Carina I. Holmberg

Insulin/IGF-1 Signaling Regulates Proteasome Activity through the Deubiquitinating Enzyme UBH-4

The proteasome plays an important role in proteostasis by carrying out controlled protein degradation in the cell. Impairments in proteasome function are associated with severe and often age-related diseases. Here, we have characterized a molecular mechanism linking insulin/IGF-1 signaling (IIS) to proteasome activity. We show that decreased IIS, which promotes proteostasis and longevity, increases proteasome activity through the FOXO transcription factor DAF-16 in C.xa0elegans. Furthermore, we reveal that DAF-16 represses expression of the proteasome-associated deubiquitinating enzyme ubh-4, which we suggest functions as a tissue-specific proteasome inhibitor. Finally, we demonstrate that proteasome activation through downregulation of the ubh-4 human ortholog uchl5 increases degradation of proteotoxic proteins in mammalian cells. In conclusion, we have established a mechanism by which the evolutionarily conserved IIS contributes to the regulation of proteasome activity in a multicellular organism.

Active transcriptomic and proteomic reprogramming in the C. elegans nucleotide excision repair mutant xpa-1

Transcription-blocking oxidative DNA damage is believed to contribute to aging and to underlie activation of oxidative stress responses and down-regulation of insulin-like signaling (ILS) in Nucleotide Excision Repair (NER) deficient mice. Here, we present the first quantitative proteomic description of the Caenorhabditis elegans NER-defective xpa-1 mutant and compare the proteome and transcriptome signatures. Both methods indicated activation of oxidative stress responses, which was substantiated biochemically by a bioenergetic shift involving increased steady-state reactive oxygen species (ROS) and Adenosine triphosphate (ATP) levels. We identify the lesion-detection enzymes of Base Excision Repair (NTH-1) and global genome NER (XPC-1 and DDB-1) as upstream requirements for transcriptomic reprogramming as RNA-interference mediated depletion of these enzymes prevented up-regulation of genes over-expressed in the xpa-1 mutant. The transcription factors SKN-1 and SLR-2, but not DAF-16, were identified as effectors of reprogramming. As shown in human XPA cells, the levels of transcription-blocking 8,5-cyclo-2-deoxyadenosine lesions were reduced in the xpa-1 mutant compared to the wild type. Hence, accumulation of cyclopurines is unlikely to be sufficient for reprogramming. Instead, our data support a model where the lesion-detection enzymes NTH-1, XPC-1 and DDB-1 play active roles to generate a genomic stress signal sufficiently strong to result in transcriptomic reprogramming in the xpa-1 mutant.

Exocyst subunits Exo70 and Exo84 cooperate with small GTPases to regulate behavior and endocytic trafficking in C. elegans.

The exocyst complex is required for cell polarity regulation and the targeting and tethering of transport vesicles to the plasma membrane. The complex is structurally well conserved, however, the functions of individual subunits and their regulation is poorly understood. Here we characterize the mutant phenotypes for the exocyst complex genes exoc-7 (exo70) and exoc-8 (exo84) in Caenorhabditis elegans. The mutants display pleiotropic behavior defects that resemble those observed in cilia mutants (slow growth, uncoordinated movement, defects in chemo-, mechano- and thermosensation). However, no obvious morphological defects in cilia were observed. A targeted RNAi screen for small GTPases identified eleven genes with enhanced phenotypes when combined with exoc-7, exoc-8 single and exoc-7;exoc-8 double mutants. The screen verified previously identified functional links between the exocyst complex and small GTPases and, in addition, identified several novel potential regulators of exocyst function. The exoc-8 and exoc-7;exoc-8 mutations caused a significant size increase in the rab-10 RNAi-induced endocytic vacuoles in the intestinal epithelial cells. In addition, exoc-8 and exoc-7;exoc-8 mutations resulted in up-regulation of RAB-10 expression and affected the accumulation of endocytic marker proteins in these cells in response to rab-10 RNAi. The findings identify novel, potential regulators for exocyst function and show that exoc-7 and exoc-8 are functionally linked to rab-10 in endosomal trafficking in intestinal epithelial cells in C. elegans.

par-1, Atypical pkc, and PP2A/B55 sur-6 Are Implicated in the Regulation of Exocyst-Mediated Membrane Trafficking in Caenorhabditis elegans

