|M.Sc Student||Bishara Jeeda|
|Subject||Characterization of the Ribosome-Associated Chaperone NAC|
Regulation in Response to Aggregation in Mammalian
|Department||Department of Medicine||Supervisor||Dr. Reut Shalgi|
|Full Thesis text|
Nascent-chain associated complex (NAC) is considered to be the first ribosome associated chaperone to contact nascent peptides. NAC is a heterodimeric complex consisting of two subunits, α-NAC and β-NAC. Importantly, it has been suggested to affect protein homeostasis during stress, as well as to have different roles outside the ribosome. Additionally, the NACA2 protein is an α-NAC homolog in higher eukaryotes, however, its function has not yet been deciphered. Here, we aim to understand the regulation of the NAC complex during proteotoxicity in human cells. Specifically, we identify changes in NAC subunits interactome, their levels and localization in response to proteotoxic stress and in disease condition, particularly in the presence of aggregated proteins.
Our results show that human NACA1, NACA2, both belong to the α-NAC family, and BTF3, which belongs to the β-NAC family, localize to the ribosomal fraction. Upon expression of mutated Huntingtin (HTT134Q), in line with previous reports, we observed that these subunits also localize to the HTT134Q protein aggregates, using several methodologies. Interestingly, we found consistent upregulation in the total amounts of all the three NAC complex subunits following expression of HTT134Q aggregate. Trying to characterize the mechanism by which NAC levels are upregulated, we found that they are not induced at the mRNA level following transfection with HTT134Q, but rather at the protein level. Additionally, we revealed that NAC components are regulated by the HECT- type E3 ligase TRIP12. Interestingly, we found that TRIP12 localizes to the insoluble HTT aggregates. Our model speculates that this localization leads to NAC upregulation during HTT aggregation conditions.
We further characterized chaperone interaction partners of each of the NAC complex members using the LUMIER assay, in ribosome preserving vs. disrupting conditions. Our results reveal several chaperones that have specific exclusive interactions with NACA2. This finding suggests specific functions for this family member that may be distinct from the traditional NAC complex. In addition, specific chaperones have ribosome-independent interactions with both BTF3 and NACA1. Moreover, we employed IP-MS to explore the interactome of BTF3 and discovered novel interactions with RNA binding proteins outside the ribosome context.
Finally, to study potential functions of the novel NACA2 subunit, we studied the effects of NACA2 ectopic expression on cell viability by performing phenotypic stress. Our results showed that NACA2 protected cells against several proteotoxic stresses including oxidative and osmotic stresses.
In summary, our work has shown that the NAC complex expression is modulated in response to protein aggregation in mammalian cells, both at their levels and localization. Furthermore, we have characterized the chaperone interactome of the NAC complex, and found that NACA2 is distinct from the other classical members, suggesting a unique role for this family member. Our ongoing studies on regulation of the complex during stress, as well as functional studies on NACA2 modulations would reveal more broadly the regulation of the complex under proteotoxic stress, and unravel potential roles for NACA2.