Jason P. DeBruyne, Ph.D. - US grants

[unreadable] DESCRIPTION (provided by applicant): The long-term goal of this proposal is to identify the components and mechanisms involved in regulating circadian patterns of clock gene expression in mammals. Mammalian circadian clock proteins are found in huge complexes, that differ in composition at various times of day. A proteomics based approach will be used to identify unknown proteins bound in these complexes, in an in vitro cellular context. Identified proteins will be evaluated for in vivo interactions to confirm the identified interactions are biologically relevant. The functions and necessity of the biologically interacting proteins within the mammalian circadian clock will then be determined. Thus, this work will identify novel circadian clock proteins, which may serve as therapeutic targets for ameliorating clock related ailments (e.g. jet lag, shift work) and more serious conditions such as circadian rhythm sleep disorders and other clock-related psychological disorders (e.g. seasonal affective disorder). [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Circadian clocks influence nearly all aspects of mammalian life, aligning our internal physiological process to optimal times of day. Understanding the molecular circuitry keeping circadian time provides insight into how the clock drives overt rhythms, and what to fix when the circadian system is disrupted (i.e. during shift work). Circadian time coded in the rhythmic regulation of clock gene expression in a negative feedback loop system. Critical to this timing system is the circadian degradation of rhythmically abundant clock proteins; however these mechanisms have remained elusive. To begin elucidating these mechanisms, we developed a new functional screening approach to identify which E3 ubiquitin ligases degrade which clock proteins and screened for E3 ligases that degrade RevErb¿/¿ proteins. Both RevErb proteins are essential for normal clock function and exhibit high amplitude abundance rhythms, but the mechanisms driving their circadian clearance are unknown. Our screen identified two novel candidate RevErb E3 ligases, Spsb4 and Siah2, and preliminary data suggest these E3s contribute directly to the rhythmic degradation of RevErb proteins. Moreover, our data suggest for the first time that the rate of circadian RevErb degradation is a determinant of circadian periodicity. Identifying the roles, mechanisms and contributions to overall clock function of Spsb4 and Siah2 is a major focus of our current research proposal. Bolstered by the fact that both hits from this screen appear to be genuine regulators of RevErb stability, the other focus of our proposal is to expand our screening efforts. Experiments proposed in Specific Aim 1 focus on elucidating the mechanisms of Siah2/Spsb4 degradation of RevErb proteins in the context of an oscillating cellular system. The experiments proposed in Specific Aim 2 delve deeper into the role of Siah2-mediated degradation of RevErb proteins in overall clock function in vivo. Experiments in Specific Aim 3 will identify candidate E3s for all of the remaining essential core clock proteins using an expanded E3 ligase cDNA screening library. Success in these aims will provide essential background and validation of our future efforts to identify protein degradation mechanisms. Overall, uncovering the novel mechanisms mediating rhythmic degradation of clock proteins will open many new avenues for treating circadian-related disorders.

Project Summary/Abstract Circadian clocks influence nearly all aspects of mammalian life, aligning our internal physiological process to optimal times of day. Understanding the molecular circuitry keeping circadian time provides insight into how the clock drives overt rhythms, and what to fix when the circadian system is disrupted (i.e. during shift work). Circadian time coded in the rhythmic regulation of ?clock gene? expression in a negative feedback loop system. Critical to this timing system is the circadian degradation of rhythmically abundant clock proteins, however these mechanisms have remained elusive. We have begun elucidating these mechanisms by developing a novel functional screening approach designed to identify which E3 ubiquitin ligases degrade which clock proteins. Screen data on three clockwork targets indicate this approach is sensitive and specific, thus operating as we hoped. A major goal of the current application is to continue to screen for, and validate, E3 ligases for other clockwork proteins as well as some key rhythmic circadian outputs. We believe identifying these E3 ligases and their roles in clock function will reveals new potential targets for therapeutic intervention of clock- related disorders. The first screen hit we have recovered, Siah2, has revealed remarkable and unexpected new insights into both clock function and how the clock regulates metabolism. We found that female mice lacking a functional Siah2 gene lost a mechanism that ?protects? against diet- induced obesity. This appears to be due to a female-specific role in the core circadian clock. These results suggest, for the first time, that female-specific clockwork mechanisms exist, and that they contribute directly to female-specific biology. Unfortunately, the vast majority of circadian data are from male mice. Therefore, we now plan to reassess clock function and circadian rhythm regulation in females to further identify and define the sex-specific clockwork mechanisms to provide a background for examining the role of Siah2 in female clocks. Understanding sex-differences in circadian clock functions will be critical as the field develops circadian-based therapeutics. !