Thorsten Hoppe

CECAD - Cluster of Excellence; Cologne, Germany University of Cologne; Cologne, Germany

Hoppe, Thorsten et al. (2013) International Worm Meeting "A Novel Link between Ubiquitin-dependent Proteolysis and Mitochondrial Metabolism."

Segref, Alexandra, Hoppe, Thorsten

[International Worm Meeting, 2013]

The ubiquitin/proteasome system (UPS) is pivotal for the elimination of damaged or regulatory proteins, and plays a crucial role in development and tissue functionality. Substrate ubiquitylation is mediated by an enzymatic cascade that involves ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2), and ubiquitin protein ligases (E3). Chains of four to six ubiquitin moieties linked via K48 of ubiquitin usually promote degradation of modified substrates by the 26S proteasome. Using green fluorescent protein (GFP)-based model substrates, we have established an in vivo degradation assay that allows rapid quantification of ubiquitin-dependent proteolysis in Caenorhabditis elegans. We used this in vivo degradation assay to delineate the physiological relevance of ubiquitin-mediated protein turnover in multicellular organisms. Beside E2 and E3 enzymes or regulators of the 26S proteasome, our main goal was to identify proteolytic factors with novel mechanistic and tissue specific functions. Therefore, we performed forward genetic screens for mutants that are defective in the degradation of GFP-based ubiquitin fusion proteins. Surprisingly, we identified mitochondrial mutants that strongly stabilize different model substrates. The corresponding genes encode the acetyl-CoA synthetase ACS-19 and the isovaleryl-CoA dehydrogenase IVD-1, which are both involved in acetyl-CoA metabolism. Substrate stabilization can be suppressed by adding the antioxidant N-acetyl cysteine (NAC), suggesting enhanced oxidative and/or metabolic stress. Additional data indicate that general defects in mitochondrial respiration interfere with the UPS. Recessive mutations in the human homolog of IVD-1 cause errors of leucine metabolism and accumulate isovaleryl-CoA derivatives linked to morbidity and mortality of the patients. Intriguingly, a related model substrate is also stabilized in patient cell lines lacking IVD-1, supporting the idea that defects in ubiquitin-mediated proteolysis might be fundamental to human mitochondrial pathology. In summary, our work offers intriguingly new mechanistic insights how ubiquitin-dependent proteolysis is fine-tuned to maintain the cellular and organismal physiology.

Thorsten Hoppe et al. (2002) European Worm Meeting "Characterisation of the Multiubiquitin Chain Assembly Factor (E4) in Caenorhabditis elegans"

Ralf Baumeister, Bianca Sperl, Thorsten Hoppe

[European Worm Meeting, 2002]

Proteins modified by multiubiquitin chains are the preferred substrates of the proteasome. Ubiquitination involves a ubiquitin-activating enzyme, E1, a ubiquitin-conjugating enzyme, E2, and often a substrate-specific ubiquitin-protein ligase, E3. In Saccharomyces cerevisiae it was shown that efficient multiubiquitination needed for proteasomal targeting of a model substrate requires an additional conjugation factor, named E4. This protein, previously known as UFD2 in yeast, binds to the ubiquitin moieties of preformed conjugates and catalyzes ubiquitin chain assembly in conjunction with E1, E2, and E3. Intriguingly, E4 defines a novel protein family that includes homologs in human, Dictyostelium, fission yeast and one homolog in C. elegans. We are interested in the function of the E4 enzyme in multicellular organisms and therefore studying the function of the C. elegans homolog of UFD2, T05H10.5. First we recombinantly expressed and purified T05H10.5. Using an in vitro ubiquitination assay, we were able to prove the binding of T05H10.5 to a ubiquitinated model substrate and its multiubiquitination, as it was previously shown for yeast UFD2. Furthermore we analysed the expression pattern of T05H10.5 in transgenic animals using GFP fusion proteins driven by the endogenous promoter. Interestingly, T05H10.5::GFP is expressed in all neurons of the worm, in some muscle cells of the pharynx and somatic muscle cells and is localised predominantly in the nucleus. Comparing early embryos and different developmental stages of the animals indicates that there might be a stage specific redistribution of T05H10.5 from the cytosol to the nucleus. Currently we are testing candidate interaction partners that might be involved in this regulated localisation by two-hybrid and RNAi analysis. In order to continue the analysis of the function of this E4 enzyme, we generated a deletion mutant of the entire corresponding gene and which we are currently phenotyping.

