ROLE OF CAVEOLIN-3 IN THE MODULATION OF HERG POTASSIUM CHANNEL

The human ether-a-go-go-related gene (hERG) encodes the pore-forming subunit of the rapidly activating delayed rectifier potassium channel (IKr) that is important for cardiac repolarization. Previously, we have discovered that hERG channels rapidly internalize in low extracellular K+ ([K+]o). In cell culture, this process is driven by the endocytic protein, caveolin-1 (Cav1), which is an integral player in the caveolae-dependant endocytosis pathway. However, in the heart, Caveolin-3 (Cav3) is, in fact, the predominant form in the myocyte, and thus may play a direct role in regulating hERG expression in the heart. Thus, I hypothesize that this reduction of hERG conductance in cardiac myocytes derives from the presence of Cav3, which is integral regulator of hERG homeostasis innately in the heart. To investigate the effect of Cav3 on hERG, I overexpressed Cav3 in human embryonic kidney 293 (HEK-293) cells stably expressing hERG channels. Cav3 overexpression significantly and specifically decreased both the hERG current amplitude and the mature channel expression in normal culture conditions. Co-immunoprecipitation analysis and confocal imaging demonstrated an association between hERG and Cav3 in HEK cells as well as rat and rabbit cardiomyocytes. Mechanistically, I discovered that Cav3 possesses a faster turnover rate compared to Cav1, and can enhance hERG degradation through up-regulating mature channel ubiquitination via the ubiquitin ligase, NEDD4-2. Knockdown of Cav3 in neonatal cardiac myocytes also enhanced hERG expression. My data indicate that Cav3 participates in hERG trafficking, and is an important regulator of hERG channel homeostasis in cardiac myocytes. This information provides a platform for future intervention of the hERG-induced type-2 long QT syndrome (LQTS).

THE HERPESVIRUS NUCLEAR EGRESS COMPLEX COMPONENT, UL31, IS RECRUITED TO SITES OF DNA DAMAGE THROUGH POLY(ADP-RIBOSE) BINDING

The herpes simplex virus (HSV) UL31 gene encodes a conserved member of the herpesvirus nuclear egress complex that not only functions in the egress of DNA-containing capsids from the nucleus, but is also required for optimal viral genome expression, replication and packaging into capsids. Here, we report that the UL31 protein from HSV-2 and the orthologous protein, ORF69, from Kaposi's sarcoma-associated herpesvirus (KSHV) are recruited to sites of DNA damage. Recruitment of UL31 to sites of DNA damage occurred in HSV-2 infected cells, but did not require other viral proteins. The N-terminus of UL31 contains sequences resembling a poly(ADP-ribose) (PAR) binding motif. As protein poly-ADP ribosylation (PARylation) is a hallmark of the DNA damage response we examined the relationship between PARylation and UL31 recruitment to DNA damage. While the PAR polymerase (PARP)1/2 inhibitor, olaparib, prevented UL31 recruitment to damaged DNA, KU55933 inhibition of signaling through the ataxia telangiectasia mutated (ATM) DNA damage response pathway had no effect. These findings were further supported by experiments demonstrating direct and specific interaction between HSV-2 UL31 and PAR using purified components. Co-transfection with the viral kinase Us3, known to phosphorylate UL31, inhibited UL31 recruitment to DNA damage but also prevented the recruitment of other proteins recruited to DNA damage sites. The viral E3 ubiquitin ligase ICP0 was observed to co-localize with UL31 in transfected cells in a manner that is independent of the PAR-binding ability of UL31. However, inhibition of PARP1/2/3 did not reduce the ability of HSV-2 to replicate and we observed reduced PAR levels in the nuclei of infected cells. This study reveals a previously unrecognized function for UL31 orthologs and may suggest that the recognition of PAR by UL31 is coupled to the nuclear egress of herpesvirus capsids, influences viral DNA replication and packaging, or possibly modulates the DNA damage response mounted by virally infected cells.