Celeste Riepe

All Publications

• Small molecule correctors divert CFTR-F508del from ERAD by stabilizing sequential folding states. Molecular biology of the cell Riepe, C., Wąchalska, M., Deol, K. K., Amaya, A. K., Porteus, M. H., Olzmann, J. A., Kopito, R. R. 2023: mbcE23080336

  Abstract

  Over 80% of people with cystic fibrosis (CF) carry the F508del mutation in the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride ion channel at the apical plasma membrane (PM) of epithelial cells. F508del impairs CFTR folding causing it to be destroyed by endoplasmic reticulum associated degradation (ERAD). Small molecule correctors, which act as pharmacological chaperones to divert CFTR-F508del from ERAD, are the primary strategy for treating CF, yet corrector development continues with only a rudimentary understanding of how ERAD targets CFTR-F508del. We conducted genome-wide CRISPR/Cas9 knockout screens to systematically identify the molecular machinery that underlies CFTR-F508del ERAD. Although the ER-resident ubiquitin ligase, RNF5 was the top E3 hit, knocking out RNF5 only modestly reduced CFTR-F508del degradation. Sublibrary screens in an RNF5 knockout background identified RNF185 as a redundant ligase and demonstrated that CFTR-F508del ERAD is robust. Gene-drug interaction experiments illustrated that correctors tezacaftor (VX-661) and elexacaftor (VX-445) stabilize sequential, RNF5-resistant folding states. We propose that binding of correctors to nascent CFTR-F508del alters its folding landscape by stabilizing folding states that are not substrates for RNF5-mediated ubiquitylation.

  View details for DOI 10.1091/mbc.E23-08-0336

  View details for PubMedID 38019608

• The herpesvirus UL49.5 protein hijacks a cellular C-degron pathway to drive TAP transporter degradation. bioRxiv : the preprint server for biology Wąhalska, M., Riepe, C., Ślusarz, M. J., Graul, M., Borowski, L. S., Qiao, W., Foltynska, M., Carette, J. E., Bieńkowska-Szewczyk, K., Szczesny, R. J., Kopito, R. R., Lipińska, A. D. 2023

  Abstract

  The transporter associated with antigen processing (TAP) is a key player in the MHC class I-restricted antigen presentation and an attractive target for immune evasion by viruses. Bovine herpesvirus 1 (BoHV-1) impairs TAP-dependent antigenic peptide transport through a two-pronged mechanism in which binding of the UL49.5 gene product to TAP both inhibits peptide transport and promotes its proteasomal degradation. How UL49.5 promotes TAP degradation is unknown. Here, we use high-content siRNA and genome-wide CRISPR-Cas9 screening to identify CLR2KLHDC3 as the E3 ligase responsible for UL49.5-triggered TAP disposal in human cells. We propose that the C-terminus of UL49.5 mimics a C-end rule degron that recruits the E3 to TAP and engages the CRL2 E3 in ER-associated degradation.

  View details for DOI 10.1101/2023.09.27.559663

  View details for PubMedID 37808699

  View details for PubMedCentralID PMC10557673

• Small molecule correctors divert CFTR-F508del from ERAD by stabilizing sequential folding states. bioRxiv : the preprint server for biology Riepe, C., Wachalska, M., Deol, K. K., Amaya, A. K., Porteus, M. H., Olzmann, J. A., Kopito, R. R. 2023

  Abstract

  Over 80% of people with cystic fibrosis (CF) carry the F508del mutation in the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride ion channel at the apical plasma membrane (PM) of epithelial cells. F508del impairs CFTR folding causing it to be destroyed by endoplasmic reticulum associated degradation (ERAD). Small molecule correctors, which act as pharmacological chaperones to divert CFTR-F508del from ERAD, are the primary strategy for treating CF, yet corrector development continues with only a rudimentary understanding of how ERAD targets CFTR-F508del. We conducted genome-wide CRISPR/Cas9 knockout screens to systematically identify the molecular machinery that underlies CFTR-F508del ERAD. Although the ER-resident ubiquitin ligase, RNF5 was the top E3 hit, knocking out RNF5 only modestly reduced CFTR-F508del degradation. Sublibrary screens in an RNF5 knockout background identified RNF185 as a redundant ligase, demonstrating that CFTR-F508del ERAD is highly buffered. Gene-drug interaction experiments demonstrated that correctors tezacaftor (VX-661) and elexacaftor (VX-445) stabilize sequential, RNF5-resistant folding states. We propose that binding of correctors to nascent CFTR-F508del alters its folding landscape by stabilizing folding states that are not substrates for RNF5-mediated ubiquitylation.SIGNIFICANCE STATEMENT: Clinically effective small molecule cystic fibrosis (CF) correctors divert mutant CFTR molecules from ER-associated degradation (ERAD). However, the mechanisms underlying CFTR ERAD are not well-understood.The authors used CRISPR knockout screens to identify ERAD machinery targeting CFTR-F508del and found that the pathway is highly buffered, with RNF185 serving as a redundant ubiquitin ligase for RNF5. Gene-drug interaction experiments demonstrated that correctors act synergistically by stabilizing sequential RNF5-resistant folding states.Inhibiting proteostasis machinery is a complementary approach for enhancing current CF corrector therapies.

