Christopher Putnam, Associate Professor of Medicine, UCSD

Genome Instability and Checkpoint Activation

Using funds provided by the SDCSB, we used a combination of bioinformatics and forward genetic screens to identify genome instability suppressing genes in S. cerevisiae. To determine the effects of genome instability on checkpoint activation, we will screen for genome instability suppressing genes using a highly sensitive marker of checkpoint activation and shutoff – ubiquitin fused to the N-terminus of a destabilized GFP (UBI-Y-GFP) resulting in a fluorescent protein with a half-life of 7 minutes. Our preliminary results suggest that the relationship between genome stability and checkpoint activation is complicated as many mutations have uncorrelated effects on genome stability and checkpoint activation. Once we have identified interesting mutants, we will use the SDCSB microfluidics core to follow checkpoint activation and shutoff in single cells of wild type and mutant strains.

Dieter Wolf, Professor, Sanford Burnham Medical Research Institute

Mechanisms of Oxidative Stress Response

Studies carried out in collaboration with the SDCSB previously suggested that oxidative stress triggers rapid translational shutdown downregulating oxidative stress-suppressed mRNAs thereby liberating ribosome capacity for translation of oxidative stress-induced mRNAs. To test this model in mammalian cells, LNCaP prostate cancer cells will be exposed to SMIP004, which stimulates oxidative stress by inhibiting mitochondrial respiration. System-wide datasets on mRNA and protein abundance, synthesis, and translation will be obtained, modeled, and compared against the yeast oxidative stress model of gene expression. Data mining will include network analysis to define cellular pathways involved. We will also determine the global correlations between mRNA abundance, translation rate and protein abundance and will assemble a mathematical model of SMIP004-mediated gene expression.

Jean Y. J. Wang, Distinguished Professor of Medicine and Biology, UCSD

The Role of Mitochondria in Cancer and Diabetes

Using funds provided by the SDCSB, we found that over-expression of a particular mitochondrial matrix adapter protein caused glucose addiction, a distinguishing feature of cancer cells, and that knock-down of this protein increased insulin sensitivity. In this seed project, we will perform a number of systems-wide analysis including proteomics, metabolomics, lipidomics and sequencing (DNA and RNA) on mitochondria isolated from epithelial cells either overexpressing this protein or with this protein knocked down. Cells will be cultured under glucose-rich or glucose-deprived conditions. We will also perform a screen using CRISPR-knockouts to map the genes that antagonize the effects of this mitochondrial matrix adapter protein on insulin sensitivity; knockouts of downstream effectors should allow MCF10A cells overexpressing this protein to survive under glucose starvation.

Suresh Subramani, Distinguished Professor of Biology, UCSD

Identifying Key Regulators of Autophagy

Autophagy is an important mechanism in eukaryotic cells for recycling proteins and organelles. To describe autophagy on a system-wide level, we generated an autophagy-specific gene interaction network, in part by merging various primary data sets. Within this network, we identified key sub-networks, studied evolutionary conservation, and predicted functionally important nodes. RNAi and an image-based screen were used to validate that CDC42, a Rho GTPase, is a novel regulator of autophagy. This study demonstrated that gene network approaches can provide insights into complex biological systems. Several other candidates, besides CDC42, were identified and we will be pursuing these in more detail.

Publications Supported by These and Previous Seed Grants: