MOL BIO COLLOQUIUM

March 3, 2023

ABSTRACTS

GRAD STUDENTS

XUANJIA YE

Muir Lab

LITag: a genetically encoded photo-proximity labeling approach for mapping protein interactomes

Studying dynamic biological processes requires approaches compatible with the lifetimes of the biochemical transactions under investigation, which can be very short. We describe a photo-proximity labeling approach that allows protein interactomes to be captured using brief exposure to visible light. The technology - Light-induced Interactome Tagging (LITag) - involves genetically fusing an engineered flavoprotein to a protein of interest. Excitation of the flavin mononucleotide cofactor leads to covalent labeling of nearby proteins with cell-permeable biotin-appended probes. The small size of the flavoprotein (~12kDa) allows the application to many different protein systems, while its intrinsic fluorescence permits correlated cellular imaging and proteomics analyses. When combined with quantitative proteomics, the system generates ‘snapshots’ of protein territories with high temporal and spatial resolution. The technology should be broadly useful in the biomedical area.

CONNOR CHAIN

Gitai Lab

A novel inhibitor of P. aeruginosa folate metabolism exploits metabolic differences for narrow-spectrum antibiotic targeting
 

Pseudomonas aeruginosa is a leading cause of hospital acquired infections for which the development of new antibiotics is urgently needed. Unlike most enteric bacteria, P. aeruginosa lacks thymidine kinase and thymidine phosphorylase activity, and thus cannot scavenge exogenous thymine. An appealing strategy to selectively target P. aeruginosa while leaving the healthy microbiome largely intact would thus be to disrupt thymidine synthesis while providing exogenous thymine. However, this approach was previously intractable because known antibiotics that perturb thymidine synthesis are largely inactive against P. aeruginosa. Here, we characterize a novel dihydrofolate reductase inhibitor, Irresistin-143 (IRS-143), that exhibits significant activity against P. aeruginosa in culture and in a mouse thigh infection model. IRS-143 is active against a wide range of clinical P. aeruginosa isolates resistant to known antibiotics, including critical antibiotic development priorities expressing the beta-lactamases KPC-5 and NDM-1. Importantly, in the presence of thymine supplementation, IRS-143 activity is selective for P. aeruginosa. Resistance to IRS-143 can emerge through overexpression of the efflux pumps MexCD-OprJ and MexEF-OprN. However, these mutants also decrease pathogenesis, in part due to increased export of quorum sensing precursors leading to decreased virulence factor production. These findings thus demonstrate how understanding species-specific genetic differences and discovery of an antibiotic with a widely conserved target can enable selective targeting of important pathogens while revealing new tradeoffs between resistance and pathogenesis.  

POSTDOC

LINDSAY BECKER

Brangwynne Lab

How do condensates regulate neuronal RNAs?

Neurons are large, complex cells that must precisely control their protein composition over time at thousands of synapses per cell to make functional neural circuits. One way neurons accomplish this is by regulating protein translation in time and space. However, the mechanisms by which most neuronal mRNAs are translationally regulated is not well understood. Emerging evidence suggests that compartmentalization within condensates can mediate mRNA transport and regulate translation. We are developing two novel genomics tools to determine the function of known translation-regulating condensates and to comprehensively map all RNA condensates in neurons. We optimized the proximity labeling technique APEXseq to sequence mRNAs within liquid-like p-body condensates to discover which neuronal functions may be regulated by p-bodies. We will next knock down p-bodies by targeting proteins necessary for their formation to see what effect this has on translation of p-body mRNAs and on neuron function. Additionally, we developed RNA-SPRITE to create a genome-wide atlas of RNA-RNA interactions in neurons. Using this technique, we can identify non-coding RNA interactions in known condensates, such as nucleoli and splicing speckles. We also observe interactions among mRNAs, including those in p-bodies and FMR1 granules. Interestingly, we observe substantially more interactions among autism-associated RNAs than other protein-coding RNAs. Next, we will investigate transcript features that can predict recruitment to specific condensates. We expect to discover novel condensates that regulate important functions in neurons, including those impaired in autism.