Biology subjects

Schug, W. J.

Publications and source records attributed to Schug, W. J..

4 recordsLinked to original sources

bioRxiv2026

Pannexin 1 Ablation Disrupts Cell Cycle Progression and Compromises the Structure of Melanoma Tumours

Pannexin 1 (PANX1) is a channel-forming glycoprotein that has been implicated in regulating melanoma cell properties such as growth, migration, and invasion. Here we report that the CRISPR-Cas9 mediated ablation of PANX1 or channel inhibition with probenecid shifted A375-P cell cycle distribution towards G2/M, while channel inhibition with spironolactone stalled cell cycle progression at the G1/S checkpoint regardless of PANX1 expression. Bulk RNA sequencing revealed cell cycle related gene expression was downregulated with PANX1 targeting strategies and this was exacerbated when PANX1 deficient cells were treated with spironolactone. When grown as spheroids, we found the deletion and inhibition of PANX1 disrupted spheroid compaction, with spheroids displaying poorly defined borders and reduced circularity. The deletion of PANX1 increased spheroid fragmentation, and this phenotype was intensified when spheroids devoid of PANX1 were treated with spironolactone, highlighting a potential synthetic lethal effect. Using liquid chromatography mass spectrometry, we sought to determine whether PANX1 had binding partners that may act to stabilize spheroid integrity and found that PANX1 co-immunoprecipitated various cell adhesion molecules such as {beta}-actin and F-actin-capping protein subunit -1, suggesting PANX1 and its interacting partners may stabilize cell-cell contacts. When control and PANX1 ablated cells were grown in chick chorioallantoic membrane xenografts to assess their tumour growth characteristics, we discovered that the deletion of PANX1 reduced tumour surface area and viability. Our findings indicate that PANX1 regulates cell cycle progression and the structural architecture of A375-P melanoma tumours, highlighting the therapeutic utility of targeting PANX1 in melanoma.

cancer biology↗

bioRxiv2026

Pannexin 1 phosphorylation sites differentially modulate channel activity and physiological outcomes

Within the vasculature, pannexin 1 (PANX1) channels in smooth muscle cells (SMCs) regulate -adrenergic constriction and blood pressure. PANX1 channel activity is regulated by phosphorylation at Y198, S205 and Y308 residues, but the physiological significance of these modifications is unknown. Here, we utilize newly developed PANX1 Y198F, S205A and Y308F phospho-dead mutant mice to test physiological changes related to hemodynamics. Radiotelemetry-measured blood pressure was decreased in Y198F, increased in Y308F, but unchanged in S205A mice at baseline. Clonidine-sensitive sympathetic-driven hypertension was observed in all mouse lines except Y198F. Pressure myography of third-order mesenteric arteries revealed -adrenergic contractile responses were decreased in Y198F, slightly enhanced in Y308F, but unchanged in S205A, with responses in Y198F vessels mimicking controls treated with PANX1 inhibitors. To understand signaling changes driving these phenotypes, we performed mesenteric artery bulk RNA sequencing, but found a minimal number of differentially expressed genes between phospho-dead mutants and controls. Similarly, co-immunoprecipitation-mass spectrometry of wildtype or phospho-dead mutant-expressing vascular SMCs revealed few interacting proteins distinct to each PANX1 variant. However, PANX1 channel activity assessments in HEK293T cells expressing the 1D-adrenergic receptor as well as each phospho-dead mutant PANX1 showed that phenylephrine-induced ATP release from Y198F channels was significantly decreased compared to wildtype, but current was unaffected. Conversely, basal and phenylephrine-induced S205A and Y308F currents were reduced, but ATP release resembled controls. Taken together, these findings indicate that distinct PANX1 phosphorylation determines PANX1 metabolite release versus current conducting properties and in turn, regulates physiological outcomes in the vasculature. One Sentence SummaryPANX1 Y198 phosphorylation-mediated ATP release is a major driver of -adrenergic vasoconstriction in vascular smooth muscle cells.

physiology↗

bioRxiv2025

Arterial iron regulates vasodilation during anemia via endothelial holo-alpha globin

Iron deficiency is a highly prevalent nutrient deficiency and the most common cause of anemia. Although iron deficiency exacerbates cardiovascular disease, the direct impact of iron deficiency on the vasculature remains unstudied. We assessed iron levels across the vascular endothelium and found resistance artery endothelial cells to have the lowest iron stores suggesting they may be especially impacted by iron deficiency. Anemia has previously been shown to increase arterial NO signaling in patients, and we have previously shown endothelial -globin (Hb) scavenges nitric oxide (NO) in the resistance artery endothelium. We hypothesize iron regulates vascular function through downregulation of endothelial Hb. To test this, we used a novel model of iron deficiency anemia (IDA). In female mice, IDA increased NO signaling which was rescued to control levels by repletion of vascular iron with ferric dextran. Despite being similarly anemic and having a similar reduction in Hb protein, there were no changes in NO signaling across groups in male mice. We further measured whether Hb was in its heme-bound (holo-Hb) or heme-free (apo-Hb) state and found males did not fully lose holo-Hb. Using endothelial specific Hb knockout mice, we show loss of endothelial Hb is necessary for increased NO signaling in IDA and for the rescue of NO signaling by ferric dextran in female mice. Altogether the data presented here demonstrate iron modulates endothelial NO signaling through the regulation of Hb.

physiology↗

bioRxiv2024

Nitrosation of CD36 regulates endothelial function and serum lipids

During obesity, endothelial cells (ECs) become lipid laden leading to endothelial dysfunction. We demonstrate endothelium downregulates caveolin-1 (Cav1) in mouse and human in response to obesity. Using an EC-specific Cav1 knockout mouse, we find mice are hyperlipidemic regardless of diet, but retain endothelial cell function. Whereas initially this was thought to be due to Cav1 mediate endocytosis, we find instead the mice have significantly increased nitric oxide (NO) in response to the lack of Cav1. The presence or absence of NO toggled inversely EC lipid content and plasma lipid in mice. We found the fatty acid translocase CD36 was directly nitrosated by endogenous NO at the same cysteines that are palmitoylated on CD36. The nitrosation of CD36 prevented its trafficking to the plasma membrane and decreased lipid uptake. The physiological effect of this mechanism was a reliance on NO for endothelial function. This work suggests that CD36 nitrosation occurs as a protective mechanism to prevent EC lipotoxicity and preserve function. TeaserNitric oxide regulates serum lipids and endothelial cell lipid content through nitrosation of CD36.

molecular biology↗