The exocyst is a conserved protein complex that is involved in tethering secretory vesicles to the plasma membrane and regulating cell polarity. Despite a large body of work, little is known how exocyst function is controlled. To identify regulators for exocyst function, we performed a targeted RNA interference (RNAi) screen in Caenorhabditis elegans to uncover kinases and phosphatases that genetically interact with the exocyst. We identified seven kinase and seven phosphatase genes that display enhanced phenotypes when combined with hypomorphic alleles of exoc-7 (exo70), exoc-8 (exo84), or an exoc-7;exoc-8 double mutant. We show that in line with its reported role in exocytotic membrane trafficking, a defective exoc-8 caused accumulation of exocytotic soluble NSF attachment protein receptor (SNARE) proteins in both intestinal and neuronal cells in C. elegans. Down-regulation of the phosphatase protein phosphatase 2A (PP2A) phosphatase regulatory subunit sur-6/B55 gene resulted in accumulation of exocytic SNARE proteins SNB-1 and SNAP-29 in wild-type and in exoc-8 mutant animals. In contrast, RNAi of the kinase par-1 caused reduced intracellular green fluorescent protein signal for the same proteins. Double RNAi experiments for par-1, pkc-3, and sur-6/B55 in C. elegans suggest a possible cooperation and involvement in postembryo lethality, developmental timing, as well as SNARE protein trafficking. Functional analysis of the homologous kinases and phosphatases in Drosophila median neurosecretory cells showed that atypical protein kinase C kinase and phosphatase PP2A regulate exocyst-dependent, insulin-like peptide secretion. Collectively, these results characterize kinases and phosphatases implicated in the regulation of exocyst function, and suggest the possibility for interplay between the par-1 and pkc-3 kinases and the PP2A phosphatase regulatory subunit sur-6 in this process.

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 10u2009years after surgery (pu2009=u20090.012). 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.

Positive cytoplasmic UCHL5 tumor expression in gastric cancer is linked to improved prognosis

Gastric cancer is the second most common cause of cancer-related mortality worldwide. Accurate prediction of disease progression is difficult, and new biomarkers for clinical use are essential. Recently, we reported that the proteasome-associated deubiquitinating enzyme UCHL5/Uch37 is a new prognostic marker in both rectal cancer and pancreatic ductal adenocarcinoma. Here, we have assessed by immunohistochemistry UCHL5 tumor expression in gastric cancer. The study cohort comprised 650 patients, who underwent surgery in Helsinki University Hospital, Finland, between 1983 and 2009. We investigated the association of cytoplasmic UCHL5 tumor expression to assess clinicopathological parameters and patient survival. Positive cytoplasmic UCHL5 tumor immunoexpression is linked to increased survival of patients with small (<5 cm) tumors (p = 0.001), disease stages I-II (p = 0.025), and age 66 years or older (p = 0.037). UCHL5 is thus a potential marker in gastric cancer with new prognostic relevance.

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 (pu2009=u20090.005). A survival benefit was also detectable in these subgroups of patients: over 65u2009years (pu2009<u20090.001), at tumor stages IIB to III (pu2009=u20090.007), or with lymph-node positivity (pu2009=u20090.006). 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 (pu2009=u20090.012). 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.

Fluorescent Tools for In Vivo Studies on the Ubiquitin-Proteasome System

The ubiquitin-proteasome system (UPS) plays a key role in maintaining proteostasis by degrading most of the cellular proteins. Traditionally, UPS activity is studied in vitro, in yeast, or in mammalian cell cultures by using short-lived GFP-based UPS reporters. Here, we present protocols for two fluorescent tools facilitating real-time imaging of UPS activity in living animals. We have generated transgenic Caenorhabditis elegans (C. elegans) expressing a photoconvertible UbG76V-Dendra2 UPS reporter, which permits measurement of reporter degradation by the proteasome independently of reporter protein synthesis, and a fluorescent polyubiquitin-binding reporter for detection of the endogenous pool of Lys48-linked polyubiquitinated proteasomal substrates. These reporter systems facilitate cell- and tissue-specific analysis of UPS activity especially in young adult animals, but can also be used for studies during development, aging, and for example stress conditions.

PIM-related kinases selectively regulate sensory functions in C. elegans

The mammalian PIM family of serine/threonine kinases regulate several cellular functions, such as cell survival and motility. Since we have observed PIM expression in the olfactory epithelium and other sensory organs of mice, this has raised the question of whether PIM kinases regulate also sensory cell functions. As our model organism to investigate this question, we used the Caenorhabditis elegans nematodes, which express two PIM-related kinases, PRK-1 and PRK-2. We demonstrated them to be true PIM orthologs with similar substrate specificity as well as sensitivity to PIM-inhibitory compounds. Furthermore, we obtained evidence to indicate that PRKs are selectively involved in regulation of olfactory sensations via AWB or AWCON neurons to volatile attractants or repellants, but do not affect gustatory sensations.

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.