Elif Filiztekin from the O'Donnell Lab and James Kuldell from the Kaplan Lab to speak
Friday, September 19th, 2022
A219B Langley Hall
12:00 PM
Titles:
α-Arrestins impact on metabolism and organelle function (Elif)
Modeling POLR2A syndrome in human cells (James)
Abstracts:
Elif:
The membrane proteome at the cell surface is controlled by vesicle-mediated protein trafficking. In response to environmental changes, such as altered nutrient supply or stressors, cells reorganize the membrane proteome to promote cell survival. The a-arrestins are master regulators of protein trafficking, helping to selectively relocalize membrane proteins from one compartment to another. A key class of membrane protein regulated by the a-arrestins are amino acid permeases, which allow for the uptake of amino acids from the extracellular milieu. The absence of α-arrestins causes retention of amino acid transporters at the plasma membrane. We hypothesize that this aberrant retention of amino acid transporters, and inappropriate storage of amino acids in the vacuole, lead to cytotoxicity due to excess uptake and accumulation of intracellular amino acids, which has been associated with cellular toxicity. Using metabolomics approaches, we find that intracellular amino acid concentrations are increased in cells lacking a-arrestins. This increase in amino acids is associated with mitochondrial malfunction in cells lacking a-arrestins, where we find that mitochondria are fragmented and non-functional and we can restore mitochondrial function by growing cells on medium lacking amino acids. Further, we find that vacuole function is impaired in some a-arrestin mutants, and this could additionally contribute to the negative impact excess amino acids has on cells. Here we begin to define a role for a-arrestins in maintaining metabolic balance as well as mitochondrial and vacuolar physiology. We work to delineate the mechanisms of amino acid cytotoxicity, a largely unaddressed question in biology for several decades.
James:
RNA polymerase II is an essential protein complex that transcribes every protein coding gene. Recently, a human disease was described where individuals had mutations in POLR2A, the largest subunit of RNA polymerase II. Haijes et al, 2019 discovered children with various neurodevelopmental and neuromuscular disorders who also had mutations in POLR2A. However, the severity of the observed phenotypes ranged between mutations and the underlying causes remain unclear. One hypothesis is the observed phenotypes result from defects in transcription during early development. To determine how these POLR2A mutations result in transcriptional defects, total and nascent transcriptome analysis will be performed on disease alleles in HEK293T cells. To discover how these transcription defects affect cellular differentiation, similar transcriptome analyses will be conducted on mutant inducible pluripotent stem cells (iPSCs) induced to differentiate. The observation of any defects from these transcriptome analyses would provide evidence for potential mechanisms. Determining if severe disease alleles result in a higher magnitude of transcription defects may explain the range of severity between observed phenotypes.