University of Pittsburgh Department of Biological Sciences Presents:
Friday Noon Seminar Series 2017-2018
Graduate Student: Samuel Estabrooks
Reconstitution of CFTR Ubiquitination and Endoplasmic Reticulum Quality Control in Vitro
Deletion of F508 (“F508del”) causes CFTR channels to misfold and become trapped in the endoplasmic reticulum (ER), where they are targeted for degradation by cellular protein quality control (PQC) systems, causing cystic fibrosis. Among these is the ubiquitin proteasome system, which attaches ubiquitin chains onto misfolded proteins, marking them for degradation by the proteasome. This process is known as ER-associated degradation (ERAD). Molecular therapies, such as Orkambi, facilitate correct folding and function of CFTR, however respiratory improvement in patients has thus far been modest. As no aspect of the therapy directly limits selection of F508del-CFTR by PQC machinery for ERAD, I hypothesize that inhibition of specific PQC factors will synergize with therapeutic compounds to further improve CFTR stability and function. To better characterize which factors preferentially guide F508del-CFTR ubiquitination, I developed an in vitro assay in which F508del-CFTR ubiquitin chain elongation can be recapitulated. In reactions containing ER-derived microsomes from CFTR-expressing human cell culture, purified enzymes required for ubiquitin chain synthesis, and iodine-125-labeled ubiquitin, wild type and F508del-CFTR were both readily ubiquitinated. Addition to reactions of an E3 ubiquitin ligase, CHIP, enhanced ubiquitination. Surprisingly, I found another disease-causing allele, N1303K-CFTR, was not modified in this assay, but could be observed as a polyubiquitinated species in human cell culture. I am currently testing how add-back of CHIP and other PQC factors affects ubiquitination of additional CFTR alleles in vitro. Ultimately, I hope to identify which factors preferentially mark mutant CFTR alleles for degradation, and are thus attractive targets for pharmacological intervention.
Friday, January 19, 2018
A219B Langley Hall
12:00 PM Seminar