Biology subjects

Govender, I.

Publications and source records attributed to Govender, I..

3 recordsLinked to original sources

bioRxiv2025

Arachidonic acid modulates fluconazole-induced responses in Candida albicans biofilms

Candida albicans is a human commensal, which causes opportunistic infections with high morbidity and mortality and is characterised by the production of resistant biofilms. Polyunsaturated fatty acids, such as arachidonic acid, can increase azole susceptibility of C. albicans biofilms significantly. However, the underlining mechanism is not known. We investigated the effect of arachidonic acid on known fluconazole resistance mechanisms namely, overexpression of ERG11, increased ergosterol content, oxidative stress resistance, as well as expression and activity of the efflux pump, Cdr1p. Upregulation of ERG11 was observed in biofilms exposed to fluconazole. However, this was reversed in the presence of arachidonic acid, even in the presence of fluconazole. Furthermore, arachidonic acid downregulated the fluconazole-induced oxidative stress response of C. albicans. Previous transcriptome data indicated a significant increase in CDR1 expression during early biofilm formation in the presence of arachidonic acid. However, we found that efflux activity was reduced in the presence of arachidonic acid, which indicates the loss of function of the membrane-associated protein. This contradictory phenomenon was further investigated by determining the effect of arachidonic acid on the localisation and phosphorylation of Cdr1p (ProteomeXchange identifier PXD070958). Our results show that the presence of arachidonic acid may cause mislocalisation and changes in the phosphorylation of Cdr1p; all of which could impact its activity. These results demonstrate that multiple mechanisms are potentially involved in the arachidonic acid-induced increased fluconazole susceptibility and allows for further exploration of lipid-mediated modulation of antifungal responses.

microbiology↗

bioRxiv2023

Mag-Net: Rapid enrichment of membrane-bound particles enables high coverage quantitative analysis of the plasma proteome

Membrane-bound particles in plasma are composed of exosomes, microvesicles, and apoptotic bodies and represent [~]1-2% of the total protein composition. Proteomic interrogation of this subset of plasma proteins augments the representation of tissue-specific proteins, representing a "liquid biopsy," while enabling the detection of proteins that would otherwise be beyond the dynamic range of liquid chromatography-tandem mass spectrometry of unfractionated plasma. We have developed an enrichment strategy (Mag-Net) using hyper-porous strong-anion exchange magnetic microparticles to sieve membrane-bound particles from plasma. The Mag-Net method is robust, reproducible, inexpensive, and requires <100 L plasma input. Coupled to a quantitative data-independent mass spectrometry analytical strategy, we demonstrate that we can collect results for >37,000 peptides from >4,000 plasma proteins with high precision. Using this analytical pipeline on a small cohort of patients with neurodegenerative disease and healthy age-matched controls, we discovered 204 proteins that differentiate (q-value < 0.05) patients with Alzheimers disease dementia (ADD) from those without ADD. Our method also discovered 310 proteins that were different between Parkinsons disease and those with either ADD or healthy cognitively normal individuals. Using machine learning we were able to distinguish between ADD and not ADD with a mean ROC AUC = 0.98 {+/-} 0.06.

biochemistry↗

bioRxiv2020

Zirconium(IV)-IMAC for phosphopeptide enrichment in phosphoproteomics

Phosphopeptide enrichment is an essential step in large-scale, quantitative phosphoproteomics studies by mass spectrometry. Several phosphopeptide affinity enrichment techniques exist, such as Immobilized Metal ion Affinity Chromatography (IMAC) and Metal Oxide Affinity Chromatography (MOAC). We compared Zirconium (IV) IMAC (Zr-IMAC) magnetic microparticles to more commonly used Titanium (IV) IMAC (Ti-IMAC) and TiO2 magnetic microparticles for phosphopeptide enrichment from simple and complex protein samples prior phosphopeptide sequencing and characterization by mass spectrometry (LC-MS/MS). We optimized sample-loading conditions to increase phosphopeptide recovery for Zr-IMAC, Ti-IMAC and TiO2 based workflows. The performance of Zr-IMAC was enhanced by 19-22% to recover up to 5173 phosphopeptides from 200 {micro}g of protein extract from HepG2/C3A cells, making Zr-IMAC the preferred method for phosphopeptide enrichment in this study. Ti-IMAC and TiO2 performance were also optimized to improve phosphopeptide numbers by 28% and 35%, respectively. Furthermore, Zr-IMAC based phosphoproteomics in the magnetic microsphere format identified 23% more phosphopeptides than HPLC-based Fe(III)-IMAC for same sample amount (200 {micro}g), thereby adding 37% more uniquely identified phosphopeptides. We conclude that Zr-IMAC improves phosphoproteome coverage and recommend that this affinity enrichment method should be more widely used in biological and biomedical studies of cell signalling and in the search for disease-biomarkers.

biochemistry↗