Browsing by Subject "proteasome"

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  • Srinivasan, Vignesh; Bruelle, Celine; Scifo, Enzo; Pham, Dan Duc; Soliymani, Rabah; Lalowski, Maciej; Lindholm, Dan Bj (2020)
    USP14 is a deubiquitinating enzyme associated with the proteasome that is important for protein degradation. Here we show that upon proteasomal inhibition or expression of the mutant W58A38 USP14, association of USP14 with the 19S regulatory particle is disrupted. MS-based interactomics revealed an interaction of USP14 with the chaperone, HSC70 in neuroblastoma cells. Proteasome inhibition enhanced binding of USP14 to HSC70, but also to XBP1u and IRE1α proteins, demonstrating a role in the unfolded protein response. Striatal neurons expressing mutant huntingtin exhibited reduced USP14 and HSC70 levels, whilst inhibition of HSC70 downregulated USP14. Furthermore, proteasome inhibition or the use of mutant W58A-USP14 facilitated the interaction of USP14 with the autophagy protein, GABARAP. Functionally, overexpression of W58A-USP14 increased GABARAP positive autophagosomes in striatal neurons and this was abrogated using the HSC70 inhibitor, VER-155008. Modulation of the USP14-HSC70 axis by various drugs may represent a potential therapeutic target in HD to beneficially influence multiple proteostasis pathways
  • Gomez, Miguel; Leung, Whinkie; Dantuluri, Swathi; Pillai, Alexander; Gani, Zyan; Hwang, Sungmin; McMillan, Lana J.; Kiljunen, Saija; Savilahti, Harri; Maupin-Furlow, Julie A. (2018)
    Halophilic archaea thrive in hypersaline conditions associated with desiccation, ultraviolet (UV) irradiation and redox active compounds, and thus are naturally tolerant to a variety of stresses. Here, we identified mutations that promote enhanced tolerance of halophilic archaea to redox-active compounds using Haloferax volcanii as a model organism. The strains were isolated from a library of random transposon mutants for growth on high doses of sodium hypochlorite (NaOCl), an agent that forms hypochlorous acid (HOCl) and other redox acid compounds common to aqueous environments of high concentrations of chloride. The transposon insertion site in each of twenty isolated clones was mapped using the following: (i) inverse nested two-step PCR (INT-PCR) and (ii) semi-random two-step PCR (ST-PCR). Genes that were found to be disrupted in hypertolerant strains were associated with lysine deacetylation, proteasomes, transporters, polyamine biosynthesis, electron transfer, and other cellular processes. Further analysis revealed a Delta psmA1 (alpha 1) markerless deletion strain that produces only the alpha 2 and beta proteins of 20S proteasomes was hypertolerant to hypochlorite stress compared with wild type, which produces alpha 1, alpha 2, and beta proteins. The results of this study provide new insights into archaeal tolerance of redox active compounds such as hypochlorite.
  • Arpalahti, Leena; Saukkonen, Kapo; Hagström, Jaana; Mustonen, Harri; Seppänen, Hanna; Haglund, Caj; Holmberg, Carina I. (2017)
    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.
  • Loppi, Sanna; Korhonen, Paula; Bouvy-Liivrand, Maria; Caligola, Simone; Turunen, Tiia A.; Turunen, Mikko P.; de Sande, Ana Hernandez; Kolosowska, Natalia; Scoyni, Flavia; Rosell, Anna; Garcia-Berrocoso, Teresa; Lemarchant, Sighild; Dhungana, Hiramani; Montaner, Joan; Koistinaho, Jari; Kanninen, Katja M.; Kaikkonen, Minna U.; Giugno, Rosalba; Heinäniemi, Merja; Malm, Tarja (2021)
    Ischemic stroke, the third leading cause of death in the Western world, affects mainly the elderly and is strongly associated with comorbid conditions such as atherosclerosis or diabetes, which are pathologically characterized by increased inflammation and are known to influence the outcome of stroke. Stroke incidence peaks during influenza seasons, and patients suffering from infections such as pneumonia prior to stroke exhibit a worse stroke outcome. Earlier studies have shown that comorbidities aggravate the outcome of stroke, yet the mediators of this phenomenon remain obscure. Here, we show that acute peripheral inflammation aggravates stroke-induced neuronal damage and motor deficits specifically in aged mice. This is associated with increased levels of plasma proinflammatory cytokines, rather than with an increase of inflammatory mediators in the affected brain parenchyma. Nascent transcriptomics data with mature microRNA sequencing were used to identify the neuron-specific miRNome, in order to decipher dysregulated miRNAs in the brains of aged animals with stroke and co-existing inflammation. We pinpoint a previously uninvestigated miRNA in the brain, miR-127, that is highly neuronal, to be associated with increased cell death in the aged, LPS-injected ischemic mice. Target prediction tools indicate that miR-127 interacts with several basally expressed neuronal genes, and of these we verify miR-127 binding to Psmd3. Finally, we report reduced expression of miR-127 in human stroke brains. Our results underline the impact of peripheral inflammation on the outcome of stroke in aged subjects and pinpoint molecular targets for restoring endogenous neuronal capacity to combat ischemic stroke.
  • Punger, Tatjana (Helsingin yliopisto, 2017)
    Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma. It arises from skeletal muscle stem cells, which fail to differentiate. Multimodal treatment approach has improved the outcome of RMS during the recent years. However, in case of relapsed or metastatic disease, the prognosis is still very poor. This indicates a high demand for novel targeted therapy approach for RMS. Prox1 transcription factor has been shown to regulate myoblast differentiation into skeletal muscle fibers. Our unpublished observations indicate that Prox1 is highly expressed in RMS tumors and that it is essential for RMS cell growth. The aim of this study was to find novel drug candidates for RMS treatment depending on Prox1 and/or its downstream signaling. High-throughput drug screening with 528 oncology compounds was tested on wild-type and Prox1 silenced rhabdomyosarcoma cells (RD cell line). Gene silencing was performed via lentiviral vectors encoding shRNA for Prox1. In the screening results, we focused on the drugs, which were more potent in Prox1 silenced cells with nano- or micromolar concentrations compared to the wild-type cells. The results revealed 7 potential groups of inhibitors, which had superior suppressive effect on RMS cell viability specifically when Prox1 was silenced. In vitro validation of high-throughput screening results by MTT and luciferase assays confirmed the results. Based on the magnitude of their inhibitory effect and information available on these compounds, three drugs were chosen for further investigation. Two of these compounds also potently inhibited the growth of patient-derived primary RMS cells, which we obtained from the Helsinki University Hospital and named KLHEL1. These drugs were also less toxic to healthy myoblasts. In addition, these two compounds significantly decreased Prox1 mRNA and protein levels in wild-type cells, and completely inhibited the ability of both RD and KLHEL1 cells to form colonies. Combinational exposure to these inhibitors further enhanced the effect compared to a single agent treatment. The present findings demonstrate a potential for repurposing of these drugs for targeted treatment in rhabdomyosarcoma expressing high Prox1 levels.
  • Arpalahti, Leena; Hagstrom, Jaana; Mustonen, Harri; Lundin, Mikael; Haglund, Caj; Holmberg, Carina I. (2017)
    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.