genome features a circular structure that facilitates replication through long-range RNA-RNA interac- tions, utilizing cyclization sequences located in the untranslated regions (UTRs). To gain new insights into the organization of the DENV genome, we purified the 5′ and 3′ UTRs of DENV in vitro and examined their structural and binding properties using various biophysical techniques combined with computational methods. Through our biophysical characterization, we deter- mined the 5′ and 3′ UTR regions to bind with an affinity of 40 nM in a 1:1 stoichiometry. By using small-angle x-ray scattering, we provide the first structural characterization of the 3′ and 5′ UTR regions, revealing several plausible conformations that the viral UTRs may adopt during replication. This comprehensive investigation revealed key features that provide mechanistic in- sights into the different structural states during DENV replication, as tracked through the accessibility of various RNA conforma- tions. Overall, our research enhances the understanding of DENV cyclization, emphasizing the structural adaptability, dynamic folding, and flexibility of these RNA molecules in solution. By uncovering details at the atomic level, we aim to contribute to the development of targeted drugs that can disrupt crucial stages of viral replication.
- ItemAssessment of HBV variants and novel viral and immune biomarkers in chronic hepatitis B patients with metabolic dysfunction associated steatotic liver disease(Wiley, 2024) Patel, Nishi H.; Lucko, Aaron; Vachon, Alicia; Doucette, Karen E.; Ramji, Alnoor; Sycuro, Laura; Patel, Trushar R.; Chadee, Kris; Raman, Maitreyi; van Marle, Guido; Osiowy, Carla; Coffin, Carla S.Co-existing chronic hepatitis B virus (CHB) infection and metabolic dysfunction associated steatotic liver disease (MASLD) can exert complex effects on hepatic metabolism, requiring mechanistic study. CHB participants were assessed for MASLD and the impact of hepatic steatosis/metabolic syndrome (MetS) on novel viral and immunological markers. In this prospective, cohort study, untreated CHB subjects were assessed for liver disease by non-invasive tests (i.e. FibroScan, controlled attenuation parameter, CAP). Subjects were tested for cytokines and IFN-γ ELISPOT assay to HBV Surface (S) and Core (C) proteins. Standard HBV serological, exploratory biomarkers and deep sequencing of HBV S and C genes were performed. In 53 subjects (median age 45 years [SD = 10.6], 35% F, 56% Asian, 20% Black, 3% White), 94% (50) HBeAg negative, 63% genotype B/C, mean HBV DNA 3.2 log10 IU/mL (SD = 1.8), quantitative HBsAg 2.9 log10 IU/mL (SD = 1.2) and HBV pgRNA 2.1 log10 copies/mL (SD = 1.3). In enrolled subjects, the mean ALT was 41.9 U/L (SD = 24.0), FibroScan was 5.7 kPa (SD = 1.9) and CAP was 306.4 dB/m (SD = 49.0). The mean BMI was 28.2 kg/m2 (SD = 4.2), 20% (11/53) had diabetes, 35% (19/53) dyslipidaemia and 24% (13/53) hypertension. Subjects with MetS and steatosis showed lower HBV markers (p < .01), higher HBV S diversity (p = .02) and greater frequency of HBV variants associated with host-anti-viral immune escape. Pro-inflammatory cytokine levels and HBV-specific cellular responses were higher in participants with hepatic steatosis. In CHB, MASLD/hepatic steatosis was associated with HBV variants and systemic immune responses potentially impacting liver disease progression despite low-level viraemia.
