Structural Biology & Biochemistry (SBB) Theme Seminar - 1) The yeast secretome: responses to cellular stressors and profiling in wine; 2) Investigating the role of nucleotide binding by Tir domains in plant innate immunity

Speaker 1: Shulei Liu

Title: The yeast secretome: responses to cellular stressors and profiling in wine

Abstract: The yeast secretome represents the small subset of proteins that are secreted outside the cell, and is functionally important in diverse biological mechanisms and yeast-based products. We use proteomics to explore the response of the yeast secretome to various cellular stressors including oxidative stress, endoplasmic reticulum (ER) stress, and heat stress. Our results reveal variation in secretome profiles under different stress conditions, with Gene Ontology (GO) enrichment analysis highlighting the key biological mechanisms involved. We observe an upregulation of the glycolysis pathway under ethanol-induced stress, indicating metabolic consumption pathway changes and activation of non-canonical secretion. Notably, under specific stress conditions we detect enhanced secretion of several ER-functional proteins including ER chaperone BiP, and show physical interactions between BiP and diverse client proteins. We propose the secretion of BiP under stress conditions allows chaperon-mediated stabilisation of client proteins even after they have left the cell.

The yeast secretome also has profound implications in the wine industry, where it can affect wine quality, particularly in clarity, stability, and sensory properties. Proteins are released from yeast during the fermentation process and are present at low concentrations in the final product which can be challenging for assessment. We develop an efficient workflow for red wine proteomics based on liquid chromatography-mass spectrometry (LC-MS/MS) using small volumes of sample and investigate commercial red wine produced from different grape varieties, with different aging times, and from various subregions, revealing the relationships between proteomic profiles and these production variables, as well as identifying the key functional proteins involved.

Bio: Shulei is a PhD candidate at The University of Queensland, under the supervision of Prof Benjamin Schulz in the School of Chemistry and Molecular Biosciences (SCMB). She uses glyco/proteomics based on liquid chromatography-mass spectrometry (LC-MS) to investigate the role of N-glycosylation in secreted glycoproteins, yeast secretome regulation, and related industrial applications.

Speaker 2: Bryan Lim

Title: Investigating the role of nucleotide binding by TIR domains in plant innate immunity

Abstract: In plants, TIR (Toll/interleukin 1 receptor) domain-containing proteins are key components involved in innate immunity. TIR domains cleave nicotinamide adenine dinucleotide (NAD+) into a large variety of nucleotide containing products, collectively termed as immunomodulatory purine nucleotides (IPNs). These products are essential for activation of downstream defence signalling, eventually leading to localised cell death. A recent study found that TIR domains form filamentous complexes upon DNA-binding and cleave DNA and RNA to produce non-canonical 2′, 3′-cAMP/cGMP compounds linked to stress signalling. Given this newly identified function, this project aims to structurally and functionally characterise various TIR domains across different plant species, to understand the prevalence of their ability to bind to and cleave nucleotides. To determine DNA binding activity, electrophoretic mobility shift assays (EMSA) and microscale thermophoresis (MST) were used. Among the various plant TIR domains tested, only L6TIR from flax plants displayed DNA or RNA binding and formed filamentous assemblies. We obtained a cryo-EM structure of a L6TIR filaments in complex with double-stranded and single-stranded DNA at a global resolution of 3.3 Å and 3.5 Å, respectively. Mutagenesis experiments showed that DNA binding requires the KKR motif identified previously. However, we could not detect the presence of  2′, 3′-cAMP/cGMP compounds based on our cryo-EM and LC-MS/MS experiments. We instead found that formation of a protein-DNA complex led to enhanced activation of NADase activity. Current and future work will utilise different substrates and mutants to further understand products formed upon DNA and RNA cleavage and how filament formation affects NADase activity.

Bio: Bryan obtained his Bachelor’s degree in molecular biotechnology (Honours) under the supervision of Prof Bostjan Kobe at The University of Queensland where he investigated the structures of bacterial solute binding protein using X-ray crystallography and their molecular interactions with their cognate ligands in 2019. He then briefly left academia to work as an Medical lab analyst in a SARS-COV2 diagnostic lab. He then started his PhD candidature at UQ in 2022 under Prof. Bostjan Kobe at SCMB focusing on the understanding the structural and molecular interactions of TIR domains in plant innate immunity. His project covers a range of research topics including protein expression and purification, enzymatic product identification, DNA:protein interactions, and cryo-EM. He is now an 3rd year PhD candidate and is interested in further understanding of the molecular interactions of biological systems through structure biology.