Person: Qiao, Yuan
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Qiao
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Yuan
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Qiao, Yuan
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Publication Detection of Lipid-Linked Peptidoglycan Precursors by Exploiting an Unexpected Transpeptidase Reaction(American Chemical Society, 2014) Qiao, Yuan; Lebar, Matthew D.; Schirner, Kathrin; Schaefer, Kaitlin; Tsukamoto, Hirokazu; Kahne, Daniel; Walker, SuzannePenicillin-binding proteins (PBPs) are involved in the synthesis and remodeling of bacterial peptidoglycan (PG). Staphylococcus aureus expresses four PBPs. Genetic studies in S. aureus have implicated PBP4 in the formation of highly cross-linked PG, but biochemical studies have not reached a consensus on its primary enzymatic activity. Using synthetic Lipid II, we show here that PBP4 preferentially acts as a transpeptidase (TP) in vitro. Moreover, it is the PBP primarily responsible for incorporating exogenous d-amino acids into cellular PG, implying that it also has TP activity in vivo. Notably, PBP4 efficiently exchanges d-amino acids not only into PG polymers but also into the PG monomers Lipid I and Lipid II. This is the first demonstration that any TP domain of a PBP can activate the PG monomer building blocks. Exploiting the promiscuous TP activity of PBP4, we developed a simple, highly sensitive assay to detect cellular pools of lipid-linked PG precursors, which are of notoriously low abundance. This method, which addresses a longstanding problem, is useful for assessing how genetic and pharmacological perturbations affect precursor levels, and may facilitate studies to elucidate antibiotic mechanism of action.Publication Chemical Genetics Identify eIF2α Kinase Heme Regulated Inhibitor as Anti-Cancer Target(2013) Chen, Ting; Ozel, Duygu; Qiao, Yuan; Harbinski, Fred; Chen, Limo; Denoyelle, Séverine; He, Xiaoying; Zvereva, Nela; Supko, Jeffrey; Chorev, Michael; Halperin, Jose; Aktas, BertalTranslation initiation plays a critical role in cellular homeostasis, proliferation, differentiation and malignant transformation. Consistently, increasing the abundance of the eIF2·GTP·Met-tRNAi translation initiation complex transforms normal cells and contributes to cancer initiation and the severity of some anemia. The chemical modifiers of the eIF2·GTP·Met-tRNAi ternary complex are therefore invaluable tools for studying its role in the pathobiology of human disorders and for determining if this complex can be pharmacologically targeted for therapeutic purposes. Using a cell based assay, we identified N,N’-diarylureas as novel inhibitors of the ternary complex abundance. Direct functional-genetics and biochemical evidence demonstrated that the N,N’-diarylureas activate heme regulated inhibitor kinase, thereby phosphorylate eIF2α and reduce abundance of the ternary complex. Using tumor cell proliferation in vitro and tumor growth in vivo as paradigms, we demonstrate that N,N’-diarylureas are potent and specific tools for studying the role eIF2·GTP·Met-tRNAi ternary complex in the pathobiology of human disorders.Publication Reconstitution of the Final Step of Peptidoglycan Assembly in Staphylococcus aureus(2016-05-10) Qiao, Yuan; Walker, Suzanne; Kahne, Daniel; Balskus, Emily P.; Stubbe, JoAnneBacterial peptidoglycan (PG) is an exoskeleton structure that maintains cell shape and protects cells from lysis. Peptidoglycan is essential in bacteria but is not found in mammalian cells. Therefore, it is the target for several classes of antibiotics, including the beta-lactam family. Beta-lactams target the transpeptidase (TP) domain of penicillin-binding proteins (PBPs), the enzymes responsible for the final step of peptidoglycan biosynthesis. Mutations of transpeptidases in Staphylococcus aureus (S. aureus) have been implicated in beta-lactam resistance in the clinic (commonly known as MRSA infections). Despite the fact that PBPs are important antibiotic targets, there have been no direct assays to monitor their enzymatic activity, primarily due to inaccessibility to appropriate substrates. The PG precursor, Lipid II, is required to study transpeptidase activity. Lipid II contains a glycopeptide attached to a pyrophosphate lipid containing 55 carbons. It has poor physical properties and is present in low abundance in bacteria. This thesis describes the reconstitution of peptidoglycan assembly in Staphylococcus aureus by the essential Class A PBP, PBP2. This was enabled by several key advances, which are also described. The first advance is the discovery that PBP4, a low-molecular weight PBP in S. aureus, can use a Lipid II analogue as a transpeptidation substrate. It can incorporate biotin-D-Lys (BDL) and other non-canonical D-amino acids into the terminal position of the stem peptide in Lipid II. BDL labeling of Lipid II with S. aureus PBP4 has enabled the second advance: the direct detection of native Lipid II extracted from bacteria. This has facilitated elucidation of cellular mechanisms of antibiotics that target cell wall biosynthesis. The third advance is a general strategy to accumulate and isolate native Lipid II in bacteria in useful quantities. Access to substantial quantities of native S. aureus Lipid II has enabled reconstitution of PBP2 transpeptidase activity, as well as characterization of several beta-lactam antibiotics by monitoring enzymatic inhibition. In sum, this work establishes important tools for studying enzymatic mechanisms of bacterial transpeptidases and for characterizing inhibitors that target bacterial peptidoglycan biosynthesis.