Biology subjects

Lui, I.

Publications and source records attributed to Lui, I..

8 recordsLinked to original sources

bioRxiv2024

Hypoxia induces extensive protein and proteolytic remodeling of the cell surface in pancreatic adenocarcinoma (PDAC) cell lines

The tumor microenvironment (TME) plays a crucial role in cancer progression. Hypoxia is a hallmark of the TME and induces a cascade of molecular events that affects cellular processes involved in metabolism, metastasis, and proteolysis. In pancreatic ductal adenocarcinoma (PDAC), tumor tissues are extremely hypoxic. Here, we leveraged mass spectrometry technologies to examine hypoxia-induced alterations in the abundance and proteolytic modifications to cell surface and secreted proteins. Across four PDAC cell lines, we discovered extensive proteolytic remodeling of cell surface proteins involved in cellular adhesion and motility. Looking outward at the surrounding secreted space, we identified hypoxia-regulated secreted and proteolytically-shed proteins that are responsible for regulating the humoral immune and inflammatory response and an upregulation of proteins involved in metabolic processing and tissue development. Combining cell surface N-terminomics and secretomics to evaluate the cellular response to hypoxia enabled us to identify significantly altered candidate proteins which may serve as potential biomarkers and therapeutic targets in PDAC. Furthermore, this approach provides a blue print for studying dysregulated extracellular proteolysis in other cancers and inflammatory diseases.

cancer biology↗

bioRxiv2023

Multi-scale photocatalytic proximity labeling reveals cell surface neighbors on and between cells

The cell membrane proteome is the primary biohub for cell communication, yet we are only beginning to understand the dynamic protein neighborhoods that form on the cell surface and between cells. Proximity labeling proteomics (PLP) strategies using chemically reactive probes are powerful approaches to yield snapshots of protein neighborhoods but are currently limited to one single resolution based on the probe labeling radius. Here, we describe a multi-scale PLP method with tunable resolution using a commercially available histological dye, Eosin Y, which upon visible light illumination, activates three different photo-probes with labeling radii ranging from [~]100 to 3000 [A]. We applied this platform to profile neighborhoods of the oncogenic epidermal growth factor receptor (EGFR) and orthogonally validated >20 neighbors using immuno-assays and AlphaFold-Multimer prediction that generated plausible binary interaction models. We further profiled the protein neighborhoods of cell-cell synapses induced by bi-specific T-cell engagers (BiTEs) and chimeric antigen receptor (CAR)T cells at longer length scales. This integrated multi-scale PLP platform maps local and distal protein networks on cell surfaces and between cells. We believe this information will aid in the systematic construction of the cell surface interactome and reveal new opportunities for immunotherapeutics.

biochemistry↗

bioRxiv2022

Computational pipeline provides mechanistic understanding of Omicron variant of concern neutralizing engineered ACE2 receptor traps

The SARS-CoV-2 Omicron variant, with 15 mutations in Spike receptor binding domain (Spike-RBD), renders virtually all clinical monoclonal antibodies against WT SARS-CoV-2 ineffective. We recently engineered the SARS-CoV-2 host entry receptor, ACE2, to tightly bind WT-Spike-RBD and prevent viral entry into host cells ("receptor traps"). Here we determine cryo-EM structures of our receptor traps in complex with full length Spike. We develop a multi-model pipeline combining Rosetta protein modeling software and cryo-EM to allow interface energy calculations even at limited resolution and identify interface side chains that allow for high affinity interactions between our ACE2 receptor traps and Spike-RBD. Our structural analysis provides a mechanistic rationale for the high affinity (0.53 - 4.2nM) binding of our ACE2 receptor traps to Omicron-RBD confirmed with biolayer interferometry measurements. Finally, we show that ACE2 receptor traps potently neutralize Omicron- and Delta-pseudotyped viruses, providing alternative therapeutic routes to combat this evolving virus.

biochemistry↗

bioRxiv2021

BRD2 inhibition blocks SARS-CoV-2 infection in vitro by reducing transcription of the host cell receptor ACE2

SARS-CoV-2 infection of human cells is initiated by the binding of the viral Spike protein to its cell-surface receptor ACE2. We conducted a targeted CRISPRi screen to uncover druggable pathways controlling Spike protein binding to human cells. We found that the protein BRD2 is required for ACE2 transcription in human lung epithelial cells and cardiomyocytes, and BRD2 inhibitors currently evaluated in clinical trials potently block endogenous ACE2 expression and SARS-CoV-2 infection of human cells, including those of human nasal epithelia. Moreover, pharmacological BRD2 inhibition with the drug ABBV-744 inhibited SARS-CoV-2 replication in Syrian hamsters. We also found that BRD2 controls transcription of several other genes induced upon SARS-CoV-2 infection, including the interferon response, which in turn regulates the antiviral response. Together, our results pinpoint BRD2 as a potent and essential regulator of the host response to SARS-CoV-2 infection and highlight the potential of BRD2 as a novel therapeutic target for COVID-19.

cell biology↗

bioRxiv2020

Bi-paratopic and multivalent human VH domains neutralize SARS-CoV-2 by targeting distinct epitopes within the ACE2 binding interface of Spike

