Cullin-RING ubiquitin ligases (CRLs) facilitate regulated degradation of many cellular proteins. Recently CRLs have been implicated in the regulation of blood pressure through degradation of with-no-lysine kinases (WNKs). Hypertension is the most common risk factor in heart disease and stroke; therefore, it is important to understand the mechanisms underlying the diseases associated with hypertension. Rare Mendelian causes of hypertension identify previously unrecognized physiological pathways and networks, which can illuminate new treatments for disease. The Mendelian syndrome Familial Hyperkalemic Hypertension (FHHt, or pseudohypoaldosteronism type II) is a monogenic disease resulting from mutations which increase expression of WNKs. A mutation in cullin 3 (CUL3), which results in skipping of exon 9 (CUL3 ?403-459), causes FHHt and was shown, by in vitro analysis, to have enhanced CUL3 neddylation, increased degradation of the substrate adaptor kelch-like 3 (KLHL3), and decreased binding to the deneddylase COP9 signalosome (CSN). The precise molecular mechanism of CUL3 ?403-459 in the pathogenesis of FHHt has yet to be revealed. CRL activity is dependent on cycling between a neddylated (active) and denneddylated (inactive) state. Therefore, we propose to utilize a genetic mouse model as well as in vitro cell culture to examine the interaction between CSN and CUL3, and the effect of enhanced CUL3 neddylation on CUL3 ligase complex proteins. Specifically, we will test the hypothesis that the FHHt CUL3 ?403-459 mutant has increased neddylation due to decreased binding to the CSN; altered CUL3 function causes ubiquitination and degradation of KLHL3 leading to increased WNK4 expression. These topics will be explored in two Specific Aims.
In Aim 1 it will be determined how enhanced neddylation of CUL3 ?403-459 alters substrate-specific CUL3 ligase function. By using a neddylation-deficient CUL3 construct in which a point mutation is introduced at the specific lysine residue for NEDD8 conjugation (K712R), the specificity and dependence of enhanced neddylation in CUL3 ?403-459 will be determined.
In Aim 2 a combination of in vitro and in vivo techniques will be used to determine whether decreased binding of CUL3 ?403-459 to CSN mediates CUL3 ligase activity. In vitro analysis will utilize HEK293 cells transfected with JAB1 siRNA to examine whether CSN inhibition activates CUL3 ligase activity. Additionally, characterization of a novel inducible and kidney-specific JAB1 knock out mouse model will be performed to determine the phenotypic effects of inhibiting JAB1 in vivo. The proposed research will help understand the mechanism involved in regulation of blood pressure through the CUL3-KLHL3-WNK4 pathway and could lead to pharmaceutical treatment of hypertension by targeting CRLs.
The work proposed in this research training plan is designed to determine how certain gene mutations, that cause high blood pressure (hypertension), specifically in the disease Familial Hyperkalemic Hypertension (FHHt), change the way that the kidneys handle salt. The kidney tubules are the main site in the body that adjusts body salt content, and by doing so, helps to set the blood pressure to an optimal level. The project investigates the molecular mechanism behind a newly discovered genetic mutation that causes FHHt, and may lead to the development of new drugs to target hypertension.
| Cornelius, Ryan J; Si, Jinge; Cuevas, Catherina A et al. (2018) Renal COP9 Signalosome Deficiency Alters CUL3-KLHL3-WNK Signaling Pathway. J Am Soc Nephrol 29:2627-2640 |
