The use of tetracycline-off system to study yeast with deficiency in both Ost3p and Ost6p

presented by Chun Zhou


Oligosaccharyltransferase (OTase), the central enzyme in the N-glycosylation pathway in eukaryotic cells, is a multimeric complex consisting of eight subunits in yeast. Ost3p and Ost6p are mutually exclusive subunits in the yeast OTase, defining two OTase isoforms with distinct polypeptide substrate-specificities. Absence of both Ost3p/6p in yeast causes underglycosylation and severe growth defect of the yeast. To study the functions of Ost3p/6p directly in glycosylation and more broadly, we constructed a tetracycline-off to conditionally knockdown OST3 in ∆ost6 yeast. This yeast strain lacks both Ost3p and Ost6p only when tetracycline is present. Interestingly, it was found that abnormally fast-growing revertants could be obtained from the OST3/6double-deficient yeast occasionally. We performed whole genome sequencing to identify possible suppressor mutations that might cause the growth rescue of the glycosylation defective revertants. This sequencing data showed that in multiple independent revertants, mutations accumulated in the tetracycline-off system rather than in native yeast genes. Together, our data suggest that single mutational events that can suppress the important roles of OST3 and OST6 in yeast are very rare, and that yeast growth is very robust to severe defects in N-glycosylation. 

NAD+ Nucleosidase activity and structure study of AbTir and PumA

presented by Sunlin Li


Bacterial Toll/interleukin-1 receptor (TIR) domain-containing proteins have been shown to mediate to the pathogenicity and anti-viral activity of bacteria. During bacterial infection, TIR domain-containing proteins may act as virulence factors to inhibit immune responses by interfering with Toll-like receptor signalling. Additionally, some bacterial TIR domain-containing proteins possess NAD+ nucleosidase activity, which is not only also related to the virulence of pathogenic bacteria, but also plays an important role in bacterial anti-viral defence 1, 2. Here we report our studies on two bacterial TIR domain-containing proteins: PumA and AbTIR. Acinetobacter baumannii TIR domain-containing (AbTir) is one of the few bacterial proteins that has been reported to produce v-cADPR after NAD+ nucleosidase. We determined the crystal structure of AbTir TIR domain and the structure of v2-cADPR. V2-cADPR is the product after NAD+ cleavage and can bind to ThsA which related to the bacterial Thoeris defence system. Furthermore, we found that addition of an inhibitor of AbTir NAD+ nucleosidase activity (3AD) induces the formation of AbTir TIR domain filaments, and appears to trap the protein in an active conformation. Using cryoEM, we successfully determined the AbTir filament structure. Recent studies showed that PumA, which is from multi-drug resistant pathogen Pseudomonas aeruginosa PA7, is essential for PA7 strain virulence 3. We determined that PumA also has NAD+ nucleosidase activity. We also saw observed AbTir TIR domain filaments upon incubation with 3AD. Further structural and functional studies will determine the structure of these filaments and investigate the role these assemblies play in bacterial virulence and anti-viral defence. 

About School research seminars

Seminars cover all aspects of chemistry and molecular biosciences and are delivered by visiting national and international academics. PhD completion seminars are also incorporated into the program.

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