Tissue-Specific Impact of Autophagy Genes on the Ubiquitin–Proteasome System in C. elegans

The ubiquitin–proteasome system (UPS) and the autophagy–lysosomal pathway (ALP) are the two main eukaryotic intracellular proteolytic systems involved in maintaining proteostasis. Several studies have reported on the interplay between the UPS and ALP, however it remains largely unknown how compromised autophagy affects UPS function in vivo. Here, we have studied the crosstalk between the UPS and ALP by investigating the tissue-specific effect of autophagy genes on the UPS at an organismal level. Using transgenic Caenorhabditis elegans expressing fluorescent UPS reporters, we show that the downregulation of the autophagy genes lgg-1 and lgg-2 (ATG8/LC3/GABARAP), bec-1 (BECLIN1), atg-7 (ATG7) and epg-5 (mEPG5) by RNAi decreases proteasomal degradation, concomitant with the accumulation of polyubiquitinated proteasomal substrates in a tissue-specific manner. For some of these genes, the changes in proteasomal degradation occur without a detectable alteration in proteasome tissue expression levels. In addition, the lgg-1 RNAi-induced reduction in proteasome activity in intestinal cells is not dependent on sqst-1/p62 accumulation. Our results illustrate that compromised autophagy can affect UPS in a tissue-specific manner, and demonstrate that UPS does not function as a direct compensatory mechanism in an animal. Further, a more profound understanding of the multilayered crosstalk between UPS and ALP can facilitate the development of therapeutic options for various disorders linked to dysfunction in proteostasis

Tissue-Specific Impact of Autophagy Genes on the Ubiquitin–Proteasome System in C. elegans

The ubiquitin–proteasome system (UPS) and the autophagy–lysosomal pathway (ALP) are the two main eukaryotic intracellular proteolytic systems involved in maintaining proteostasis. Several studies have reported on the interplay between the UPS and ALP, however it remains largely unknown how compromised autophagy affects UPS function in vivo. Here, we have studied the crosstalk between the UPS and ALP by investigating the tissue-specific effect of autophagy genes on the UPS at an organismal level. Using transgenic Caenorhabditis elegans expressing fluorescent UPS reporters, we show that the downregulation of the autophagy genes lgg-1 and lgg-2 (ATG8/LC3/GABARAP), bec-1 (BECLIN1), atg-7 (ATG7) and epg-5 (mEPG5) by RNAi decreases proteasomal degradation, concomitant with the accumulation of polyubiquitinated proteasomal substrates in a tissue-specific manner. For some of these genes, the changes in proteasomal degradation occur without a detectable alteration in proteasome tissue expression levels. In addition, the lgg-1 RNAi-induced reduction in proteasome activity in intestinal cells is not dependent on sqst-1/p62 accumulation. Our results illustrate that compromised autophagy can affect UPS in a tissue-specific manner, and demonstrate that UPS does not function as a direct compensatory mechanism in an animal. Further, a more profound understanding of the multilayered crosstalk between UPS and ALP can facilitate the development of therapeutic options for various disorders linked to dysfunction in proteostasis

Tissue-Specific Impact of Autophagy Genes on the Ubiquitin–Proteasome System in C. elegans

