Biology subjects

Desai, U. R.

Publications and source records attributed to Desai, U. R..

4 recordsLinked to original sources

bioRxiv2025

A Robust Proteomics-Based Method for Identifying Preferred Protein Targets of Synthetic Glycosaminoglycan Mimetics

A robust technology is critically needed for identifying preferred protein targets of glycosaminoglycans (GAGs), and synthetic mimetics thereof, in biological milieu. We present a robust 10-step strategy for identification and validation of preferred protein targets of highly sulfated, synthetic, small, GAG-like molecules using diazirine-based photoaffinity labeling- proteomics approach. Our work reveals that optimally designed, homogeneous probes based on minimalistic photoactivation and affinity pulldown groups coupled with rigorous proteomics, biochemical and orthogonal validation steps offer excellent potential to identify preferred targets of GAG mimetics from the potentially numerous possible targets that cloud GAG interaction studies. Application of this 10-step strategy for a promising highly sulfated, small GAG mimetic led to identification of only a handful of preferred targets in human plasma. This new robust strategy will greatly aid drug discovery and development efforts involving GAG sequences, or sulfated small mimetics thereof, as leads.

biochemistry↗

bioRxiv2022

The alphaherpesvirus conserved pUS10 is important for natural infection and its expression is regulated by the conserved Herpesviridae protein kinase (CHPK)

Conserved Herpesviridae protein kinases (CHPK) are conserved among all members of the Herpesviridae. Herpesviruses lacking CHPK propagate in cell culture at varying degrees, depending on the virus and cell culture system. CHPK is dispensable for Mareks disease herpesvirus (MDV) replication in cell culture and experimental infection in chickens; however, CHPK--particularly its kinase activity--is essential for horizontal transmission in chickens, also known as natural infection. To address the importance of CHPK during natural infection in chickens, we used liquid chromatography-tandem mass spectrometry (LC-MS/MS) based proteomics of samples collected from live chickens. Comparing modification of viral proteins in feather follicle epithelial (FFE) cells infected with wildtype or a CHPK-null virus, we identified the US10 protein (pUS10) as a potential target for CHPK in vivo. When expression of pUS10 was evaluated in cell culture and in FFE skin cells during in vivo infection, pUS10 was severely reduced or abrogated in cells infected with CHPK mutant or CHPK-null viruses, respectively, indicating a potential role for pUS10 in transmission. To test this hypothesis, US10 was deleted from the MDV genome, and the reconstituted virus was tested for replication, horizontal transmission, and disease induction. Our results showed that removal of US10 had no effect on the ability of MDV to transmit in experimentally infected chickens, but disease induction in naturally infected chickens was significantly reduced. These results show CHPK is necessary for pUS10 expression both in cell culture and in the host, and pUS10 is important for disease induction during natural infection. Author SummaryMareks disease herpesvirus (MDV) is an important pathogen in the poultry industry. Current vaccines reduce disease but do not protect chickens from infection. Understanding natural infection provides vital information for developing potential therapies to protect against Mareks disease, while also providing a robust natural virus-host model to study herpesvirus pathogenesis. The conserved Herpesviridae protein kinase (CHPK) of MDV is essential for the initiation of natural infection, and we identified the virion protein US10 as a potential target for CHPK during natural infection. Our results showed that MDV CHPK was required for expression of pUS10 in both cell culture and in chickens. Although MDV lacking pUS10 replicated and spread in experimentally infected chickens, it was less virulent implicating an important role of pUS10 in natural infection of the host. These results emphasize the importance of studying specific viral proteins and their functions during infection of the natural host.

microbiology↗

bioRxiv2020

Preferential recognition and antagonism of SARS-CoV-2 spike glycoprotein binding to 3-O-sulfated heparan sulfate

The COVID-19 pandemic caused by SARS-CoV-2 is in immediate need of an effective antidote. Although the Spike glycoprotein (SgP) of SARS-CoV-2 has been shown to bind to heparins, the structural features of this interaction, the role of a plausible heparan sulfate proteoglycan (HSPG) receptor, and the antagonism of this pathway through small molecules remain unaddressed. Using an in vitro cellular assay, we demonstrate HSPGs modified by the 3-O-sulfotransferase isoform-3, but not isoform-5, preferentially increased SgP-mediated cell-to-cell fusion in comparison to control, unmodified, wild-type HSPGs. Computational studies support preferential recognition of the receptor-binding domain of SgP by 3-O-sulfated HS sequences. Competition with either fondaparinux, a 3-O-sulfated HS-binding oligopeptide, or a synthetic, non-sugar small molecule, blocked SgP-mediated cell-to-cell fusion. Finally, the synthetic, sulfated molecule inhibited fusion of GFP-tagged pseudo SARS-CoV-2 with human 293T cells with sub-micromolar potency. Overall, overexpression of 3-O-sulfated HSPGs contribute to fusion of SARS-CoV-2, which could be effectively antagonized by a synthetic, small molecule.

biochemistry↗

bioRxiv2019

Molecular Dynamics Simulations of Glycosaminoglycan Oligosaccharide Using Newer Force Fields

Heparin/heparan sulfate (H/HS) are ubiquitous biopolymers that interact with many proteins to induce myriad biological functions. It is critical to understand conformational properties of H/HS in solution so as to identify their preferred protein targets. Unfortunately, the massive heterogeneity of H/HS precludes the use of solution-based experimental techniques for the thousands of sequences that occur in nature. Computational simulations offer an attractive alternative and several all-atom force fields have been developed to understand their conformational properties. Recently, CHARMM36 carrying parameters for N-sulfamate was developed. This work compares molecular dynamics simulations of a hexasaccharide (HS06) using two all-atom force fields - CHARMM36 and GLYCAM06. We also introduce two new straightforward parameters, including end-to-end distance and minimum volume enclosing ellipsoid, to understand the conformational behavior of HS06. In addition, we analyzed inter- and intra-molecular hydrogen bonds and intermediate water bridges formed for HS06 using both force fields. Overall, CHARMM36 and GLYCAM06 gave comparable results, despite few, small differences. The MD simulations show that HS06 samples a range of conformations in solution with more than one nearly equivalent global minima, which contrasts with the assumed single conformation conclusion derived on the basis of 1HPN structure. A key reason for the stability of multiple low energy conformations was the contribution of intermediate water bridges, which is usually not evaluated in most MD studies of H/HS.

biophysics↗