A study on the novel regulators of proteasome

Abstract

The proteasome is a large protein complex that degrades diverse proteins in ubiquitine-proteasome system (UPS). Numerous substrates which play roles in many signaling to maintain homeostasis are known to be degraded by the complicated degradation processes. In addition, aberrant regulation in UPS and of this complex is associated with various diseases such as cancer, disorder of immune response and neurodegenerative disease. However, it is not known whether and how this elaborate machinery is regulated by diverse cellular signaling. Thus, discovery of novel proteasome regulators is important to understand UPS-associated cellular function and the pathogenesis of various diseases related to proteasome malfunction. To identify new proteasome modulators regulating the proteasome activity, a cell-based functional screening was established using Degron-GFP and a collection of cDNA library. In this study, I have isolated iRhom1 as a stimulator of proteasome activity from genome-wide functional screening using cDNA expression and an unstable GFP-degron. Expression level of iRhom1 regulated enzymatic activity and assembly of proteasome complexes. iRhom1 expression was induced by endoplasmic reticulum (ER) stressors, leading to the enhancement of proteasome activity, especially in ER-containing microsomes. iRhom1 interacted with PAC1 and PAC2, the 20S proteasome assembly chaperones, affecting their protein stability by dimerization of them. In addition, iRhom1 deficiency in D. melanogaster accelerated the rough-eye phenotype of mutant Huntingtin, while transgenic flies expressing either human iRhom1 or Drosophila iRhom showed rescue of the rough-eye phenotype. S5b was previously identified as a proteasome-assembly chaperone in yeast and a negative regulator of 26S proteasome in mammalian. Although regulation of GRK2 is considered as one of cell death mediators in neuronal cells, the regulation of GRK2 expression is not known. Here, I show that GRK2 is regulated by S5b in neuronal cells and mouse model. GRK2 is down-regulated in the cortex and hippocampus of S5b transgenic mice, a chronic inflammation model and also reduced by S5b expression in HT22 mouse hippocampal cells. Conversely, knockdown of S5b expression increases GRK2 level through increasing the stability of GRK2 protein, independent of its ability to impair proteasome activity. GRK2 and GRK2 K220R, a kinase dead mutant, similarly interacts with S5b in the mouse cortex and HT22 cells through its C-terminal domain, and this domain also decreases GRK2 level. Membrane targeting of GRK2 is affected by S5b expression, as assessed with immunocytochemistry, fractionation, and surface biotinylation assays. In addition, neurotoxic effect of S5b is suppressed by overexpression of GRK2 but not by GRK2 K220R. Thus, S5b may exert its toxic effect through down-regulation of GRK2, a neurotoxic mediator, in neuronal cells, showing an aberrant role of S5b as a negative regulator of GRK2 in neuronal cell death. In addition, Psmd5/S5b knockout mouse was successfully generated by the Cas9/CRISPR-mediated Psmd5/S5b knockout cassette and show enhanced proteasome activity compared to aged matched littermates. Together, S5b plays a diverse role in the regulation of proteasome activity under pathologic condition and in neuronal cell death through GRK2. In conclusion, I suggest a novel stress signaling pathway responsible for proteasome regulation and critical role of S5b in neuronal cell death independent of its inhibitory function of proteasome.