Abstract MP49: Central Angiotensin II Mediates Neurogenic Hypertension Through Hif1-Alpha/NMDAR Axis In The Paraventricular Nucleus Of The Hypothalamus (PVN)

Central activation of the renin-angiotensin system and subsequent sympathoexcitation plays a pivotal role in the pathogenesis of hypertension. The specific neural substrates and the possible molecular mechanisms for the sympathoexcitation remain unclear. The glutamatergic activation via N-methyl-d-aspartate receptors (NMDAR) in the PVN enhances sympathetic outflow in hypertension. Previously, using Chromatin immunoprecipitation assay (ChiP) we have shown that Hypoxia-Inducible Factor-1 (heterodimer of HIF-1 α and HIF-1 β subunits ) binds to HREs elements in the NMDA-NR1(structural subunit of NMDAR) promoter in nucleus and increases transcription. In this study, central infusion of Ang II (20 ng/min, 14days, 0.5μl/h, i.c.v) through osmotic mini-pumps in Sprague-Dawley rats increased renal sympathetic nerve activity (20.5 ± 2.3 vs. 6.4 ± 1.9 % of Max) and mean arterial pressure (126 ± 9 vs. 84 ± 4 mmHg). At the same time, there was an increased expression of HIF-1α mRNA (2.79 fold) and HIF-1α protein (1.54 fold), as well as the expression of NMDA-NR1 mRNA (3.17 fold) and NMDA-NR1 protein levels (1.52 fold) in the PVN of Ang II infused group. Direct application of Ang II (1μM) induced an increase in the expression of HIF-1α protein (0.74 ± 0.03* vs. 0.50 ± 0.04) as well as decreased expression of prolyl hydroxylase domain protein 2 (an enzyme hydroxylating the proline residues of HIF-α, inducing subsequent ubiquitination and proteasomal degradation) (1.47 ± 0.14* vs. 0.50 ± 0.03) in NG108-15 neuronal cells. Tandem Ubiquitin-Binding Entities assay showed decreased HIF-α -Ub conjugates in Ang II-treated cells (0.65 ± 0.09* vs. 1.31 ± 0.10) suggesting a role for Ang II in post-translational stabilization of HIF-α. Further, silencing of HIF-1α (~60%) with siRNA in NG108-15 cells leads to a decrease in the expression of NMDA-NR1 induced by Ang II, compared with scrambled siRNA (0.47 ± 0.05* vs 0.80 ± 0.04). Taken together, these studies suggest that elevated central Ang II upregulates the expression of HIF-1α at transcriptional as well as post-translational level, which potentiates glutamatergic tone by enhanced expression of NMDA-NR1 in the PVN leading to heightened glutamatergic tone with a consequent increase in sympathetic outflow resulting in hypertension.

A Novel Luminescence-Based High-Throughput Approach for Cellular Resolution of Protein Ubiquitination Using Tandem Ubiquitin Binding Entities (TUBEs)

Protein turnover is highly regulated by the posttranslational process of ubiquitination. Deregulation of the ubiquitin proteasome system (UPS) has been implicated in cancer and neurodegenerative diseases, and modulating this system has proven to be a viable approach for therapeutic intervention. The development of novel technologies that enable high-throughput studies of substrate protein ubiquitination is key for UPS drug discovery. Conventional approaches for studying ubiquitination either have high protein requirements or rely on exogenous or modified ubiquitin moieties, thus limiting their utility. In order to circumvent these issues, we developed a high-throughput live-cell assay that combines the NanoBiT luminescence-based technology with tandem ubiquitin binding entities (TUBEs) to resolve substrate ubiquitination. To demonstrate the effectiveness and utility of this assay, we studied compound-induced ubiquitination of the G to S Phase Transition 1 (GSPT1) protein. Using this assay, we characterized compounds with varying levels of GSPT1 ubiquitination activity. This method provides a live-cell-based approach for assaying substrate ubiquitination that can be adapted to study the kinetics of ubiquitin transfer onto a substrate protein of interest. In addition, our results show that this approach is portable for studying the ubiquitination of target proteins with diverse functions.

Properties of natural and artificial proteins displaying multiple ubiquitin-binding domains

Ubiquitylation provides a rapid alternative to control the activity of crucial cellular factors through the remodelling of a target protein. Diverse ubiquitin chains are recognized by domains with affinity for UBDs (ubiquitin-binding domains) present in receptor/effector proteins. Interestingly, some proteins contain more than one UBD and the preservation of this structure in many species suggests an evolutionary advantage for this topology. Here, we review some typical proteins that naturally contain more than one UBD and emphasize how such structures contribute to the mechanism they mediate. Characteristics such as higher affinities for polyubiquitin chains and chain-linkage preferences can be replicated by the TUBEs (tandem ubiquitin-binding entities). Furthermore, TUBEs show two additional properties: protection of ubiquitylated substrates from deubiquitylating enzymes and interference with the action of the proteasome. Consequently, TUBEs behave as ‘ubiquitin traps’ that efficiently capture endogenous ubiquitylated proteins. Interpretations and hypothetical models proposed by different groups to understand the synchronous action of multiple UBDs are discussed herein.