Biology subjects

Wijeratne, H. S.

Publications and source records attributed to Wijeratne, H. S..

3 recordsLinked to original sources

bioRxiv2025

Application of Whole Proteome Thermal Shift Assays to Define PERK-dependent Changes in Protein Homeostasis during the Unfolded Protein Response

The Unfolded Protein Response (UPR) is a cellular pathway activated by sensory proteins, including the protein kinase PERK (EIF2AK3), that monitors perturbations in the endoplasmic reticulum (ER). Using tunicamycin, which induces ER stress by thwarting N-glycosylation, we monitored system-wide changes in the proteome using PISA (Proteome Integral Solubility Alteration) and abundance analysis. Global proteomics revealed precise changes in membrane- and ER-associated proteins through widespread induction of ER-associated degradation (ERAD) while normalized PISA (nPISA) analyses selectively identified pathway changes associated with drug mechanism of action. nPISA analysis following tunicamycin treatment in cells, in combination with genetic disruption of PERK, facilitated identification of novel proteins involved in PERK-dependent and -independent processes and how those changes intersect with PERK function specifically during the ER stress response. Overall, protein-centered multiomics analyses defined the precise proteome alterations in tunicamycin-induced ER stress, highlighting the consequences of PERK disruption on ER-mitochondrial homeostasis.

systems biology↗

bioRxiv2024

IB-DNQ and Rucaparib dual treatment alters cell cycle regulation and DNA repair in triple negative breast cancer cells

Triple negative breast cancer (TNBC) is a highly aggressive breast cancer that is unresponsive to hormonal therapies. One potential TNBC-specific therapeutic target is NQO1, as it is highly expressed in many TNBC patients and lowly expressed in non-cancer tissues. Here we use a derivative of DNQ, isobutyl-deoxynyboquinone (IB-DNQ) that is more potent and specific in killing TNBC cells than NQO1-activator {beta}-lapachone while displaying strong NQO1-dependence. We evaluated the cellular signaling changes that occur following 4-hour treatment of TNBC cells with either single agent or combination IB-DNQ and / or PARP inhibitor (Rucaparib). Short treatments (4 hours) with IB-DNQ alone or combined with the PARP inhibitor Rucaparib revealed few changes in protein abundance but significant rapid alterations in protein phosphorylation and thermal stability, with clear synergy in the combination treatment. Key phosphorylated targets linked to RNA Polymerase II inhibition and DNA damage response were altered during our short time treatment. Thermal proteome profiling (TPP) identified novel, combination-specific changes in protein biophysical state suggesting new therapeutic vulnerabilities in TNBC cells. Our findings highlight how even brief treatments can uncover distinct biophysical protein changes via TPP, offering a resource for mechanistic studies of IB-DNQ mechanism of action and the development of NQO1-activated therapeutics for TNBC treatment.

cancer biology↗

bioRxiv2022

Obtaining Increased Functional Proteomics Insights from Thermal Proteome Profiling through Optimized Melt Shift Calculation and Statistical Analysis

Thermal Proteome Profiling (TPP) is an invaluable tool for functional proteomics studies that has been shown to discover changes associated with protein-ligand, protein- protein, and protein-RNA interaction dynamics along with changes in protein stability resulting from cellular signaling. The increasing number of reports employing this assay has not been met concomitantly with advancements and improvements in the quality and sensitivity of the corresponding data analysis. The gap between data acquisition and data analysis tools is even more apparent as TPP findings have reported more subtle melt shift changes related to protein post-translational modifications. In this study, we have improved the Inflect data analysis pipeline (now referred to as InflectSSP, available at https://CRAN.R-project.org/package=InflectSSP) to increase the sensitivity of detection for both large and subtle changes in the proteome as measured by TPP. Specifically, InflectSSP now has integrated statistical and bioinformatic functions to improve objective functional proteomics findings from the quantitative results obtained from TPP studies through increasing both the sensitivity and specificity of the data analysis pipeline. To benchmark InflectSSP, we have reanalyzed two publicly available datasets to demonstrate the performance of this publicly available R based program for TPP data analysis. Additionally, we report new findings following temporal treatment of human cells with the small molecule Thapsigargin which induces the unfolded protein response (UPR). InflectSSP analysis of our UPR study revealed highly reproducible target engagement over time while simultaneously providing new insights into the dynamics of UPR induction.

biophysics↗