Biology subjects

Gangadharan, B.

Publications and source records attributed to Gangadharan, B..

5 recordsLinked to original sources

bioRxiv2025

A biochemical mechanism for Stu2/XMAP215-family microtubule polymerases

Defining quantitative biochemical mechanisms of microtubule dynamics and regulation is a current challenge. Stu2/XMAP215-family polymerases use tubulin-binding TOG domains to catalyze microtubule growth, but how polymerase activity results from the number and tubulinbinding properties of TOGs is not understood. We tested whether an enzyme-like biochemical model for the unrelated actin polymerase Ena/VASP could be applied to quantitatively relate Stu2 microtubule polymerase activity to the number of its TOGs and the rate constants governing their interactions with tubulin. Stu2 activity displayed enzyme-like characteristics consistent with the biochemical model: Stu2 stimulated microtubule growth rates with hyperbolic dependence on tubulin concentration, and the amount of Stu2 on the microtubule end did not vary with tubulin concentration (microtubule growth rate). Complementary measurements of TOG:tubulin binding revealed high affinity (10 nM) and slow dissociation (0.03 s-1). The polymerase and binding measurements can be unified within the biochemical model: Stu2 operates with high efficiency, acting as a tubulin-shuttling antenna on the microtubule end that is primarily limited by the rate of tubulin:TOG association. Our work thus provides a quantitative biochemical mechanism for TOG-based polymerases. That unrelated microtubule and actin polymerases use the same enzyme-like mechanism provides an example of convergent evolution in the cytoskeleton.

cell biology↗

bioRxiv2023

Altered hepatic metabolism mediates sepsis preventive effects of reduced glucose supply in infected preterm newborns

Preterm infants are susceptible to neonatal sepsis, a syndrome of pro-inflammatory activity, organ damage and altered metabolism following infection. Given the unique metabolic challenges and poor glucose regulatory capacity of preterm infants, their glucose intake during infection may have a high impact on the degree metabolism dysregulation and organ damage. Using a preterm pig model of neonatal sepsis, we previously showed that a drastic restriction in glucose supply during infection protects against sepsis via suppression of glycolysis-induced inflammation, but results in severe hypoglycemia. Now we explored clinically relevant options of reducing glucose intake to decrease sepsis risk, without causing hypoglycemia and further explore the involvement of the liver in these protective effects. We found that a reduced glucose regime during infection increased survival via reduced pro-inflammatory response, while maintaining normoglycemia. Mechanistically, this intervention enhanced hepatic oxidative phosphorylation and possibly gluconeogenesis, and dampened both circulating and hepatic inflammation. However, switching from a high to a reduced glucose supply after debut of clinical symptoms did not prevent sepsis, suggesting metabolic conditions at the start of infection are key in driving the outcome. Finally, an early therapy with purified human inter-alpha inhibitor protein, a liver derived anti-inflammatory protein, partially reversed the effects of low parenteral glucose provision, likely by inhibiting neutrophil functions that mediate pathogen clearance. Our findings suggest a clinically relevant regime of reduced glucose supply for infected preterm infants could prevent or delay the development of sepsis in vulnerable neonates.

immunology↗

bioRxiv2022

Stimulating microtubule growth is not the essential function of the microtubule polymerase Stu2

ch-TOG family proteins, including the budding yeast Stu2, are essential for spindle formation and chromosome segregation. Such functions depend on an array of activities ranging from microtubule nucleation, polymerization and depolymerization, to conferring tension sensitivity to kinetochores. This functional diversity makes it challenging to dissect these various functions and understand their relative importance. Here, we developed separation-of-function mutants and used artificial tethering tools to elucidate several important mechanistic insights into Stu2s essential role. We show that Stu2s microtubule polymerization activity depends on its basic linker region but is surprisingly dispensable for viability; that in fact, Stu2 carries out an essential kinetochore-associated function; and finally, that Stu2s precise location within the kinetochore is critical for its function, suggesting a spatial separation mode of action may underlie its ability to confer tension sensitivity. Our findings highlight the significance of Stu2s kinetochore role and provide insights into the molecular mechanisms by which it performs its various functions.

