Biology subjects

Orr, A.

Publications and source records attributed to Orr, A..

4 recordsLinked to original sources

bioRxiv2025

Integrated Metabolic Complex Genetic Interaction 1 Network of Chromosome 4p Loss in Basal Breast Cancer

Basal breast cancer subtype is enriched for triple-negative breast cancer (TNBC) and exhibits a recurrent large chromosomal deletion in chromosome 4p (chr4p). Chr4p loss is associated with poor survival, evolves early in tumorigenesis and confers on cells a proliferative state. Here, we map the integrated metabolic complex genetic interaction network of chr4p in basal breast cancer to identify targetable vulnerabilities. Differential gene expression analysis of patient derived xenografts and cancer cell models revealed that chr4p loss is associated with changes in cellular energetics and reduction/oxidation balance. Analysis of DepMap pooled genome-wide CRISPR-Cas9 screens identified complex genetic interactions specific to chr4p deletion in basal breast cancer cell models. Functional assays revealed that chr4p loss is associated with disrupted mitochondrial respiratory function and reduced glycolytic capacity, suggesting metabolic rewiring. Increased reactive oxygen species and lipid peroxidation compromised antioxidant defense mechanisms. Ultimately, this study sheds light on targeted therapies for basal breast cancer harboring large chromosomal deletions.

genomics↗

bioRxiv2024

Membrane fusion reactions limited by defective SNARE zippering or stiff lipid fatty acyl composition have distinct requirements for Sec17, Sec18, and adenine nucleotide

Intracellular membrane fusion is catalyzed by SNAREs, Rab GTPases, SM proteins, tethers, Sec18/NSF and Sec17/SNAP. Membrane fusion has been reconstituted with purified vacuolar proteins and lipids to address 3 salient questions: whether ATP hydrolysis by Sec18 affects its promotion of fusion, whether fusion promotion by Sec17 and Sec18 is only seen with mutant SNAREs or can also be seen with wild-type SNAREs, and whether Sec17 and Sec18 only promote fusion when they work together or whether they can each work separately. Fusion is driven by two engines, completion of SNARE zippering (which does not need Sec17/Sec18) and Sec17/Sec18-mediated fusion (needing SNAREs but not the energy from their complete zippering). Sec17 is required to rescue fusion that is blocked by incomplete zippering, though optimal rescue also needs the ATPase Sec18. ATP is an essential Sec18 ligand, but at limiting Sec17 levels Sec18 ATP hydrolysis also drives release of Sec17 without concomitant trans-SNARE complex disassembly. At higher (physiological) Sec17 levels, or without ATP hydrolysis, fusion prevails over Sec17 release. Stiff 16:0, 18:1 fatty acyl chain lipids provide an alternative route to suppressing fusion, with entirely wild-type SNAREs and without impediment to zippering. In this case, Sec17 and Sec18 restore comparable fusion with either ATP or a nonhydrolyzable analog. Fusion blocked by impaired zippering can be restored by concentrated Sec17 alone (but not by Sec18), while fusion inhibited by stiff fatty acyl chains is partially restored by Sec18 alone (but not by Sec17). With distinct fusion impediments, Sec18 and Sec17 have both shared roles and independent roles in promoting fusion. SignificanceSec17/SNAP and Sec18/NSF catalyze cis-SNARE complex disassembly through ATP hydrolysis, but also drive fusion itself without ATP hydrolysis. Fusion can be inhibited by either "stiff" fatty acyl chains while allowing full zippering of entirely wild-type SNAREs or by incomplete SNARE zippering. In either case, Sec17 and Sec18 restore full fusion, but with clear differences. With defective zippering, high levels of Sec17 can restore fusion without Sec18. With stiff fatty acyl chains, Sec18 alone can restore fusion but Sec17 alone will not. In both cases, Sec17 and Sec18 are more efficient together. SNARE-dependent membrane fusion thus relies on two energy sources, complete SNARE zippering and interactions with Sec17 and Sec18.

biochemistry↗

bioRxiv2024

Daxx mediated histone H3.3 deposition on HSV-1 DNA restricts genome decompaction and the progression of immediate-early transcription

Herpesviruses are ubiquitous pathogens that cause a wide range of disease. Upon nuclear entry, their genomes associate with histones and chromatin modifying enzymes that regulate the progression of viral transcription and outcome of infection. While the composition and modification of viral chromatin has been extensively studied on bulk populations of infected cells by chromatin immunoprecipitation, this key regulatory process remains poorly defined at single-genome resolution. Here we use high-resolution quantitative imaging to investigate the spatial proximity of canonical and variant histones at individual Herpes Simplex Virus 1 (HSV-1) genomes within the first 90 minutes of infection. We identify significant population heterogeneity in the stable enrichment and spatial proximity of canonical histones (H2A, H2B, H3.1) at viral DNA (vDNA) relative to established promyelocytic leukaemia nuclear body (PML-NB) host factors that are actively recruited to viral genomes upon nuclear entry. We show the replication-independent histone H3.3/H4 chaperone Daxx to cooperate with PML to mediate the enrichment and spatial localization of variant histone H3.3 at vDNA that limits the rate of HSV-1 genome decompaction to restrict the progress of immediate-early (IE) transcription. This host response is counteracted by the viral ubiquitin ligase ICP0, which degrades PML to disperse Daxx and variant histone H3.3 from vDNA to stimulate the progression of viral genome expansion, IE transcription, and onset of HSV-1 replication. Our data support a model of intermediate and sequential histone assembly initiated by Daxx that limits the rate of HSV-1 genome decompaction independently of the stable enrichment of histones H2A and H2B at vDNA required to facilitate canonical nucleosome assembly. We identify HSV-1 genome decompaction upon nuclear infection to play a key role in the initiation and functional outcome of HSV-1 lytic infection, findings pertinent to the transcriptional regulation of many nuclear replicating herpesvirus pathogens.

microbiology↗

bioRxiv2021

Sec17/Sec18 can support membrane fusion without help from completion of SNARE zippering

Membrane fusion requires R-, Qa-, Qb-, and Qc-family SNAREs that zipper into RQaQbQc coiled coils, driven by the sequestration of apolar amino acids. Zippering has been thought to provide all the force driving fusion. Sec17/SNAP can form an oligomeric assembly with SNAREs with the Sec17 C-terminus bound to Sec18/NSF, the central region bound to SNAREs, and a crucial apolar loop near the N-terminus poised to insert into membranes. Though Sec17 aids zippering, we now report that Sec17 and Sec18 will drive robust fusion without requiring zippering completion. Zippering-driven fusion is blocked by deleting the C-terminal quarter of any Q-SNARE domain or by replacing the apolar amino acids of the Qa-SNARE which face the center of the 4-SNARE coiled coils with polar residues. These blocks to fusion, singly or combined, are bypassed by Sec17 and Sec18, and SNARE-dependent fusion is restored without help from completing zippering.

biochemistry↗