  View details for DOI 10.1101/2023.09.15.556420

  View details for PubMedID 37745470

• Double stranded DNA breaks and genome editing trigger loss of ribosomal protein RPS27A FEBS JOURNAL Riepe, C., Zelin, E., Frankino, P. A., Meacham, Z. A., Fernandez, S. G., Ingolia, N. T., Corn, J. E. 2022; 289 (11): 3101-3114

  Abstract

  DNA damage activates a robust transcriptional stress response, but much less is known about how DNA damage impacts translation. The advent of genome editing with Cas9 has intensified interest in understanding cellular responses to DNA damage. Here, we find that DNA double-strand breaks (DSBs), including those induced by Cas9, trigger the loss of ribosomal protein RPS27A from ribosomes via p53-independent proteasomal degradation. Comparisons of Cas9 and dCas9 ribosome profiling and mRNA-seq experiments reveal a global translational response to DSBs that precedes changes in transcript abundance. Our results demonstrate that even a single DSB can lead to altered translational output and ribosome remodeling, suggesting caution in interpreting cellular phenotypes measured immediately after genome editing.

  View details for DOI 10.1111/febs.16321

  View details for Web of Science ID 000740693000001

  View details for PubMedID 34914197

• Ribosomal protein RPL26 is the principal target of UFMylation PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Walczak, C. P., Leto, D. E., Zhang, L., Riepe, C., Muller, R. Y., DaRosa, P. A., Ingolia, N. T., Elias, J. E., Kopito, R. R. 2019; 116 (4): 1299–1308

  View details for DOI 10.1073/pnas.1816202116

  View details for Web of Science ID 000456336100035

• Multi-endpoint, High-Throughput Study of Nanomaterial Toxicity in Caenorhabditis elegans ENVIRONMENTAL SCIENCE & TECHNOLOGY Jung, S., Qu, X., Aleman-Meza, B., Wang, T., Riepe, C., Liu, Z., Li, Q., Zhong, W. 2015; 49 (4): 2477–85

  Abstract

  The booming nanotechnology industry has raised public concerns about the environmental health and safety impact of engineered nanomaterials (ENMs). High-throughput assays are needed to obtain toxicity data for the rapidly increasing number of ENMs. Here we present a suite of high-throughput methods to study nanotoxicity in intact animals using Caenorhabditis elegans as a model. At the population level, our system measures food consumption of thousands of animals to evaluate population fitness. At the organism level, our automated system analyzes hundreds of individual animals for body length, locomotion speed, and lifespan. To demonstrate the utility of our system, we applied this technology to test the toxicity of 20 nanomaterials at four concentrations. Only fullerene nanoparticles (nC60), fullerol, TiO2, and CeO2 showed little or no toxicity. Various degrees of toxicity were detected from different forms of carbon nanotubes, graphene, carbon black, Ag, and fumed SiO2 nanoparticles. Aminofullerene and ultraviolet-irradiated nC60 also showed small but significant toxicity. We further investigated the effects of nanomaterial size, shape, surface chemistry, and exposure conditions on toxicity. Our data are publicly available at the open-access nanotoxicity database www.QuantWorm.org/nano.

  View details for DOI 10.1021/es5056462

  View details for Web of Science ID 000349806400061

  View details for PubMedID 25611253

  View details for PubMedCentralID PMC4336152

• QuantWorm: A Comprehensive Software Package for Caenorhabditis elegans Phenotypic Assays PLOS ONE Jung, S., Aleman-Meza, B., Riepe, C., Zhong, W. 2014; 9 (1): e84830

  Abstract

  Phenotypic assays are crucial in genetics; however, traditional methods that rely on human observation are unsuitable for quantitative, large-scale experiments. Furthermore, there is an increasing need for comprehensive analyses of multiple phenotypes to provide multidimensional information. Here we developed an automated, high-throughput computer imaging system for quantifying multiple Caenorhabditis elegans phenotypes. Our imaging system is composed of a microscope equipped with a digital camera and a motorized stage connected to a computer running the QuantWorm software package. Currently, the software package contains one data acquisition module and four image analysis programs: WormLifespan, WormLocomotion, WormLength, and WormEgg. The data acquisition module collects images and videos. The WormLifespan software counts the number of moving worms by using two time-lapse images; the WormLocomotion software computes the velocity of moving worms; the WormLength software measures worm body size; and the WormEgg software counts the number of eggs. To evaluate the performance of our software, we compared the results of our software with manual measurements. We then demonstrated the application of the QuantWorm software in a drug assay and a genetic assay. Overall, the QuantWorm software provided accurate measurements at a high speed. Software source code, executable programs, and sample images are available at www.quantworm.org. Our software package has several advantages over current imaging systems for C. elegans. It is an all-in-one package for quantifying multiple phenotypes. The QuantWorm software is written in Java and its source code is freely available, so it does not require use of commercial software or libraries. It can be run on multiple platforms and easily customized to cope with new methods and requirements.

  View details for DOI 10.1371/journal.pone.0084830

  View details for Web of Science ID 000329862500176

  View details for PubMedID 24416295

  View details for PubMedCentralID PMC3885606