Biology subjects

Wang, M. L.

Publications and source records attributed to Wang, M. L..

7 recordsLinked to original sources

bioRxiv2026

TXNDC15 modulated quality control at the endoplasmic reticulum shapes ciliogenesis

At the endoplasmic reticulum (ER), membrane protein quality control is tightly regulated to ensure excess subunits are recognized and degraded to protect cellular homeostasis. Using genome wide CRISPR screens, we identified a factor of unknown function, thioredoxin domain containing protein 15 (TXNDC15), and showed that it regulates membrane protein stability by tuning the activity of the E3-ubiquitin ligase, MARCHF6. TXNDC15 modulates MARCHF6 in two opposing ways: first, it enhances the binding, ubiquitination, and degradation of membrane protein subunits with soluble cytosolic domains; and second, it prevents the inappropriate recruitment and ubiquitination of subunits with globular lumenal domains. Patient mutations to TXNDC15 that cause the ciliopathy Meckel-Gruber syndrome, disrupted its binding to MARCHF6, allowing degradation of critical ciliary proteins as they transit through the ER leading to defects in ciliogenesis. The regulatory function of TXNDC15 therefore exemplifies how protein quality control maintains the integrity of the proteome to prevent disease.

molecular biology↗

bioRxiv2025

A multiplex extracellular interactome screening method employing high-avidity nanoparticles

Metazoan cells signal to each other via direct contact between cell surface proteins (CSPs) and by interactions of CSP receptors with secreted ligands. CSP extracellular domain (ECD) interactions control organ development and physiology and are perturbed in disease states. However, because they cannot be accurately assessed using standard high-throughput screening techniques, they are underrepresented in protein interaction databases. Many ECD interactions are of low affinity, and their detection in vitro requires taking advantage of avidity effects, typically by multimerization of fusion proteins. Assays that test only one or a few interactions in each binding reaction are inadequate for global interactome screening. Here we describe a new multiplex method that uses purified dimeric ECD fusion proteins coupled to 60-mer nanoparticles as soluble prey, and the same dimers coupled to spectrally distinguishable fluorescent microspheres (beads) as bait. We add one prey to a mixture of up to 500 baits in a single well, then use a Luminex FLEXMAP 3D (FM3D) instrument to read out bait identity and prey binding. The FM3D measures the fluorescent dye ratio for each bead and simultaneously determines the amount of epitope-tagged prey bound to that bead. We use the method, denoted as the Multiplex Interactome Assay (MPIA), to analyze a proof-of-concept (PoC) set of 41 CSPs and secreted protens that is derived from larger collections examined in two interactome screens that used ELISA-based binding assays. By analyzing interactions among PoC proteins, we compared the MPIA with earlier screening methods. The MPIA has a dynamic range that is at least 30-fold greater than ELISA-based assays and appears to be more sensitive. By coupling the MPIA to an automated protein production and purification platform, we hope to be able to conduct a screen for interactions among thousands of human CSPs and secreted ligands.

molecular biology↗

bioRxiv2025

An Automated End-to-End Workflow for Production of Secreted Proteins in Transfected Mammalian Cells

The advancement of automation technologies has helped to enable a surge in large-scale screening efforts across fields such as molecular biology, protein biochemistry, cell biology, and structural biology. In the context of this "omics"-driven research, there is a need to generate automation platforms that are more flexible and less expensive, so that they can be utilized for basic research conducted by small groups. A key challenge in automation lies in developing methods that can replicate fine motor techniques that are normally performed manually by researchers at the bench. We are engaged in a large-scale project to map interactions among human cell-surface and secreted proteins and assess their effects on cells. This project involves production of a library of more than 2000 recombinant His-tagged fusion proteins secreted from transfected Expi293 cells. To execute such a project with a small group at an academic institution required construction of an affordable automated system that could also be used by other investigators. This led us to develop a high-throughput, 96-well format automation platform for end-to-end protein production. The workflow includes transformation of E. coli, plasmid DNA preparation, transient transfection, protein purification, desalting and buffer exchange, protein quantification, and normalization of protein concentrations, resulting in assay-ready proteins. The system is built around an in-house engineered modular robotic platform that integrates liquid handling with a suite of interchangeable plug-and-play mobile enclosed device modules. Housed within a BSL-2 sterile environment, the platform enables flexible, fully automated workflows and can be readily customized for diverse user-defined protocols.