The ubiquitin–proteasome system (UPS) and the autophagy–lysosomal pathway (ALP) are the two main eukaryotic intracellular proteolytic systems involved in maintaining proteostasis. Several studies have reported on the interplay between the UPS and ALP, however it remains largely unknown how compromised autophagy affects UPS function in vivo. Here, we have studied the crosstalk between the UPS and ALP by investigating the tissue-specific effect of autophagy genes on the UPS at an organismal level. Using transgenic Caenorhabditis elegans expressing fluorescent UPS reporters, we show that the downregulation of the autophagy genes lgg-1 and lgg-2 (ATG8/LC3/GABARAP), bec-1 (BECLIN1), atg-7 (ATG7) and epg-5 (mEPG5) by RNAi decreases proteasomal degradation, concomitant with the accumulation of polyubiquitinated proteasomal substrates in a tissue-specific manner. For some of these genes, the changes in proteasomal degradation occur without a detectable alteration in proteasome tissue expression levels. In addition, the lgg-1 RNAi-induced reduction in proteasome activity in intestinal cells is not dependent on sqst-1/p62 accumulation. Our results illustrate that compromised autophagy can affect UPS in a tissue-specific manner, and demonstrate that UPS does not function as a direct compensatory mechanism in an animal. Further, a more profound understanding of the multilayered crosstalk between UPS and ALP can facilitate the development of therapeutic options for various disorders linked to dysfunction in proteostasis.The ubiquitin–proteasome system (UPS) and the autophagy–lysosomal pathway (ALP) are the two main eukaryotic intracellular proteolytic systems involved in maintaining proteostasis. Several studies have reported on the interplay between the UPS and ALP, however it remains largely unknown how compromised autophagy affects UPS function in vivo. Here, we have studied the crosstalk between the UPS and ALP by investigating the tissue-specific effect of autophagy genes on the UPS at an organismal level. Using transgenic Caenorhabditis elegans expressing fluorescent UPS reporters, we show that the downregulation of the autophagy genes lgg-1 and lgg-2 (ATG8/LC3/GABARAP), bec-1 (BECLIN1), atg-7 (ATG7) and epg-5 (mEPG5) by RNAi decreases proteasomal degradation, concomitant with the accumulation of polyubiquitinated proteasomal substrates in a tissue-specific manner. For some of these genes, the changes in proteasomal degradation occur without a detectable alteration in proteasome tissue expression levels. In addition, the lgg-1 RNAi-induced reduction in proteasome activity in intestinal cells is not dependent on sqst-1/p62 accumulation. Our results illustrate that compromised autophagy can affect UPS in a tissue-specific manner, and demonstrate that UPS does not function as a direct compensatory mechanism in an animal. Further, a more profound understanding of the multilayered crosstalk between UPS and ALP can facilitate the development of therapeutic options for various disorders linked to dysfunction in proteostasis.Peer reviewe

Immunohistochemical analysis reveals variations in proteasome tissue expression in C. elegans

The ubiquitin-proteasome system (UPS) plays a crucial part in normal cell function by mediating intracellular protein clearance. We have previously shown that UPS-mediated protein degradation varies in a cell type-specific manner in C. elegans. Here, we use formalin-fixed, paraffin-embedded C. elegans sections to enable studies on endogenous proteasome tissue expression. We show that the proteasome immunoreactivity pattern differs between cell types and within subcellular compartments in adult wild-type (N2) C. elegans. Interestingly, widespread knockdown of proteasome subunits by RNAi results in tissue-specific changes in proteasome expression instead of a uniform response. In addition, long-lived daf-2 (e1370) mutants with impaired insulin/IGF-1 signaling (IIS) display similar proteasome tissue expression as aged-matched wild-type animals. Our study emphasizes the importance of alternate approaches to the commonly used whole animal lysate-based methods to detect changes in proteasome expression occurring at the sub-cellular, cell or tissue resolution level in a multicellular organism.Peer reviewe

Tissue-specific effects of temperature on proteasome function

Variation in ambient growth temperature can cause changes in normal animal physiology and cellular functions such as control of protein homeostasis. A key mechanism for maintaining proteostasis is the selective degradation of polyubiquitinated proteins, mediated by the ubiquitin-proteasome system (UPS). It is still largely unsolved how temperature changes affect the UPS at the organismal level. Caenorhabditis elegans nematodes are normally bred at 20 °C, but for some experimental conditions, 25 °C is often used. We studied the effect of 25 °C on C. elegans UPS by measuring proteasome activity and polyubiquitinated proteins both in vitro in whole animal lysates and in vivo in tissue-specific transgenic reporter strains. Our results show that an ambient temperature shift from 20 to 25 °C increases the UPS activity in the intestine, but not in the body wall muscle tissue, where a concomitant accumulation of polyubiquitinated proteins occurs. These changes in the UPS activity and levels of polyubiquitinated proteins were not detectable in whole animal lysates. The exposure of transgenic animals to 25 °C also induced ER stress reporter fluorescence, but not the fluorescence of a heat shock responsive reporter, albeit detection of a mild induction in hsp-16.2 mRNA levels. In conclusion, C. elegans exhibits tissue-specific responses of the UPS as an organismal strategy to cope with a rise in ambient temperature.Peer reviewe

