Human Biology Division

2013 Poster Winners Abstracts

Kris Blair, PhD, Salama Lab

Image: Kris Blair

Helicobacter pylori is a carcinogenic bacterial pathogen that chronically infects more than half of the global human population and is a risk factor for the development of stomach cancer in a subset of those infected. It is contracted primarily during childhood and can persist for decades if left untreated. H. pylori colonization is facilitated by a postulated corkscrew mechanism that enables the bacteria to escape the inhospitable acidic lumen, traverse through the viscous mucus lining of the stomach and intimately associate with the epithelial surface of the stomach. Non-helical H. pylori mutants are defective for efficient colonization and our lab has identified many proteins that ­contribute to normal helical cell shape. These cell-shape-determinant (Csd) proteins modify the bacterial cell wall through direct and indirect actions on peptidoglycan (PG); PG is a singular molecule that encases the cell and which determines the shape of nearly all bacteria. My overarching hypothesis states that during growth, Csd proteins must be localized in both time and space to modify the peptidoglycan in a way that generates the asymmetrical helical morphology characteristic of H. pylori. I will take a multidisciplinary approach to solving this four dimensional problem by coupling structural, biochemical and evolutionary analyses of two proteins that likely function in concert to promote helical cell shape. I will investigate the molecular determinants of Csd4 and Csd5 function and investigate a direct protein-protein interaction between them. I will employ domain deletion studies, quantitative cell shape and molecular evolutionary analyses to test our hypothesis that the non-enzymatic Csd5 protein orchestrates localized modification of PG to generate the helical asymmetry. My long term goal is to unify our understanding of the interplay between all of the known and yet to be discovered Csd proteins in H. pylori.

Phil Calses, PhD, Taniguchi Lab

Image: Phil Calses

The DGCR8-mediated UV-response signaling pathway connects the microRNA biogenesis machinery and DNA repair

Philamer Calses1,2, Yemin Wang1, and Toshiyasu Taniguchi 1
1 Howard Hughes Medical Institute, Fred Hutchinson Cancer Research Center, 2Molecular and Cellular Biology Program, University of Washington, Seattle, WA, USA

Ultraviolet (UV) radiation generates toxic DNA lesions such as cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts (6-4PPs). MicroRNAs have been implicated in cellular response to UV. The expression of a subset of microRNAs is altered in response to UV. Furthermore, depletion of Dicer and Ago2, factors required for microRNA biogenesis, leads to hypersensitivity to UV. However, mechanisms connecting the microRNA biogenesis machinery and UV response are largely unknown. To address this, we focused on DGCR8, an RNA binding protein in the microRNA processing Drosha-DGCR8 complex.

We found that treatment with UV (UV-B and -C) induced phosphorylation on Serine 153 (S153) of DGCR8 in human cells in a time- and dose-dependent manner. DGCR8-depleted cells were highly sensitive to UV. Re-introduction of wild type DGCR8 (or S153D phospho-mimetic mutant) restored UV resistance, while S153A mutant failed to restore UV resistance in DGCR8-depleted cells, suggesting that S153 phosphorylation is critical for UV resistance. Surprisingly, the RNA-binding domain DGCR8 mutant restored UV resistance of DGCR8-depleted cells, suggesting that microRNA processing function of DGCR8 is not important for UV resistance. DGCR8 depletion delayed the repair of UV-induced CPDs and 6-4PPs, indicating that repair of UV-induced DNA lesions is dependent on DGCR8. DGCR8 and genes involved in transcription-coupled nucleotide excision repair (TC-NER), such as XPA, CSA and CSB, were epistatic in terms of UV sensitivity, suggesting that DGCR8 is involved in TC-NER. Currently, we are identifying the responsible kinase for S153 phosphorylation by performing a siRNA kinome library screening.

Taken together, we propose a novel DGCR8-mediated UV-response signaling pathway, which connects the microRNA biogenesis machinery and DNA repair.

Supported by NIH/NIGMS PHS NRSA T32 GM07270 and HHMI.

Jaki Braggin, PhD, Geballe Lab

Image: Jaki Braggin

Title: Roles of TRS1 in HCMV Replication

Accumulation of double-stranded RNA (dsRNA) during viral infection activates the antiviral Protein Kinase R (PKR), which inhibits protein synthesis and viral replication. In response, many viruses have evolved PKR antagonists, such as human cytomegalovirus (HCMV) TRS1 and IRS1. HCMV lacking both genes (HMCV[ΔIΔT]) activates the PKR pathway and is unable to replicate, except in fibroblasts expressing a TRS1 transgene (HF-TRS1). Although these results suggest that TRS1 or IRS1 is essential in order to antagonize PKR, HCMV[ΔIΔT] does not replicate in cells in which PKR has been knocked down (PKR-kd). Thus, TRS1 may have another essential role(s) in replication or, alternatively, the low level of PKR expressed in PKR-kd cells might be sufficient to block HMCV[ΔIΔT] replication. To distinguish between these possibilities, we evaluated the functional state of the PKR pathway after viral infection of PKR-kd and control cells. Vaccinia virus lacking its major PKR antagonist, E3L (VVΔE3L), replicated a log more efficiently in PKR-kd cells than in control cells, demonstrating that PKR was reduced in the PKR-kd cells to a level permitting vaccinia virus replication. After HMCV[ΔIΔT] infection, overall protein synthesis in PKR-kd cells was intermediate between the low level found in control cells and the high level in HF-TRS1. Notably, the level of phosphorylated (activated) PKR in PKR-kd cells was considerably less even than that in HF-TRS1, suggesting that PKR activation may not account for the failure of HMCV[ΔIΔT] to replicate in PKR-kd cells. A dsRNA binding-deficient TRS1 mutant that is unable to support VVDE3L replication was recently found to maintain the ability to bind PKR and other TRS1-interacting factors. These studies, combined with previously published data showing that vaccinia E3L rescues HCMV[ΔIΔT] replication, suggest that the essential roles of TRS1 depend on its ability to block dsRNA-activated host defenses including but possibly not limited to PKR.

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