Pei Zhou

Podisus maculiventris thanatin has been reported as a potent antimicrobial peptide with antibacterial and antifungal activity. Its antibiotic activity has been most thoroughly characterized against E. coli and shown to interfere with multiple pathways, such as the lipopolysaccharide transport (LPT) pathway comprised of seven different Lpt proteins. Thanatin binds to E. coli LptA and LptD, thus disrupting the LPT complex formation and inhibiting cell wall synthesis and microbial growth. Here, we performed a genomic database search to uncover novel thanatin orthologs, characterized their binding to E. coli LptA using bio-layer interferometry, and assessed their antimicrobial activity against E. coli. We found that thanatins from Chinavia ubica and Murgantia histrionica bound tighter (by 3.6- and 2.2-fold respectively) to LptA and exhibited more potent antibiotic activity (by 2.1- and 2.8-fold respectively) than the canonical thanatin from P. maculiventris. We crystallized and determined the LptA-bound complex structures of thanatins from C. ubica (1.90 Å resolution), M. histrionica (1.80 Å resolution), and P. maculiventris (2.43 Å resolution) to better understand their mechanism of action. Our structural analysis revealed that residues A10 and I21 in C. ubica and M. histrionica thanatin are important for improving the binding interface with LptA, thus overall improving the potency of thanatin against E. coli. We also designed a stapled variant of thanatin that removes the need for a disulfide bond but retains the ability to bind LptA and antibiotic activity. Our discovery presents a library of novel thanatin sequences to serve as starting scaffolds for designing more potent antimicrobial therapeutics.

SPINDLY (SPY) in Arabidopsis thaliana is a novel nucleocytoplasmic protein O-fucosyltransferase (POFUT), which regulates diverse developmental processes. Sequence analysis indicates that SPY is distinct from ER-localized POFUTs and contains N-terminal tetratricopeptide repeats (TPRs) and a C-terminal catalytic domain resembling the O-linked-N-acetylglucosamine (GlcNAc) transferases (OGTs). However, the structural feature that determines the distinct enzymatic selectivity of SPY remains unknown. Here we report the cryo-electron microscopy (cryo-EM) structure of SPY and its complex with GDP-fucose, revealing distinct active-site features enabling GDP-fucose instead of UDP-GlcNAc binding. SPY forms an antiparallel dimer instead of the X-shaped dimer in human OGT, and its catalytic domain interconverts among multiple conformations. Analysis of mass spectrometry, co-IP, fucosylation activity, and cryo-EM data further demonstrates that the N-terminal disordered peptide in SPY contains trans auto-fucosylation sites and inhibits the POFUT activity, whereas TPRs 1-5 dynamically regulate SPY activity by interfering with protein substrate binding.

DNA damage tolerance and mutagenesis are hallmarks and enabling characteristics of neoplastic cells that drive tumorigenesis and allow cancer cells to resist therapy. The 'Y-family' trans-lesion synthesis (TLS) DNA polymerases enable cells to replicate damaged genomes, thereby conferring DNA damage tolerance. Moreover, Y-family DNA polymerases are inherently error-prone and cause mutations. Therefore, TLS DNA polymerases are potential mediators of important tumorigenic phenotypes. The skin cancer-propensity syndrome xeroderma pigmentosum-variant (XPV) results from defects in the Y-family DNA Polymerase Pol eta (Polη) and compensatory deployment of alternative inappropriate DNA polymerases. However, the extent to which dysregulated TLS contributes to the underlying etiology of other human cancers is unclear. Here we consider the broad impact of TLS polymerases on tumorigenesis and cancer therapy. We survey the ways in which TLS DNA polymerases are pathologically altered in cancer. We summarize evidence that TLS polymerases shape cancer genomes, and review studies implicating dysregulated TLS as a driver of carcinogenesis. Because many cancer treatment regimens comprise DNA-damaging agents, pharmacological inhibition of TLS is an attractive strategy for sensitizing tumors to genotoxic therapies. Therefore, we discuss the pharmacological tractability of the TLS pathway and summarize recent progress on development of TLS inhibitors for therapeutic purposes.