Thorsten Hoppe et al. (2003) International Worm Meeting "UBO-1 and UBO-2 form a functional complex regulating multiubiquitination of a specific substrate"

Thorsten Hoppe, Bianca Sperl, Ralf Baumeister

[International Worm Meeting, 2003]

Proteins modified by multiubiquitin chains are the preferred substrates of the proteasome. Ubiquitination involves a ubiquitin-activating enzyme, E1, a ubiquitin-conjugating enzyme, E2, and often a substrate-specific ubiquitin-protein ligase, E3. In Saccharomyces cerevisiae, efficient multiubiquitination needed for proteasomal targeting of a model substrate requires an additional conjugation factor, named E4. This protein, previously known as UFD2 in yeast, binds to the ubiquitin moieties of preformed conjugates and catalyzes ubiquitin chain assembly in conjunction with E1, E2, and E3. Intriguingly, E4 defines a novel protein family that includes homologs in human, Dictyostelium, fission yeast and in C. elegans. We are interested in the function of the E4 enzyme in multicellular organisms and therefore studying the function of the C. elegans homolog of UFD2, which we named UBO-2 (U-box domain protein-2). First we recombinantly expressed and purified UBO-2. Using an in vitro ubiquitination assay, we verified the binding of UBO-2 to a ubiquitinated model substrate as it was previously shown for yeast UFD2. Additionally UBO-2 is able to selfubiquitinate in vitro and therefore its C-terminal U-box domain is required. Furthermore we analyzed the expression pattern of ubo-2 in transgenic animals using GFP fusion proteins driven by the endogenous promoter. Interestingly, UBO-2::GFP is expressed in all neurons of the worm, in some muscle cells of the pharynx and somatic muscle cells and is localized predominantly in the nucleus. Using the two-hybrid system we identified one specific binding partner. Interestingly, this interactor also bears a U-box domain like UBO-2; therefore we named it UBO-1 (U-box domain protein-1). UBO-1 shows selfubiquitination in vitro and thereby collaborates with the same E2-enzyme like UBO-2. In further biochemical studies we identified that both enzymes function in the same pathway and we identified one specific substrate. UBO-1 and UBO-2 form a trimeric complex with this substrate resulting in its multiubiquitination. To continue the analysis of both enzymes genetically, we generated deletion mutants of ubo-1 and ubo-2. We will present biochemical results including the in vitro ubiquitination assay and an epistasis analysis between our deletion mutants and conditional alleles of the substrate gene. Our data define a novel pathway and a novel mechanism involved in protein turnover during development.

WBAntibody00003045

anti-CDC-48.1 (Hoppe-lab/Biogenes Berlin, custom antibody, 1:50,000) cdc-48.1

Ravanelli, Sonia et al. (2021) International Worm Meeting "Role of branched chain amino acid metabolism in ubiquitin-dependent proteolysis"

Ravanelli, Sonia, Hoppe, Thorsten

[International Worm Meeting, 2021]

