Biology subjects

Fairn, G. D.

Publications and source records attributed to Fairn, G. D..

4 recordsLinked to original sources

bioRxiv2021

Anisotropy of plasmalemmal sterols and cell mating require StARkin domain proteins Ysp2 and Lam4.

In the budding yeast S. cerevisiae Cdc42 is required for polarized growth and the formation of mating projections (shmoos). Negatively charged lipids including phosphatidylserine and phosphatidylinositol 4,5-bisphosphate support a positive feedback loop that recruits Cdc42 effectors and MAP kinase scaffolds, many of which contain polybasic patches that directly interact with the membrane. Here, using genetically encoded sterol sensor ALOD4 we find that ergosterol is accumulated in the cytosolic leaflet of buds and shmoos. The accumulation of ergosterol in the plasma membrane requires both Osh and Lam proteins however cells lacking Ysp2/Lam2 and Lam4 displayed a reversal in the polarity of ergosterol. The redistribution of ergosterol impairs the polarization of phosphatidylserine and phosphatidylinositol 4,5-bisphophate which further impacts shmoo formation, MAPK signaling and mating efficiency. Our observations demonstrate that the ability of Lam proteins to deliver ergosterol from the plasma membrane to the ER helps maintain a gradient of ergosterol which in turn supports robust cell polarity. SummaryThe sterol sensor ALOD4 is enriched at sites of polarized growth. Elimination of the Osh proteins solubilized the ALOD4 whereas elimination of Ysp2 and Lam4 reversed ALOD4 polarization. Cells lacking Ysp2 and Lam4 have defects in mating and MAP kinase signaling.

cell biology↗

bioRxiv2021

Phosphatidylinositol 3-kinase-independent synthesis of PtdIns(3,4)P2 by a phosphotransferase

Despite their comparatively low abundance, phosphoinositides play a central role in membrane traffic and signalling. PtdIns(3,4,5)P3 and PtdIns(3,4)P2 are uniquely important, as they promote cell growth, survival, and migration. Pathogenic organisms have developed means to subvert phosphoinositide metabolism to promote successful infection and their survival within host organisms. We demonstrate that PtdIns(3,4)P2 is generated in host cells by effectors of the enteropathogenic bacteria Salmonella and Shigella. Pharmacological, gene silencing and heterologous expression experiments revealed that, remarkably, the biosynthesis of PtdIns(3,4)P2 occurs independently of phosphoinositide 3-kinases. Instead, we found that the Salmonella effector SopB, heretofore believed to be a phosphatase, generates PtdIns(3,4)P2 de novo via a phosphotransferase/phosphoisomerase mechanism. Recombinant SopB is capable of generating PtdIns(3,4)P2 from PtdIns(4,5)P2 in a cell-free system. Through a remarkable instance of convergent evolution, bacterial effectors acquired the ability to synthesize 3-phosphorylated phosphoinositides by an ATP- and kinase-independent mechanism, thereby subverting host signaling to gain entry and even provoke oncogenic transformation.

cell biology↗

bioRxiv2021

Multiscale interactome analysis coupled with off-target drug predictions reveals drug repurposing candidates for human coronavirus disease

The COVID-19 pandemic has led to an urgent need for the identification of new antiviral drug therapies that can be rapidly deployed to treat patients with this disease. COVID-19 is caused by infection with the human coronavirus SARS-CoV-2. We developed a computational approach to identify new antiviral drug targets and repurpose clinically-relevant drug compounds for the treatment of COVID-19. Our approach is based on graph convolutional networks (GCN) and involves multiscale host-virus interactome analysis coupled to off-target drug predictions. Cellbased experimental assessment reveals several clinically-relevant repurposing drug candidates predicted by the in silico analyses to have antiviral activity against human coronavirus infection. In particular, we identify the MET inhibitor capmatinib as having potent and broad antiviral activity against several coronaviruses in a MET-independent manner, as well as novel roles for host cell proteins such as IRAK1/4 in supporting human coronavirus infection, which can inform further drug discovery studies.

cell biology↗

bioRxiv2020

Fatty Acid Synthase inhibition prevents palmitoylation of SARS-CoV2 SpikeProtein and improves survival of mice infected with murine hepatitis virus.

The Spike protein of SARS-CoV2 and other coronaviruses mediate host cell entry and are S-acylated on multiple phylogenetically conserved cysteine residues. Multiple protein acyltransferase enzymes of the ZDHHC family have been reported to modify Spike proteins post-translationally. Using resin-assisted capture mass spectrometry, we demonstrate that the Spike protein is S-acylated in SARS-CoV2 infected human and monkey cells. We further show that increased abundance of the human acyltransferase ZDHHC5 results in increased S-acylation of the SARS-CoV2 Spike protein, whereas ZDHHC5 knockout cells had a 40% reduction in the incorporation of an alkynyl-palmitate using click chemistry detection. We also find that the S-acylation of the Spike protein is not limited to palmitate, as clickable versions of myristate and stearate were also found on the immunocaptured protein. Yet, ZDHHC5 was highly selective for palmitate, suggesting that other ZDHHC enzymes mediated the incorporation of other fatty acyl chains. Thus, since multiple ZDHHC isoforms may modify the Spike protein, we examined the ability of the fatty acid synthase inhibitor TVB-3166 to prevent the S-acylation of the Spike proteins of SARS-CoV-2 and human CoV-229E. Treating cells with TVB-3166 inhibited S-acylation of ectopically expressed SARS-CoV2 Spike and attenuated the ability of SARS-CoV2 and human CoV-229E to spread in vitro. Additionally, treatment of mice with a comparatively low dose of TVB-3166 promoted survival from an otherwise fatal murine coronavirus infection. Our findings further substantiate the necessity of CoV Spike protein S-acylation and the potential use of fatty acid synthase inhibitors.

cell biology↗