Biology subjects

Perez-Leal, O.

Publications and source records attributed to Perez-Leal, O..

3 recordsLinked to original sources

bioRxiv2022

Identification of tubulin polymerization inhibitors with a CRISPR-edited cell line with endogenous fluorescent tagging of β-tubulin and Histone 1

Tubulin is an essential protein to maintain the cellular structure and for the cell division process. Inhibiting tubulin polymerization has proven to be an effective method for slowing cancer cell growth. Traditionally, identifying tubulin polymerization inhibitors involved using pure tubulin for in vitro assays or procedures using cells that require cell fixing and anti-tubulin antibody staining. This study explores using a cell line developed via CRISPR genome editing as a cell model to identify tubulin polymerization inhibitors with live cells without using exogenous staining. The cell line has endogenous tagging with fluorescent proteins of {beta}-tubulin and a nuclear protein to facilitate image cellular segmentation by high-content imaging analysis (HCI). The cells were treated with known tubulin polymerization inhibitors, colchicine and vincristine, and the presence of phenotypic changes that indicate tubulin polymerization inhibition were confirmed via HCI. A library of 429 kinase inhibitors was screened to discover tubulin polymerization inhibitors and three compounds that inhibit tubulin polymerization were found (ON-01910, HMN-214, and KX2-391). Live cell tracking analysis confirms that depolymerization of tubulin occurs rapidly after compound treatments. These results suggest that CRISPR-edited cells with fluorescent endogenous tagging of {beta}-tubulin can be used to screen larger compound libraries containing diverse chemical families to identify novel tubulin polymerization inhibitors.

cancer biology↗

bioRxiv2022

Secreted folate receptor-gamma drives fibrogenesis in nonalcoholic steatohepatitis by amplifying TGFβ signaling in hepatic stellate cells

Hepatic fibrosis is the primary determinant of mortality in nonalcoholic steatohepatitis (NASH) patients. Antagonism of transforming growth factor {beta} (TGF{beta}), a master profibrogenic cytokine, is a promising therapeutic target that has not yet been translated into an effective therapy, due in part to the lack of animal models resembling the human phenotype of NASH. Here we have identified that soluble secreted folate receptor gamma (FOLR3), expressed in humans but not rodents, is a secreted protein that is elevated in livers of NASH subjects but not in subjects with nonalcoholic fatty liver, type II diabetes, or healthy subjects. FOLR3, based on global proteomics, was the most highly expressed NASH-specific protein and positively correlated with increasing fibrosis stages, suggesting an impact on activated hepatic stellate cells (HSCs), the key fibrogenic cell in the liver. Exposure of stellate cells to exogenous FOLR3 led to elevated extracellular matrix (ECM) protein production, an effect synergistic with TGF{beta}1. Structurally, FOLR3 interacts with serine protease HTRA1, which downregulates TGF{beta} signaling through the degradation of its receptor TGFBR2. Administration of human FOLR3 to mice induced severe bridging fibrosis and an ECM pattern resembling human NASH. Our study uncovers a novel role of FOLR3 in enhancing fibrosis and identifies FOLR3 as a potential therapeutic target in NASH fibrosis. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=143 SRC="FIGDIR/small/500829v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@1ac3ce3org.highwire.dtl.DTLVardef@d1945corg.highwire.dtl.DTLVardef@1632eb1org.highwire.dtl.DTLVardef@8e8d21_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2020

A versatile vector system for the fast generation of knock-in cell lines with CRISPR

Until recent advancements in genome editing via CRISPR/Cas9 technology, understanding protein function typically involved artificially overexpressing proteins of interest. Despite that CRISPR/Cas9 has ushered in a new era of possibilities for modifying endogenous genes with labeling tags (knock-in) to more accurately study proteins under physiological conditions, the technique is largely underutilized due to its tedious, multi-step process. Here we outline a homologous recombination system (FAST-HDR) to be used in combination with CRISPR/Cas9 that significantly simplifies and accelerates this process while introducing multiplexing to allow live-cell studies of 3 endogenous proteins within the same cell line. Furthermore, the recombination vectors are assembled in a single reaction that is enhanced for eliminating false positives and reduces the overall creation time for the knockin cell line from ~8 weeks to <15 days. Finally, the system utilizes a modular construction to allow for seamlessly swapping labeling tags to ensure flexibility according to the area under study. We validated this new methodology by developing advanced cell lines with 3 fluorescent-labeled endogenous proteins that support high-content phenotypic drug screening without using antibodies or exogenous staining. Therefore, Fast-HDR cell lines provide a robust alternative for studying multiple proteins of interest in live cells without artificially overexpressing labeled proteins.

molecular biology↗