The role of ubiquitin in mitochondrial surveillance is increasingly gaining attention with numerous stress pathways being recently described. Our group observed that mitochondrial stress affects ubiquitin-dependent proteolysis both in C. elegans as well as in mammalian cells. We performed a candidate screen and identified that metabolic defects could result in reduced UPS functionality independently of the mitochondrial unfolded protein response (UPRmt). Especially defects in the catabolism of branched chain amino acids (BCAAs) affects the ubiquitin-dependent turnover of a GFP-based model substrate in C. elegans. Surprisingly, a newly generated mutant of the enzyme responsible for the first step of the BCAA catabolism, bcat-1 (hh58), rescues this defect. We have indications that this mutation impairs mitochondrial import of BCAT-1, resulting in increased levels of this enzyme in the cytosol, where, its mammalian homolog BCAT1 is reported to be localized, in contrast to the other homolog BCAT2, which localizes in mitochondria. Through multiple omics analysis, we explored the general transcriptional, translational and metabolic regulation deriving from BCAA defects in combination with mislocalization of BCAT-1. Considering that balanced regulation of proteolysis and metabolism is crucial for organismal health, understanding the mechanisms underlying bcat-1 (hh58) increased proteolytic capacity might represent a novel starting point for the development of new therapeutic strategies or nutritional guidelines to treat metabolic disorders as well as neurodegenerative diseases.

Finger, Fabian et al. (2015) International Worm Meeting "Regulation of protein homeostasis by microRNAs in Caenorhabditis elegans."

Finger, Fabian, Hoppe, Thorsten

[International Worm Meeting, 2015]

Maintenance of a functional set of proteins (proteome) is a highly dynamic process and crucial for any organism in terms of longevity and viability. A sophisticated machinery fulfills these demands in order to establish protein homeostasis (proteostasis) but its functionality declines with age. This machinery involves transcription, translation, protein folding, and degradation pathways, such as autophagy and the ubiquitin proteasome system (UPS). In this context, microRNAs, a class of short non-coding RNAs, might represent an elegant mechanism to control proteostasis. microRNAs are implicated in diverse biological processes including longevity; however, their influence on proteostasis mechanisms is not sufficiently investigated.Therefore, we started RNAi experiments inhibiting the biogenesis of microRNAs via knockdown of the essential factor PASH-1 and the argonaute proteins ALG-1 and ALG-2, respectively. Strikingly, downregulation of microRNA biogenesis results in a functional impairment of various proteostasis mechanisms, as indicated by reporter strains. Here, we especially made use of a ubiquitin fusion degradation (UFD) substrate, which is represented by a noncleavable Ubiquitin-GFP fusion protein. Accumulation of this substrate is easily detectable and strongly implicates malfunction of the UPS. In addition, ER-associated degradation (ERAD) and the unfolded protein response in the ER (UPRER) are also negatively affected upon downregulation of microRNA biogenesis. In order to identify microRNAs important for functional proteostasis, as well as facilitating downstream factors, we used a comparative approach that includes microRNA sequencing, microarray analysis, and microRNA target prediction algorithms. The comparison between unstressed and proteotoxic stress conditions allowed us to discover microRNAs involved in proteostasis mechanisms. Moreover, we are able to distinguish microRNAs with a broad influence from those affecting only specific pathways of the proteostasis machinery. Taken into account that regulatory processes can occur either cell-autonomously or cell-nonautonomously, we also tested tissue-specificity with particular attention to respective expression profiles.Our current observations suggest that we are able to identify microRNA families, as well as individual microRNAs that regulate diverse proteostasis mechanisms which might be the underlying cause of a functional impaired proteome influencing protein aggregation diseases and aging.

BR2823

Caenorhabditis elegans

Finger, Fabian et al. MicroPubl Biol

Finger, Fabian, Ottens, Franziska, Hoppe, Thorsten

[MicroPubl Biol, 2021]

The conserved Argonaute-family members ALG-1 and ALG-2 are known to regulate processing and maturation of microRNAs to target mRNAs for degradation or translational inhibition (Bouasker and Simard 2012; Meister 2013). Consequently, depletion of alg-1 and alg-2 results in multiple phenotypes. Our data describe a role of microRNA-regulation in stress resistance and proteostasis with special emphasis on ubiquitin-dependent degradation pathways, such as ubiquitin fusion degradation (UFD) and endoplasmic reticulum (ER)-associated protein degradation (ERAD).

EE86

Caenorhabditis elegans