cell biology↗

bioRxiv2022

Deep proteomics network and machine learning analysis of human cerebrospinal fluid in Japanese encephalitis virus infection

Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus, and leading cause of neurological infection in Asia and the Pacific, with recent emergence in multiple territories in Australia in 2022. Patients may experience devastating socioeconomic consequences; JEV infection (JE) predominantly affects children in poor rural areas, has a 20-30% case fatality rate, and 30-50% of survivors suffer long-term disability. JEV RNA is rarely detected in patient samples, and the standard diagnostic test is an anti-JEV IgM ELISA with sub-optimal specificity; there is no means of detection in more remote areas. We aimed to test the hypothesis that there is a diagnostic protein signature of JE in human cerebrospinal fluid (CSF), and contribute to understanding of the host response and predictors of outcome during infection. We retrospectively tested a cohort of 163 patients recruited as part of the Laos central nervous system infection study. Application of liquid chromatography and tandem mass spectrometry (LC-MS/MS), using extensive offline fractionation and tandem mass tag labelling, enabled a comparison of the CSF proteome in 68 JE patient vs 95 non-JE neurological infections. 5,070 proteins were identified, including 4,805 human proteins and 265 pathogen proteins. We incorporated univariate analysis of differential protein expression, network analysis and machine learning techniques to build a ten-protein diagnostic signature of JE with >99% diagnostic accuracy. Pathways related to JE infection included neuronal damage, anti-apoptosis, heat shock and unfolded protein responses, cell adhesion, macrophage and dendritic cell activation as well as a reduced acute inflammatory response, hepatotoxicity, activation of coagulation, extracellular matrix and actin regulation. We verified the results by performing DIA LC-MS/MS in 16 (10%) of the samples, demonstrating 87% accuracy using the same model. Ultimately, antibody-based validation will be required, in a larger group of patients, in different locations and in field settings, to refine the list to 2-3 proteins that could be harnessed in a rapid diagnostic test. Author summaryJapanese encephalitis virus (JEV) is a leading cause of brain infection in Asia and the Pacific, with recent introduction in multiple territories in Australia in 2022. Patients may experience devastating socioeconomic consequences; JEV infection (JE) predominantly affects children in poor rural areas, has a 20-30% case fatality rate, and 30-50% of survivors suffer long-term disability. The disease is difficult to diagnose, and there are no rapid tests that may be performed in remote areas that it exists such that we remain unclear of the burden of disease and the effects of control measures. We aimed to apply a relatively novel method to analyse the proteins in patients with JE as compared to other neurological infections, to see if this could be useful for making a diagnosis. We tested the brain fluid of 163 patients recruited as part of the Laos central nervous system infection study. We used a method, liquid chromatography mass spectrometry that does not require prior knowledge of the proteins present, that is you do not target any specific protein. Over 5,000 proteins were identified, and these were analysed by various methods. We grouped the proteins into different clusters that provided insight into their function. We also filtered the list to 10 proteins that predicted JE as compared to other brain infections. Future work will require confirmation of the findings in a larger group of patients, in different locations and in field settings, to refine the list to 2-3 proteins that could be harnessed in a rapid diagnostic test.

microbiology↗

bioRxiv2020

Analysis of SARS-CoV-2 spike glycosylation reveals shedding of a vaccine candidate

Severe acute respiratory syndrome coronavirus 2 is the causative pathogen of the COVID-19 pandemic which as of Nov 15, 2020 has claimed 1,319,946 lives worldwide. Vaccine development focuses on the viral trimeric spike glycoprotein as the main target of the humoral immune response. Viral spikes carry glycans that facilitate immune evasion by shielding specific protein epitopes from antibody neutralisation. Immunogen integrity is therefore important for glycoprotein-based vaccine candidates. Here we show how site-specific glycosylation differs between virus-derived spikes and spike proteins derived from a viral vectored SARS-CoV-2 vaccine candidate. We show that their distinctive cellular secretion pathways result in different protein glycosylation and secretion patterns, which may have implications for the resulting immune response and future vaccine design.

biochemistry↗