C. elegans dss-1 is functionally conserved and required for oogenesis and larval growth

<p>Abstract</p> <p>Background</p> <p>Dss1 (or Rpn15) is a recently identified subunit of the 26S proteasome regulatory particle. In addition to its function in the protein degradation machinery, it has been linked to BRCA2 (breast cancer susceptibility gene 2 product) and homologous DNA recombination, mRNA export, and exocytosis. While the fungal orthologues of Dss1 are not essential for viability, the significance of Dss1 in metazoans has remained unknown due to a lack of knockout animal models.</p> <p>Results</p> <p>In the current study deletion of <it>dss-1 </it>was studied in <it>Caenorhabditis elegans </it>with a <it>dss-1 </it>loss-of-function mutant and <it>dss-1 </it>directed RNAi. The analysis revealed an essential role for <it>dss-1 </it>in oogenesis. In addition, <it>dss-1 </it>RNAi caused embryonic lethality and larval arrest, presumably due to loss of the <it>dss-1 </it>mRNA maternal contribution. DSS-1::GFP fusion protein localised primarily in the nucleus. No apparent effect on proteasome function was found in <it>dss-1 </it>RNAi treated worms. However, expression of the <it>C. elegans dss-1 </it>in yeast cells deleted for its orthologue <it>SEM1 </it>rescued their temperature-sensitive growth phenotype, and partially rescued the accumulation of polyubiquitinated proteins in these cells.</p> <p>Conclusion</p> <p>The first knockout animal model for the gene encoding the proteasome subunit DSS-1/Rpn15/Sem1 is characterised in this study. In contrast to unicellular eukaryotes, the <it>C. elegans dss-1 </it>encodes an essential protein, which is required for embryogenesis, larval growth, and oogenesis, and which is functionally conserved with its yeast and human homologues.</p

Structural properties and neuronal toxicity of amyotrophic lateral sclerosis–associated Cu/Zn superoxide dismutase 1 aggregates

The appearance of protein aggregates is a characteristic of protein misfolding disorders including familial amyotrophic lateral sclerosis, a neurodegenerative disease caused by inherited mutations in Cu/Zn superoxide dismutase 1 (SOD1). Here, we use live cell imaging of neuronal and nonneuronal cells to show that SOD1 mutants (G85R and G93A) form an aggregate structure consisting of immobile scaffolds, through which noninteracting cellular proteins can diffuse. Hsp70 transiently interacts, in a chaperone activity-dependent manner, with these mutant SOD1 aggregate structures. In contrast, the proteasome is sequestered within the aggregate structure, an event associated with decreased degradation of a proteasomal substrate. Through the use of time-lapse microscopy of individual cells, we show that nearly all (90%) aggregate-containing cells express higher levels of mutant SOD1 and died within 48 h, whereas 70% of cells expressing a soluble mutant SOD1 survived. Our results demonstrate that SOD1 G85R and G93A mutants form a distinct class of aggregate structures in cells destined for neuronal cell death

UCHL5 expression associates with improved survival in lymph-node-positive rectal cancer

