Biology subjects

O'Loughlin, T.

Publications and source records attributed to O'Loughlin, T..

4 recordsLinked to original sources

bioRxiv2024

A multi-subunit autophagic capture complex facilitates degradation of ER stalled MHC-I in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDA) evades immune detection partly via autophagic capture and lysosomal degradation of major histocompatibility complex class I (MHC-I). Why MHC-I is susceptible to capture via autophagy remains unclear. By synchronizing exit of proteins from the endoplasmic reticulum (ER), we show that PDAC cells display prolonged retention of MHC-I in the ER and fail to efficiently route it to the plasma membrane. A capture-complex composed of NBR1 and the ER-phagy receptor TEX264 facilitates targeting of MHC-I for autophagic degradation, and suppression of either receptor is sufficient to increase total levels and re-route MHC-I to the plasma membrane. Binding of MHC-I to the capture complex is linked to antigen presentation efficiency, as inhibiting antigen loading via knockdown of TAP1 or beta 2-Microglobulin led to increased binding between MHC-I and the TEX264-NBR1 capture complex. Conversely, expression of ER directed high affinity antigenic peptides led to increased MHC-I at the cell surface and reduced lysosomal degradation. A genome-wide CRISPRi screen identified NFXL1, as an ER-resident E3 ligase that binds to MHC-I and mediates its autophagic capture. High levels of NFXL1 are negatively correlated with MHC-I protein expression and predicts poor patient prognosis. These data highlight an ER resident capture complex tasked with sequestration and degradation of non-conformational MHC-I in PDAC cells, and targeting this complex has the potential to increase PDAC immunogenicity.

cancer biology↗

bioRxiv2023

Higher-order combinatorial chromatin perturbations by engineered CRISPR-Cas12a for functional genomics

Multiplexed genetic perturbations are critical for testing functional interactions among coding or non-coding genetic elements. Compared to double-stranded DNA cutting, repressive chromatin formation using CRISPR interference (CRISPRi) avoids genotoxicity and is more effective for perturbing non-coding regulatory elements in pooled assays. However, current CRISPRi pooled screening approaches are limited to targeting 1-3 genomic sites per cell. To develop a tool for higher-order (>3) combinatorial targeting of genomic sites with CRISPRi in functional genomics screens, we engineered an Acidaminococcus Cas12a variant -- referred to as multiplexed transcriptional interference AsCas12a (multiAsCas12a). multiAsCas12a incorporates a key mutation, R1226A, motivated by the hypothesis of nicking-induced stabilization of the ribonucleoprotein:DNA complex for improving CRISPRi activity. multiAsCas12a significantly outperforms prior state-of-the-art Cas12a variants in combinatorial CRISPRi targeting using high-order multiplexed arrays of lentivirally transduced CRISPR RNAs (crRNA), including in high-throughput pooled screens using 6-plex crRNA array libraries. Using multiAsCas12a CRISPRi, we discover new enhancer elements and dissect the combinatorial function of cis-regulatory elements. These results instantiate a group testing framework for efficiently surveying potentially numerous combinations of chromatin perturbations for biological discovery and engineering.

genetics↗

bioRxiv2023

In vivo perturb-seq of cancer and immune cells dissects oncologic drivers and therapy response

Genetic perturbation screens with single cell readouts have enabled rich phenotyping of gene function and regulatory networks. These approaches have been challenging in vivo, especially in adult disease models such as cancer, which include mixtures of malignant and microenvironment cells. Glioblastoma (GBM) is a fatal cancer, and methods of systematically interrogating gene function and therapeutic targets in vivo, especially in combination with standard of care treatment such as radiotherapy, are lacking. Here, we iteratively develop a multiplex in vivo perturb-seq CRISPRi platform for single cell genetic screens in cancer and tumor microenvironment cells that leverages intracranial convection enhanced delivery (CED) of sgRNA libraries into models of GBM. Our platform enables potent silencing of drivers of in vivo growth and tumor maintenance, as well as genes that sensitize GBM to radiotherapy. We find radiotherapy rewires transcriptional responses to genetic perturbations in an in vivo dependent manner, revealing heterogenous patterns of treatment sensitization or resistance in GBM. Furthermore, we demonstrate targeting of genes that function in the tumor microenvironment, enabling alterations of ligand-receptor interactions between immune/stromal cells following in vivo CRISPRi perturbations. In sum, we demonstrate the utility of multiplexed perturb-seq for in vivo single cell dissection of adult cancer and normal tissue biology across multiple cell types in the context of therapeutic intervention, a platform with potential for broad application.

genomics↗

bioRxiv2019

OPTN translocates to an ATG9A-positive compartment to regulate innate immune signalling and cytokine secretion

Optineurin (OPTN) is a multifunctional protein involved in autophagy, secretion as well as NF-{kappa}B and IRF3 signalling and mutations are associated with several human diseases including primary open-angle glaucoma (POAG), amyotrophic lateral sclerosis (ALS), Pagets disease of bone (PDB) and Crohns disease (CD). Here we show that, in response to viral RNA, OPTN translocates to foci in the perinuclear region, where it negatively regulates NF-{kappa}B and IRF3 signalling pathways and downstream pro-inflammatory cytokine secretion. These OPTN foci consist of a tight cluster of small membrane vesicles, which are positive for marker proteins of the trans-Golgi network/recycling compartment - most notably ATG9A. Disease mutations linked to POAG cause aberrant formation of this compartment in the absence of stimuli, which correlates with the ability of OPTN to inhibit signalling. Using proximity labelling proteomics (BioID), we identify the linear ubiquitin assembly complex (LUBAC), CYLD and TBK1 as part of the OPTN interactome and show that these proteins, along with NEMO, are recruited to this OPTN-positive perinuclear compartment. Together, we propose OPTN might be responsible for dampening the NF-{kappa}B and IRF3 signalling responses through the sequestration of LUBAC and other positive regulators of these pathways in this dsRNA-induced compartment leading to altered pro-inflammatory cytokine secretion.\n\nSummaryDisease associated OPTN mutations impact on the formation of the perinuclear compartment and result in hypo- or hyper-activation of the immune response, which could drive the development of human diseases such as POAG, ALS and also Pagets disease of bone.

cell biology↗