Neutralizing agents against SARS-CoV-2 are urgently needed for treatment and prophylaxis of COVID-19. Here, we present a strategy to rapidly identify and assemble synthetic human variable heavy (VH) domain binders with high affinity toward neutralizing epitopes without the need for high-resolution structural information. We constructed a VH-phage library and targeted a known neutralizing site, the angiotensin-converting enzyme 2 (ACE2) binding interface of the trimeric SARS-CoV-2 Spike receptor-binding domain (Spike-RBD). Using a masked selection approach, we identified 85 unique VH binders to two non-overlapping epitopes within the ACE2 binding site on Spike-RBD. This enabled us to systematically link these VH domains into multivalent and bi-paratopic formats. These multivalent and bi-paratopic VH constructs showed a marked increase in affinity to Spike (up to 600-fold) and neutralization potency (up to 1400-fold) on pseudotyped SARS-CoV-2 virus when compared to the standalone VH domains. The most potent binder, a trivalent VH, neutralized authentic SARS-CoV-2 with half-minimal inhibitory concentration (IC50) of 4.0 nM (180 ng/mL). A cryo-EM structure of the trivalent VH bound to Spike shows each VH domain bound an RBD at the ACE2 binding site, explaining its increased neutralization potency and confirming our original design strategy. Our results demonstrate that targeted selection and engineering campaigns using a VH-phage library can enable rapid assembly of highly avid and potent molecules towards therapeutically important protein interfaces.

bioengineering↗

bioRxiv2020

Engineered ACE2 receptor traps potently neutralize SARS-CoV-2

An essential mechanism for SARS-CoV-1 and -2 infection begins with the viral spike protein binding to the human receptor protein angiotensin-converting enzyme II (ACE2). Here we describe a stepwise engineering approach to generate a set of affinity optimized, enzymatically inactivated ACE2 variants that potently block SARS-CoV-2 infection of cells. These optimized receptor traps tightly bind the receptor binding domain (RBD) of the viral spike protein and prevent entry into host cells. We first computationally designed the ACE2-RBD interface using a two-stage flexible protein backbone design process that improved affinity for the RBD by up to 12-fold. These designed receptor variants were affinity matured an additional 14-fold by random mutagenesis and selection using yeast surface display. The highest affinity variant contained seven amino acid changes and bound to the RBD 170-fold more tightly than wild-type ACE2. With the addition of the natural ACE2 collectrin domain and fusion to a human Fc domain for increased stabilization and avidity, the most optimal ACE2 receptor traps neutralized SARS-CoV-2 pseudotyped lentivirus and authentic SARS-CoV-2 virus with half-maximal inhibitory concentrations (IC50) in the 10-100 ng/ml range. Engineered ACE2 receptor traps offer a promising route to fighting infections by SARS-CoV-2 and other ACE2-utilizing coronaviruses, with the key advantage that viral resistance would also likely impair viral entry. Moreover, such traps can be pre-designed for viruses with known entry receptors for faster therapeutic response without the need for neutralizing antibodies isolated or generated from convalescent patients.

bioengineering↗

bioRxiv2020

Trimeric SARS-CoV-2 Spike interacts with dimeric ACE2 with limited intra-Spike avidity

A serious public health crisis is currently unfolding due to the SARS-CoV-2 pandemic. SARS-CoV-2 viral entry depends on an interaction between the receptor binding domain of the trimeric viral Spike protein (Spike-RBD) and the dimeric human angiotensin converting enzyme 2 (ACE2) receptor. While it is clear that strategies to block the Spike/ACE2 interaction are promising as anti-SARS-CoV-2 therapeutics, our current understanding is insufficient for the rational design of maximally effective therapeutic molecules. Here, we investigated the mechanism of Spike/ACE2 interaction by characterizing the binding affinity and kinetics of different multimeric forms of recombinant ACE2 and Spike-RBD domain. We also engineered ACE2 into a split Nanoluciferase-based reporter system to probe the conformational landscape of Spike-RBDs in the context of the Spike trimer. Interestingly, a dimeric form of ACE2, but not monomeric ACE2, binds with high affinity to Spike and blocks viral entry in pseudotyped virus and live SARS-CoV-2 virus neutralization assays. We show that dimeric ACE2 interacts with an RBD on Spike with limited intra-Spike avidity, which nonetheless contributes to the affinity of this interaction. Additionally, we demonstrate that a proportion of Spike can simultaneously interact with multiple ACE2 dimers, indicating that more than one RBD domain in a Spike trimer can adopt an ACE2-accessible "up" conformation. Our findings have significant implications on the design strategies of therapeutic molecules that block the Spike/ACE2 interaction. The constructs we describe are freely available to the research community as molecular tools to further our understanding of SARS-CoV-2 biology.

biochemistry↗

bioRxiv2020

Mapping proteolytic neo-N termini at the surface of living cells

N terminomics is a powerful strategy for profiling proteolytic neo-N termini, but its application to cell surface proteolysis has been limited by the low relative abundance of plasma membrane proteins. Here we apply plasma membrane-targeted subtiligase variants to efficiently and specifically capture cell surface N termini in live cells. Using this approach, we sequenced 807 cell surface N termini and quantified changes in their abundance in response to stimuli that induce proteolytic remodeling of the cell surface proteome. This technology will facilitate greater understanding of extracellular protease biology and reveal neo-N termini biomarkers and targets in disease.

biochemistry↗