- ItemIntrinsic disorder in the partitioning protein KorB persists after co-operative complex formation with operator DNA and KorA(Portland Press, 2017) Hyde, Eva I.; Callow, Philip; Rajasekar, Karthik V.; Timmins, Peter; Patel, Trushar R.; Siligardi, Giuliano; Hussain, Rohanah; White, Scott A.; Thomas, Christopher M.; Scott, David J.The ParB protein, KorB, from the RK2 plasmid is required for DNA partitioning and transcriptional repression. It acts co-operatively with other proteins, including the repressor KorA. Like many multifunctional proteins, KorB contains regions of intrinsically disordered structure, existing in a large ensemble of interconverting conformations. Using NMR spectroscopy, circular dichroism and small-angle neutron scattering, we studied KorB selectively within its binary complexes with KorA and DNA, and within the ternary KorA/KorB/DNA complex. The bound KorB protein remains disordered with a mobile C-terminal domain and no changes in the secondary structure, but increases in the radius of gyration on complex formation. Comparison of wild-type KorB with an N-terminal deletion mutant allows a model of the ensemble average distances between the domains when bound to DNA. We propose that the positive co-operativity between KorB, KorA and DNA results from conformational restriction of KorB on binding each partner, while maintaining disorder.
- ItemRegulation of platelet derived growth factor signaling by leukocyte common antigen-related (LAR) protein tyrosine phosphatase: a quantitative phosphoproteomics study(Elsevier, 2016) Sarhan, Adil R.; Patel, Trushar R.; Creese, Andrew J.; Tomlinson, MIchael G.; Hellberg, Carina; Heath, John K.; Hotchin, Neil A.; Cunningham, Debbie L.Intracellular signaling pathways are reliant on protein phosphorylation events that are controlled by a balance of kinase and phosphatase activity. Although kinases have been extensively studied, the role of phosphatases in controlling specific cell signaling pathways has been less so. Leukocyte common antigen-related protein (LAR) is a member of the LAR subfamily of receptor-like protein tyrosine phosphatases (RPTPs). LAR is known to regulate the activity of a number of receptor tyrosine kinases, including platelet-derived growth factor receptor (PDGFR). To gain insight into the signaling pathways regulated by LAR, including those that are PDGF-dependent, we have carried out the first systematic analysis of LAR-regulated signal transduction using SILAC-based quantitative proteomic and phosphoproteomic techniques. We haveanalyzed differential phosphorylation between wild-type mouse embryo fibroblasts (MEFs) and MEFs in which the LAR cytoplasmic phosphatase domains had been deleted (LARΔP), and found a significant change in abundance of phosphorylation on 270 phosphosites from 205 proteins because of the absence of the phosphatase domains of LAR. Further investigation of specific LAR-dependent phosphorylation sites and enriched biological processes reveal that LAR phosphatase activity impacts on a variety of cellular processes, most notably regulation of the actin cytoskeleton. Analysis of putative upstream kinases that may play an intermediary role between LAR and the identified LAR-dependent phosphorylation events has revealed a role for LAR in regulating mTOR and JNK signaling.
- ItemStructural studies of RNA-protein complexes: a hybrid approach involving hydrodynamics, scattering, and computational methods(Elsevier, 2017) Patel, Trushar R.; Chojnowski, Grzegorz; Astha; Koul, Amit; McKenna, Sean A.; Bujnicki, Janusz M.The diverse functional cellular roles played by ribonucleic acids (RNA) have emphasized the need to develop rapid and accurate methodologies to elucidate the relationship between the structure and function of RNA. Structural biology tools such as X-ray crystallography and Nuclear Magnetic Resonance are highly useful methods to obtain atomic-level resolution models of macromolecules. However, both methods have sample, time, and technical limitations that prevent their application to a number of macromolecules of interest. An emerging alternative to high-resolution structural techniques is to employ a hybrid approach that combines low-resolution shape information about macromolecules and their complexes from experimental hydrodynamic (e.g. analytical ultracentrifugation) and solution scattering measurements (e.g., solution X-ray or neutron scattering), with computational modeling to obtain atomic-level models. While promising, scattering methods rely on aggregation-free, monodispersed preparations and therefore the careful development of a quality control pipeline is fundamental to an unbiased and reliable structural determination. This review article describes hydrodynamic techniques that are highly valuable for homogeneity studies, scattering techniques useful to study the low-resolution shape, and strategies for computational modeling to obtain high-resolution 3D structural models of RNAs, proteins, and RNA-protein complexes.