Biology subjects

Al-Seragi, M.

Publications and source records attributed to Al-Seragi, M..

5 recordsLinked to original sources

bioRxiv2026

A genome-wide CRISPR screen reveals cancer-specific regulators of hyaluronan binding and cellular invasion

Metastatic spread of cancer cells is driven by binding between the cell-surface receptor CD44 and hyaluronan (HA) in the extracellular matrix. The specific oncogenes that drive increased CD44-HA binding in cancer remain poorly defined. Using a fluorescently labeled hyaluronan probe, we performed a genome-wide CRISPR screen to identify genes whose knockdown disrupts HA binding in breast cancer cells. We subsequently developed a bioinformatic analysis pipeline that enabled stratification and prioritization of cancer-specific regulators. This work provides a first-in-class resource for the identification of druggable targets to inhibit HA binding. We further validate RAB4A, a top hit from our screen, as a novel regulator of this process. Mechanistically, RAB4A KO dramatically reduces CD44 expression and inhibits the invasion of breast cancer cells through HA-rich matrices. This study validates a novel strategy for identifying regulators of cancer cell invasion and identifies immediate actionable targets for anti-metastatic therapy.

cancer biology↗

bioRxiv2025

CRISPR activation screens map the genomic landscape of cancer glycome remodeling

Many cancer types upregulate expression of sialic acid-containing glycans. These oligosaccharides subsequently engage inhibitory Siglec receptors on immune cells, allowing cancer cells to evade immune surveillance. The genetic mechanisms by which this glycome remodeling occurs remain poorly defined. Understanding the ways that cancer cells change their cell surface glycosylation is critical for identification of biomarkers and targets for glycan-directed immunotherapy. In this study, we performed multiple gain-of-function CRISPR activation (CRISPRa) screens to broadly define genetic pathways that regulate expression of Siglec-binding glycans. We show that Siglec ligand expression is largely controlled through genetic competition between genes that catalyze 2-3 sialylation and GlcNAcylation of galactose residues. Perturbation of enzyme expression at this key biosynthetic node provides multiple "paths" by which cancers can acquire elevated expression of Siglec ligands. We further show that cancer glycome remodeling is aided by overexpression of novel "professional ligands" that facilitate Siglec-glycan binding. Notably, we also find that expression of the CD24 gene is genetically dispensable for cell-surface binding of the inhibitory receptor Siglec-10. Finally, by integrating our functional genetic model with clinical tumor genomic data, we identify the sulfotransferase enzyme GAL3ST4 as a potential novel driver of immune evasion in glioma cells. Taken together, this study provides a first-in-class genomic atlas to aid understanding of cancer-associated glycosylation and identifies immediately actionable targets for cancer immunotherapy.

genomics↗

bioRxiv2024

YibN, a bona fide interactor of YidC with implications in membrane protein insertion and membrane lipid production

YidC, a prominent member of the Oxa1 superfamily, is essential for the biogenesis of the bacterial inner membrane, significantly influencing its protein composition and lipid organization. It interacts with the Sec translocon, aiding the proper folding of multi-pass membrane proteins. It also functions independently, serving as an insertase and lipid scramblase, augmenting the insertion of smaller membrane proteins while contributing to the organization of the bilayer. Despite the wealth of structural and biochemical data available, how YidC operates remains unclear. To investigate this, we employed proximity-dependent biotin labeling (BioID), leading to the identification of YibN as a crucial component within the YidC protein environment. We then demonstrated the association between YidC and YibN by affinity purification-mass spectrometry assays conducted on native membranes, with further confirmation using on-gel binding assays with purified proteins. Co-expression studies and in vitro assays indicated that YibN enhances the production and membrane insertion of YidC substrates, such as M13 and Pf3 phage coat proteins, ATP synthase subunit c, and various small membrane proteins like SecG. Additionally, the overproduction of YibN was found to stimulate membrane lipid production and promote inner membrane proliferation, perhaps by interfering with YidC lipid scramblase activity. Consequently, YibN emerges as a significant physical and functional interactor of YidC, influencing membrane protein insertion and lipid organization.

biochemistry↗

bioRxiv2024

Membrane mimetic thermal proteome profiling (MM-TPP) towards mapping membrane protein-ligand dynamics

Integral membrane proteins (IMPs) are central targets for small-molecule therapeutics, yet robust, unbiased, and detergent-free approaches to assess their on- and off-target interactions remain limited. Previously, we introduced the Peptidisc membrane mimetic (MM) for water-soluble stabilization of the membrane proteome and interactome (Carlson et al., eLife, 2019). In this work, we combine the Peptidisc with thermal proteome profiling (TPP) to establish membrane mimetic thermal proteome profiling (MM-TPP), a method that enables proteome-wide mapping of membrane protein-ligand interactions. Using a membrane protein library derived from mouse liver tissue, we detected the specific effects of ATP and orthovanadate on the thermal stability of ATP-binding cassette (ABC) transporters, as well as stability shifts driven by the hydrotropic effect of ATP and its by-products on G protein-coupled receptors (GPCRs). In contrast, detergent-based TPP (DB-TPP) with ATP-VO failed to yield specific enrichment of ATP-binding proteins, underscoring the unique capacity of MM-TPP. To further validate the approach, we demonstrated the ability of MM-TPP to detect specific ligand-induced stabilization of cognate targets, exemplified by the selective thermal stabilization of the P2RY12 receptor by 2-methylthio-ADP. Together, these findings position MM-TPP as a robust platform for uncovering both on- and off-target effects of small molecules, providing insights into the druggable membrane proteome and its stability in consequence of changing dynamic ligands.

biochemistry↗

bioRxiv2024

Hemoglobin Rothschild: Structural Rationalization of Decreased O2 Affinity as a Consequence of a β37(C3)Trp->Arg Mutation

Hemoglobin Rothschild is characterized by a {beta}37(C3)Trp[->]Arg mutation that severely impairs wildtype hemoglobin function. This mutation has previously been documented to diminish conformational cooperativity, and thereby uppercut oxygen affinity. While the mutation is known to have direct implications on the hinge region at the 1{beta}2 interface, the immediate and indirect manifestations of this mutation have not been rendered using high-resolution molecular visualization software. Further unexplored is whether low O2 affinity in HbR is an outcome of a stabilized, unliganded, tetrameric T-state, a liganded, dimerized R-state deprived of quaternary enhancement, or a combination of both. Herein, PyMOL is used to rationalize the structural artifacts of the Rothschild variant that govern decreased O2 affinity via a stabilized, tetrameric T-state HbR, and decreased O2 affinity via HbR dimerization and loss of cooperative binding in the R-state. Molecular docking simulations were then performed to determine on what grounds O2 affinity is most attenuated. The result shows that, at the 95% confidence level, reduced O2 affinity in HbR is just as much an outcome of a stabilized tetrameric T-state as it is a dimerized R-state lacking quaternary, subunit cooperativity. The work described here builds a statistical framework to accommodate further, pair-wise comparison of low O2 affinity hemoglobin variants to build intuition on which primary sequence mutations pose the largest clinical consequences.

biochemistry↗