In Vitro Studies of Steriodogenic Factor-1 Sumoylation, a Post-translational Modification Critical for Adrenal and Gonadal Development

Steroidogenic Factor-1 (SF-1) is a constitutively active nuclear hormone receptor, thus its post-translational modifications play an even more important role in its regulation. SF-1 is sumoylated at two sites, K119 and K194 of which the former has been shown to determine SF-1 binding specificity to sumo-sensitive response elements. The basic enzymology behind sumoylation of SF-1 K119 sumoylation is not fully understood nor optimized with the reaction taking well over 8 hours while still not reaching completion. Similarly, sensitive visualization assays are not currently available in order to facilitate kinetic studies of this in vitro reaction. This graduate work focuses on sumoylation of the SF-1 DBD with the goal of developing a more robust and sensitive in vitro system in order to assay new potential SF-1 binding sequences and apply the revamped protocol to other sumoylation substrates. Our work demonstrates that the rate-limiting step of the enzymatic reaction is the loading of the E2 enzyme, Ubc9, and pre-loading this enzyme prior to incubation with substrate dramatically enhances the rate of sumoylation. Finally, we attempt to determine how the putative E3 ligase, PIASγ, functions to enhance SF-1 sumoylation in vitro

Intracellular Targeting Signals Contribute to Localization of Coronavirus Spike Proteins near the Virus Assembly Site

Coronavirus budding at the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) requires accumulation of the viral envelope proteins at this point in the secretory pathway. Here we demonstrate that the spike (S) protein from the group 3 coronavirus infectious bronchitis virus (IBV) contains a canonical dilysine endoplasmic reticulum retrieval signal (-KKXX-COOH) in its cytoplasmic tail. This signal can retain a chimeric reporter protein in the ERGIC and when mutated allows transport of the full-length S protein as well as the chimera to the plasma membrane. Interestingly, the IBV S protein also contains a tyrosine-based endocytosis signal in its cytoplasmic tail, suggesting that any S protein that escapes the ERGIC will be rapidly endocytosed when it reaches the plasma membrane. We also identified a novel dibasic motif (-KXHXX-COOH) in the cytoplasmic tails of S proteins from group 1 coronaviruses and from the newly identified coronavirus implicated in severe acute respiratory syndrome. This dibasic motif also retained a reporter protein in the ERGIC, similar to the dilysine motif in IBV S. The cytoplasmic tails of S proteins from group 2 coronaviruses lack an intracellular localization signal. The inherent differences in S-protein trafficking could point to interesting variations in pathogenesis of coronaviruses, since increased levels of surface S protein could promote syncytium formation and direct cell-to-cell spread of the infection

Barth Syndrome: Psychosocial Impact and Quality of Life Assessment

Background: Barth syndrome (BTHS) is a rare X-linked genetic disease that affects multiple systems and leads to complex clinical manifestations. Although a considerable amount of research has focused on the physical aspects of the disease, less has focused on the psychosocial impact and quality of life (QoL) in BTHS. Methods: The current study investigated caregiver- (n = 10) and self-reported (n = 16) psychological well-being and QoL in a cohort of BTHS-affected patients and families. Participants completed the depression and anxiety components of the Patient-Reported Outcomes Information System (PROMIS) Short Form 8A and Health-related quality of life (HRQoL) surveys at enrollment and again during a follow-up period ranging from 6 to 36 months after baseline. Results: Quality of life changed significantly over time and the various domains with some improvement and some decline. Among the available caregiver-patient dyad data, there was a trend toward discordance between caregiver and self-reported outcomes. Most notably, patients reported improvement in HRQoL, while caregivers reported declines. This suggests that there may be differences in perceived quality of life between the patients and parents, though our study is limited by small sample size. Conclusion: Our study provides valuable insights into the impacts of psychosocial and mental health aspects of BTHS. Implications of these findings include incorporating longitudinal assessment of QoL and screening for psychological symptoms in BTHS care to identify interventions that may drastically impact health status and the course of the disease