Stella-Rita Ibeawuchi

RGS6, a Modulator of Parasympathetic Activation in Heart

Rationale: Parasympathetic regulation of heart rate is mediated by acetylcholine binding to G protein–coupled muscarinic M2 receptors, which activate heterotrimeric Gi/o proteins to promote G protein–coupled inwardly rectifying K+ (GIRK) channel activation. Regulator of G protein signaling (RGS) proteins, which function to inactivate G proteins, are indispensable for normal parasympathetic control of the heart. However, it is unclear which of the more than 20 known RGS proteins function to negatively regulate and thereby ensure normal parasympathetic control of the heart. Objective: To examine the specific contribution of RGS6 as an essential regulator of parasympathetic signaling in heart. Methods and Results: We developed RGS6 knockout mice to determine the functional impact of loss of RGS6 on parasympathetic regulation of cardiac automaticity. RGS6 exhibited a uniquely robust expression in the heart, particularly in sinoatrial and atrioventricular nodal regions. Loss of RGS6 provoked dramatically exaggerated bradycardia in response to carbachol in mice and isolated perfused hearts and significantly enhanced the effect of carbachol on inhibition of spontaneous action potential firing in sinoatrial node cells. Consistent with a role of RGS6 in G protein inactivation, RGS6-deficient atrial myocytes exhibited a significant reduction in the time course of acetylcholine-activated potassium current (IKACh) activation and deactivation, as well as the extent of IKACh desensitization. Conclusions: RGS6 is a previously unrecognized, but essential, regulator of parasympathetic activation in heart, functioning to prevent parasympathetic override and severe bradycardia. These effects likely result from actions of RGS6 as a negative regulator of G protein activation of GIRK channels.

Hypertension-causing Mutations in Cullin3 Protein Impair RhoA Protein Ubiquitination and Augment the Association with Substrate Adaptors

Background: Cullin3 ubiquitin ligase regulates protein turnover by promoting the ubiquitination of substrates. Results: Ubiquitination of RhoA is impaired by mutations in Cullin3. Conclusion: Disease-causing Cullin3 mutations impair the turnover of RhoA protein and may sequester substrates adaptors. Significance: Mutations in Cullin3 cause reduced ubiquitination and elevation of RhoA levels, which may enhance RhoA and Rho kinase signaling in a variety of cell types and could potentially contribute to hypertension. Cullin-Ring ubiquitin ligases regulate protein turnover by promoting the ubiquitination of substrate proteins, targeting them for proteasomal degradation. It has been shown previously that mutations in Cullin3 (Cul3) causing deletion of 57 amino acids encoded by exon 9 (Cul3Δ9) cause hypertension. Moreover, RhoA activity contributes to vascular constriction and hypertension. We show that ubiquitination and degradation of RhoA is dependent on Cul3 in HEK293T cells in which Cul3 expression is ablated by either siRNA or by CRISPR-Cas9 genome editing. The latter was used to generate a Cul3-null cell line (HEK293TCul3KO). When expressed in these cells, Cul3Δ9 supported reduced ubiquitin ligase activity toward RhoA compared with equivalent levels of wild-type Cul3 (Cul3WT). Consistent with its reduced activity, binding of Cul3Δ9 to the E3 ubiquitin ligase Rbx1 and neddylation of Cul3Δ9 were impaired significantly compared with Cul3WT. Conversely, Cul3Δ9 bound to substrate adaptor proteins more efficiently than Cul3WT. Cul3Δ9 also forms unstable dimers with Cul3WT, disrupting dimers of Cul3WT complexes that are required for efficient ubiquitination of some substrates. Indeed, coexpression of Cul3WT and Cul3Δ9 in HEK293TCul3KO cells resulted in a decrease in the active form of Cul3WT. We conclude that Cul3Δ9-associated ubiquitin ligase activity toward RhoA is impaired and suggest that Cul3Δ9 mutations may act dominantly by sequestering substrate adaptors and disrupting Cul3WT complexes.

