Abstract: PO1248
Unraveling Fundamental Mechanisms of UMOD Quality Control and Their Role in UMOD-Associated CKD
Session Information
- Cystic Kidney Disease - I
November 04, 2021 | Location: On-Demand, Virtual Only
Abstract Time: 10:00 AM - 12:00 PM
Category: Genetic Diseases of the Kidneys
- 1001 Genetic Diseases of the Kidneys: Cystic
Authors
- Bazua-Valenti, Silvana, Broad Institute of MIT and Harvard, Cambridge, Massachusetts, United States
- Sidhom, Eriene-Heidi, Broad Institute of MIT and Harvard, Cambridge, Massachusetts, United States
- Dvela levitt, Moran, Broad Institute of MIT and Harvard, Cambridge, Massachusetts, United States
- Padovano, Valeria, Broad Institute of MIT and Harvard, Cambridge, Massachusetts, United States
- Roignot, Julie, Broad Institute of MIT and Harvard, Cambridge, Massachusetts, United States
- Keller, Keith H., Broad Institute of MIT and Harvard, Cambridge, Massachusetts, United States
- Himmerkus, Nina, Department of Physiology, Physiology of Membrane Transport, Christian-Albrechts-Universität, Kiel, Germany
- Bleich, Markus, Department of Physiology, Physiology of Membrane Transport, Christian-Albrechts-Universität, Kiel, Germany
- Mutig, Kerim, Department of Functional Anatomy, Charité-Universitätsmedizin, Berlin, Germany
- Bachmann, Sebastian, Department of Functional Anatomy, Charité-Universitätsmedizin, Berlin, Germany
- Weins, Astrid, Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts, United States
- Shaw, Jillian, Broad Institute of MIT and Harvard, Cambridge, Massachusetts, United States
- Marshall, Jamie L., Broad Institute of MIT and Harvard, Cambridge, Massachusetts, United States
- Alimova, Maria, Broad Institute of MIT and Harvard, Cambridge, Massachusetts, United States
- Greka, Anna, Broad Institute of MIT and Harvard, Cambridge, Massachusetts, United States
Background
UMOD is a GPI-anchored protein expressed in the Thick Ascending Limb of the Loop of Henle. When properly folded, it transits from the ER to the membrane via the Golgi. Little is known about UMOD's specific trafficking partners and its quality control mechanisms in the early secretory pathway. Mutations in UMOD disrupt protein folding and promote ER retention, triggering ER-stress pathways and cell death that causes UMOD-related autosomal-dominant tubulointerstitial kidney disease. It was recently shown that mutant misfolded MUC1-fs is trapped in TMED9-containing vesicles and that treatment with compound BRD4780, releases it to the lysosome. We hypothesize that a similar pathogenic quality control mechanism is active in ADTKD-UMOD.
Methods
Co-immunoprecipitation (co-IP) of UMOD and TMED cargo-receptors was assessed in HEK293 cells transfected with wild type (wt) or mutant (C126R) human UMOD. Next, we performed untargeted Affinity Purification Mass Spectrometry followed by tandem MS to generate a list of UMOD interactors. We also conducted in vivo studies in UMOD+/C125R mice.
Results
We identified distinct wt and mutant UMOD interactomes using an unbiased AP-MS proteomics approach. Several interactors, including members of the TMED family, were significantly enriched in the mutant UMOD interactome. Targeted co-IP in lysates of HEK293 cells transfected with UMOD and interacting protein candidates confirmed the results from proteomics studies. Interestingly, when pulling down TMEDs, we found abundant immature non-glycosylated UMOD, suggesting entrapment in early secretory compartments. In vivo, treatment with BRD4780 was suggestive of disrupted interactions between mutant UMOD and interacting partners responsible for toxic entrapment in the ER, as assessed by immunoblots of kidney lysates and immunofluorescence microscopy of tissue from UMOD+/C125R mice.
Conclusion
Our results suggest that UMOD interacts with TMED cargo-receptors and other proteins that may mediate the pathogenic quality control mechanisms responsible for toxic ER-retention and accumulation. Shedding light on these new molecular mechanisms may unmask new therapeutic strategies for the treatment of ADTKD-UMOD.
Funding
- Private Foundation Support