biochemistry↗

bioRxiv2023

Role of a holo-insertase complex in the biogenesis of biophysically diverse ER membrane proteins

Mammalian membrane proteins perform essential physiologic functions that rely on their accurate insertion and folding at the endoplasmic reticulum (ER). Using forward and arrayed genetic screens, we systematically studied the biogenesis of a panel of membrane proteins, including several G-protein coupled receptors (GPCRs). We observed a central role for the insertase, the ER membrane protein complex (EMC), and developed a dual-guide approach to identify genetic modifiers of the EMC. We found that the back of sec61 (BOS) complex, a component of the multipass translocon, was a physical and genetic interactor of the EMC. Functional and structural analysis of the EMC*BOS holocomplex showed that characteristics of a GPCRs soluble domain determine its biogenesis pathway. In contrast to prevailing models, no single insertase handles all substrates. We instead propose a unifying model for coordination between the EMC, multipass translocon, and Sec61 for biogenesis of diverse membrane proteins in human cells.

cell biology↗

bioRxiv2023

A dual sgRNA library design to probe genetic modifiers using genome-wide CRISPRi screens

Mapping genetic interactions is essential for determining gene function and defining novel biological pathways. We report a simple to use CRISPR interference (CRISPRi) based platform, compatible with Fluorescence Activated Cell Sorting (FACS)-based reporter screens, to query epistatic relationships at scale. This is enabled by a flexible dual-sgRNA library design that allows for the simultaneous delivery and selection of a fixed sgRNA and a second randomized guide, comprised of a genome-wide library, with a single transduction. We use this approach to identify epistatic relationships for a defined biological pathway, showing both increased sensitivity and specificity than traditional growth screening approaches.

genomics↗

bioRxiv2022

MTCH2 is a mitochondrial outer membrane protein insertase

In the mitochondrial outer membrane, tail-anchored (TA) proteins play critical roles in cytoplasmic-mitochondrial communication. Using genome-wide CRISPRi screens, we identify factors involved in mitochondrial TA biogenesis in human cells. We show that MTCH2, and its paralog MTCH1, are required for insertion of biophysically diverse mitochondrial TAs, but not outer membrane {beta}-barrel proteins. In a reconstituted system, purified MTCH2 is sufficient to mediate insertion into proteoliposomes. Functional and mutational studies reveal that MTCH2 uses membrane-embedded hydrophilic residues to function as a gatekeeper for outer membrane protein biogenesis, controlling mislocalization of TAs into the endoplasmic reticulum and the sensitivity of leukemia cells to apoptosis. Our identification of MTCH2 as an insertase provides a mechanistic explanation for the diverse phenotypes and disease states associated with MTCH2 dysfunction. One-Sentence SummaryMTCH2 is both necessary and sufficient for insertion of diverse -helical proteins into the mitochondrial outer membrane, and is the defining member of a family of insertases that have co-opted the SLC25 transporter fold.

cell biology↗

bioRxiv2019

Enhancing Intracellular Concentration and Target Engagement of PROTACs with Reversible Covalent Chemistry

Current efforts in the proteolysis targeting chimera (PROTAC) field mostly focus on choosing an appropriate E3 ligase for the target protein, improving the binding affinities towards the target protein and the E3 ligase, and optimizing the PROTAC linker. However, due to the large molecular weights of PROTACs, their cellular uptake remains an issue. Through comparing how different warhead chemistry, reversible noncovalent (RNC), reversible covalent (RC), and irreversible covalent (IRC) binders, affects the degradation of Brutons Tyrosine Kinase (BTK), we serendipitously discover that cyano-acrylamide-based reversible covalent chemistry can significantly enhance the intracellular accumulation and target engagement of PROTACs and develop RC-1 as a reversible covalent BTK PROTAC with a high target occupancy as its corresponding kinase inhibitor and effectiveness as a dual functional inhibitor and degrader, a different mechanism-of-action for PROTACs. Importantly, this reversible covalent strategy is generalizable to improve other PROTACs, opening a path to enhance PROTAC efficacy.

pharmacology and toxicology↗