Colorectal cancer is among the three most common cancer types for both genders, with a rising global incidence. To date, prognostic evaluation is difficult and largely dependent on early detection and successful surgery. UCHL5/Uch37 is an integral part of the protein homeostasis network as one of the three deubiquitinating enzymes associated with the 26S proteasome. Here, we have investigated in colorectal cancer the possible association of UCHL5 tumor expression and patient survival. UCHL5 tumor expression was evaluated by immunohistochemistry in 779 surgically treated colorectal cancer patients from Helsinki University Hospital, Finland, with assessment of clinicopathological parameters and the effect of UCHL5 expression on patient survival. High and undetectable UCHL5 expression both correlated with increased overall disease-specific survival in the subgroup of patients with lymph-node-positive (Dukes C/stage III) rectal cancer. Within this subgroup of 105 stage-III rectal cancer patients, none of the 7 with high UCHL5 expression died of colorectal cancer within 10 years after surgery (p = 0.012). A similar, though less prominent, survival trend occurred throughout the whole patient cohort. In conclusion, UCHL5 is a promising novel prognostic marker in lymph-node-positive rectal cancer. Our results also advance the currently limited knowledge of biomarkers in colorectal cancer treatment.Peer reviewe

BAP1 Malignant Pleural Mesothelioma Mutations in Caenorhabditis elegans Reveal Synthetic Lethality between ubh-4/BAP1 and the Proteasome Subunit rpn-9/PSMD13

The deubiquitinase BAP1 (BRCA1-associated protein 1) is associated with BAP1 tumor predisposition syndrome (TPDS). BAP1 is a tumor suppressor gene whose alterations in cancer are commonly caused by gene mutations leading to protein loss of function. By CRISPR-Cas, we have generated mutations in ubh-4, the BAP1 ortholog in Caenorhabditis elegans, to model the functional impact of BAP1 mutations. We have found that a mimicked BAP1 cancer missense mutation (UBH-4 A87D; BAP1 A95D) resembles the phenotypes of ubh-4 deletion mutants. Despite ubh-4 being ubiquitously expressed, the gene is not essential for viability and its deletion causes only mild phenotypes without affecting 20S proteasome levels. Such viability facilitated an RNAi screen for ubh-4 genetic interactors that identified rpn-9, the ortholog of human PSMD13, a gene encoding subunit of the regulatory particle of the 26S proteasome. ubh-4[A87D], similarly to ubh-4 deletion, cause a synthetic genetic interaction with rpn-9 inactivation affecting body size, lifespan, and the development of germ cells. Finally, we show how ubh-4 inactivation sensitizes animals to the chemotherapeutic agent Bortezomib, which is a proteasome inhibitor. Thus, we have established a model to study BAP1 cancer-related mutations in C. elegans, and our data points toward vulnerabilities that should be studied to explore therapeutic opportunities within the complexity of BAP1 tumors

Nuclear ubiquitin C-terminal hydrolase L5 expression associates with increased patient survival in pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma is a lethal disease with an overall 5-year survival of less than 5%. Prognosis among surgically treated patients is difficult and identification of new biomarkers is essential for accurate prediction of patient outcome. As part of one of the major cellular protein degradation systems, the proteasome plays a fundamental role in both physiological and pathophysiological conditions including cancer. The proteasome-associated deubiquitinating enzyme ubiquitin C-terminal hydrolase L5 (UCHL5)/Uch37 is a modulator of proteasome activity with cancer prognostic marker potential. Cytoplasmic and nuclear immunoexpression of UCHL5 was evaluated in 154 surgical specimens from pancreatic ductal adenocarcinoma patients treated at Helsinki University Hospital, Finland, in 2000-2011. UCHL5 expression in relation to clinicopathological parameters and the association between UCHL5 In this study, positive expression and patient survival were assessed. Positive nuclear UCHL5 expression was associated with increased patient survival (p = 0.005). A survival benefit was also detectable in these subgroups of patients: over 65 years (p <0.001), at tumor stages IIB to III (p = 0.007), or with lymph-node positivity (p = 0.006). In stages IIB to III disease, patients with positive nuclear UCHL5 expression showed a twofold increase in 5-year cancer-specific survival compared to those with negative expression. Multivariate analysis identified positive nuclear UCHL5 expression as an independent prognostic factor (p = 0.012). In conclusion, UCHL5 expression could function as a prognostic marker in pancreatic ductal adenocarcinoma, particularly at disease stages IIB to III. As UCHL5 is one of the few markers predicting increased survival, our results may be of clinical relevance.Peer reviewe