Pispa, Johanna et al. (2021) International Worm Meeting "Identification of a potential regulator of proteasome nuclear localisation"

Pispa, Johanna, Mikkonen, Elisa, Arpalahti, Leena, Holmberg, Carina I

[International Worm Meeting, 2021]

The proteasome is the major enzymatic machinery of the ubiquitin-proteasome system (UPS), which is required for degradation of misfolded, aged and unnecessary proteins. It is a multisubunit complex, consisting of a core particle, 20S, capped by regulatory particles, 19S. The barrel-shaped 20S particle is responsible for the proteolytic activity, whereas the 19S particles bind polypeptide substrates via polyubiquitin chains, remove the ubiquitins for recycling, and unfold the polypeptides prior to transferring them into the lumen of the core particle. Recent studies by us and others have shown that the proteasome's expression and activity are differentially regulated in specific spatial, temporal and physiological contexts in vivo to facilitate its essential function in maintenance of cellular proteostasis. Subcellularly, the proteasome is localised both to the cytoplasm and in the nucleus. The nuclear role of the proteasome is not fully understood, but it may involve also functions not related to its proteolytic activity. In order to discover new regulators of the proteasome, we have performed a genome-wide C. elegans RNAi screen using a transgenic intestinal polyubiquitin-binding reporter strain previously published by our laboratory. This fluorescent reporter has been shown to respond to changes in the endogenous amount of polyubiquitinated proteins, and can be used as a tool for identifying disturbances in protein degradation mechanisms in live animals. After exclusion of hits affecting protein synthesis or having a developmental phenotype, we obtained 33 potential candidate genes. Of these, downregulation of 19 hits increased the fluorescence level of the polyubiquitin-binding reporter animals whereas 14 hits decreased the fluorescence. Particularly, one RNAi clone stood out as it not only resulted in increased reporter fluorescence but also changed the reporter's subcellular localisation from being expressed throughout the intestinal cell into a predominantly nuclear location. Immunostaining for proteasome 20S subunits showed that the intestinal nuclei have reduced amount of the proteasome, which could cause the accumulation of polyubiquitinated substrates. Further characterization of the potential regulator will be presented.

Matilainen, Olli et al. (2013) International Worm Meeting "Insulin/IGF-1 Signaling Regulates Proteasome Activity through the Deubiquitinating Enzyme UBH-4."

Matilainen, Olli, Holmberg, Carina I, Hautaniemi, Sampsa, Arpalahti, Leena, Rantanen, Ville

[International Worm Meeting, 2013]

The proteasome executes most of the controlled protein degradation in the cell. It has important role in regulation of protein homeostasis and dysfunctions in proteasomal degradation have been linked to many severe disorders, including several neurodegenerative diseases and cancers. Although the importance of the proteasome for cellular viability is well recognized, it is still not known how proteasome activity is regulated in a multicellular organism. Insulin/IGF-1 signaling (IIS) is a well-characterized signaling pathway that regulates e.g. protein homeostasis and lifespan in many organisms. Here we have studied if IIS regulates proteasome activity by using in vitro assays, our previously established in vivo photoconvertible UPS activity reporter system as well as our novel in vivo polyubiquitin reporter. Our results show that reduced IIS enhances proteasome activity in a DAF-16 dependent manner. Furthermore, we demonstrate that DAF-16 represses expression of the proteasome-associated deubiquitinating enzyme ubh-4, which we propose to function as a tissue-specific proteasome inhibitor and a modulator of lifespan. The role of UBH-4 appears to be well-conserved, as downregulation of its mammalian ortholog, UCHL-5, increases proteasome activity and degradation of proteotoxic proteins in mammalian cells. Altogether, our results establish a novel molecular mechanism linking IIS to proteasome activity and add one more branch to IIS mediated maintenance of cellular protein homeostasis.