Cullin-3 mutation causes arterial stiffness and hypertension through a vascular smooth muscle mechanism

Cullin-3 (CUL3) mutations (CUL3Δ9) were previously identified in hypertensive patients with pseudohypoaldosteronism type-II (PHAII), but the mechanism causing hypertension and whether this is driven by renal tubular or extratubular mechanisms remains unknown. We report that selective expression of CUL3Δ9 in smooth muscle acts by interfering with expression and function of endogenous CUL3, resulting in impaired turnover of the CUL3 substrate RhoA, increased RhoA activity, and augmented RhoA/Rho kinase signaling. This caused vascular dysfunction and increased arterial pressure under baseline conditions and a marked increase in arterial pressure, collagen deposition, and vascular stiffness in response to a subpressor dose of angiotensin II, which did not cause hypertension in control mice. Inhibition of total cullin activity increased the level of CUL3 substrates cyclin E and RhoA, and expression of CUL3Δ9 decreased the level of the active form of endogenous CUL3 in human aortic smooth muscle cells. These data indicate that selective expression of the Cul3Δ9 mutation in vascular smooth muscle phenocopies the hypertension observed in Cul3Δ9 human subjects and suggest that mutations in CUL3 cause human hypertension in part through a mechanism involving smooth muscle dysfunction initiated by a loss of CUL3-mediated degradation of RhoA.

Optimum sensor placement for source monitoring under log-normal shadowing in three dimensions

In source localization one estimates the location of a source using a variety of relative position information. Such relative position information is often provided by the received signal strength (RSS) which is in turn affected by log normal shadowing. A related issue is to place sensors around a localized source in a manner in which they can optimally monitor it. This paper considers optimal sensor placement in three dimensions so that a source can be monitored optimally from the RSS at various non-coplanar sensors. The mathematical problem becomes one of maximizing the smallest eigenvalue or the determinant of an underlying Fisher Information matrix, or minimizing the trace of the inverse of the FIM, subject to the constraint that no sensor be closer than a specified distance from the source. We show that optimality is achieved if and only if the underlying Fisher Information Matrix is a scaled diagonal, and provide methods for achieving this condition.

Optimum Sensor Placement for Source Monitoring under Log-Normal Shadowing

In source localization one estimates the location of a source using a variety of relative position information. Such relative position information is often provided by the received signal strength (RSS) which is in turn affected by log normal shadowing. A related issue is to place sensors around a localized source in a manner in which they can optimally monitor it. This paper considers optimal sensor placement in three dimensions so that a source can be monitored optimally from the RSS at various non-coplanar sensors. The mathematical problem becomes one of maximizing the smallest eigenvalue or the determinant of an underlying Fisher Information matrix, or minimizing the trace of the inverse of the FIM, subject to the constraint that no sensor be closer than a specified distance from the source. We show that optimality is achieved if and only if the underlying Fisher Information Matrix is a scaled diagonal, and provide methods for achieving this condition.

Retinol-binding protein 7 is an endothelium-specific PPARγ cofactor mediating an antioxidant response through adiponectin

Impaired PPARγ activity in endothelial cells causes oxidative stress and endothelial dysfunction which causes a predisposition to hypertension, but the identity of key PPARγ target genes that protect the endothelium remain unclear. Retinol-binding protein 7 (RBP7) is a PPARγ target gene that is essentially endothelium specific. Whereas RBP7-deficient mice exhibit normal endothelial function at baseline, they exhibit severe endothelial dysfunction in response to cardiovascular stressors, including high-fat diet and subpressor angiotensin II. Endothelial dysfunction was not due to differences in weight gain, impaired glucose homeostasis, or hepatosteatosis, but occurred through an oxidative stress-dependent mechanism which can be rescued by scavengers of superoxide. RNA sequencing revealed that RBP7 was required to mediate induction of a subset of PPARγ target genes by rosiglitazone in the endothelium including adiponectin. Adiponectin was selectively induced in the endothelium of control mice by high-fat diet and rosiglitazone, whereas RBP7 deficiency abolished this induction. Adiponectin inhibition caused endothelial dysfunction in control vessels, whereas adiponectin treatment of RBP7-deficient vessels improved endothelium-dependent relaxation and reduced oxidative stress. We conclude that RBP7 is required to mediate the protective effects of PPARγ in the endothelium through adiponectin, and RBP7 is an endothelium-specific PPARγ target and regulator of PPARγ activity.

Optimum sensor placement for localization under log-normal shadowing

In source localization one estimates the location of a source using a variety of relative position information. Such relative position information is often provided by the received signal strength (RSS) which is in turn affected by log normal shadowing. This paper considers optimal sensor placement in two dimensions so that a source can be localized optimally from the RSS at various non-collinear sensors. We assume that the source to be localized is uniformly distributed in a circle. The goal is to optimally place sensors outside a larger concentric circle, by maximizing the smallest eigenvalue or the determinant of the expectation of an underlying Fisher Information matrix, or minimizing the trace of its inverse. We show that optimality is achieved if and only if the the expected value of the FIM is a scaled identity, and provide methods for achieving this condition.

RGS6 is an essential tumor suppressor that prevents bladder carcinogenesis by promoting p53 activation and DNMT1 downregulation

Urinary bladder cancer (UBC) is largely caused by exposure to toxic chemicals including those in cigarette smoke (i.e. BBN). An activating SNP in RGS6 is associated with a pronounced reduction in UBC risk, especially among smokers. However, the mechanism underlying this reduction remains unknown. Here we demonstrate that RGS6 is robustly expressed in human urothelium, where urothelial cell carcinoma originates, and is downregulated in human UBC. Utilizing RGS6−/− mice we interrogated a possible role for RGS6 as a tumor suppressor using the BBN-induced bladder carcinogenesis model that closely recapitulates human disease. As in humans, RGS6 is robustly expressed in mouse urothelium. RGS6 loss dramatically accelerates BBN-induced bladder carcinogenesis, with RGS6−/− mice consistently displaying more advanced pathological lesions than RGS6+/+ mice. Furthermore, BBN treatment promotes urothelial RGS6 mRNA and protein downregulation. RGS6 loss impairs p53 activation and promotes aberrant accumulation of oncogenic protein DNMT1 in urothelium. Tumor suppressor RASSF1A, a DNMT1-regulated gene, is also silenced, likely via methylation of its promoter during BBN exposure. We hypothesize that this BBN-induced RGS6 loss represents a critical hit in UBC as it irrevocably impairs the anti-proliferative actions of the ATM/p53 and RASSF1A pathways. Consistent with these findings, RGS6−/− mice treated with CP-31398, a p53-stablizing agent, and/or 5-Aza, a DNMT1 inhibitor, are protected from BBN-induced tumorigenesis. Together, our data identify RGS6 as a master tumor suppressor modulating two critical signaling pathways that are often dysregulated in UBC; therefore, RGS6 represents a potential novel biomarker for UBC diagnosis/prognosis and an appealing new target in its treatment.

Source localization using a maximum likelihood/semidefinite programming hybrid

This paper considers source localization using Received Signal Strength (RSS) values at sensor locations, under the assumption of lognormal shadowing. It is known that such localization can be sensitive to path loss parameter estimates. We derive a cost function whose global minimum provides the ML estimate of the source localization. It turns out that this cost function is manifested with multiple local minima, leading to potentially poor gradient descent performance. The contribution of this paper are two fold. First, we show that in the noise free case the local minima are insensitive to the path loss parameter value. Traditional nonlinear stability theory suggests that this would imply an insensitivity of the ML algorithm to the value of the path loss parameter. Second, we propose a SDP based algorithm to initialize the ML minimization algorithm, that provides good performance, by avoiding local minima.