Browsing by Subject "unfolded protein response"

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  • Hentila, Jaakko; Nissinen, Tuuli A.; Korkmaz, Ayhan; Lensu, Sanna; Silvennoinen, Mika; Pasternack, Arja; Ritvos, Olli; Atalay, Mustafa; Hulmi, Juha J. (2019)
    Muscle wasting in cancer cachexia can be alleviated by blocking activin receptor type 2 (ACVR2) ligands through changes in protein synthesis/degradation. These changes in cellular and protein metabolism may alter protein homeostasis. First, we elucidated the acute (1-2 days) and 2-week effects of blocking ACVR2 ligands by soluble activin receptor 2B (sACVR2B-Fc) on unfolded protein response (UPR), heat shock proteins (HSPs) and redox balance in a healthy mouse skeletal muscle. Second, we examined UPR, autophagy and redox balance with or without sACVR2B-Fc administration in muscle and liver of C26 tumor-bearing mice. The indicators of UPR and HSPs were not altered 1-2 days after a single sACVR2B-Fc administration in healthy muscles, but protein carbonyls increased (p <0.05). Two weeks of sACVR2B-Fc administration increased muscle size, which was accompanied by increased UPR markers: GRP78 <0.05), phosphorylated elF2 alpha <0.01) and HSP47 (p <0.01). Additionally, protein carbonyls and reduced form of glutathione increased (GSH) (p <0.05). On the other hand, C26 cancer cachexia manifested decreased UPR markers (p-elF2 alpha, HSP47, p-JNK; p <0.05) and antioxidant GSH (p <0.001) in muscle, whereas the ratio of oxidized to reduced glutathione increased (GSSG/GSH; p <0.001). Administration of sACVR2B-Fc prevented the decline in GSH and increased some of the UPR indicators in tumor-bearing mice. Additionally, autophagy markers LC3II/I (p <0.05), Beclin-1 (p <0.01), and P62 (p <0.05) increased in the skeletal muscle of tumor-bearing mice. Finally, indicators of UPR, PERK, p-elF2 alpha and GRP78, increased (p <0.05), whereas ATF4 was strongly decreased (p <0.01) in the liver of tumor-bearing mice while sACVR2B-Fc had no effect. Muscle GSH and many of the altered UPR indicators correlated with tumor mass, fat mass and body mass loss. In conclusion, experimental cancer cachexia is accompanied by distinct and tissue-specific changes in proteostasis. Muscle hypertrophy induced by blocking ACVR2B ligands may be accompanied by the induction of UPR and increased protein carbonyls but blocking ACVR2B ligands may upregulate antioxidant protection.
  • Sket, Tina (Helsingin yliopisto, 2020)
    Endoplasmic reticulum (ER) stress is caused by the accumulation of unfolded proteins in the ER, which leads to the activation of unfolded protein response (UPR) through three transmembrane protein sensors located in the ER membrane. The sensors correspond to three branches of the UPR, namely protein kinase RNA-like endoplasmic reticulum kinase (PERK), activating transcription factor 6 (ATF6), and inositol-requiring enzyme 1 (IRE1) branches. Upon ER stress, IRE1 dimerizes and oligomerizes, and its endonuclease domain is activated. It specifically targets X-box-binding protein 1 (XBP1) mRNA, from which a 26 nt intron is spliced. This allows a complete translation of spliced XBP1 mRNA into a functional protein that acts as a transcription factor. Together with the other pathways, the UPR leads to a decrease in the protein folding load by causing a reduction in the general level of protein translation, and by inducing the expression of protein folding machinery. However, if the UPR is activated continuously for a long time, the apoptotic pathway will be triggered, and the cell will die. ER stress and UPR are associated with various disorders, such as some types of cancer, diabetes, chronic inflammatory syndromes, and particularly neurodegeneration. For example, in Parkinson’s disease, it was suggested that prolonged ER stress induces the extensive apoptosis of dopaminergic neurons in substantia nigra pars compacta region of the midbrain. This hinders the normal functioning of the nigrostriatal pathway, and hence results in the progressive development of Parkinson’s motor symptoms. In order to study the regulation or IRE1 branch of the UPR, and to identify the ER-stress-modulating compounds, a human luciferase reporter cell line (XBP1-NLuc) was created in this work. The reporter was expressed when IRE1 splicing was activated, since the XBP1 intron fragment was fused to the Nano luciferase gene. The expression of the reporter was observed with luciferase assay at several time points during treatments. The treatments were done with ER stress inducers thapsigargin and tunicamycin, and with IRE1 inhibitors KIRA6 and 4μ8c, or the combination of those. Quantitative PCR (qPCR) was used to validate the expression of the reporter and to monitor the expression of the other branches of the UPR. Additionally, the oligomerization of IRE1 was observed with IRE1-GFP cell line that was treated identically to the XBP1-NLuc cell line, fixed, stained for nuclei, and imaged with fluorescent microscopy. After imaging, the IRE1-GFP clusters were analysed and quantified with CellProfiller and CellAnalyst softwares. Both cell lines were used to test the effect of neurotrophic factors CDNF, MANF, and MANF mutant isomers on the UPR with and without tunicamycin treatment. Collectively, the experiments confirmed that XBP1-NLuc cell line was created successfully and that it accurately reports IRE1 splicing activity. As expected, ER stress treatment increased the reporter expression, while IRE1 inhibitors decreased the expression of the reporter. qPCR revealed that the other observed UPR markers were activated as well upon thapsigargin treatment, however, they were not decreased with the treatment with IRE1 specific inhibitors. In line with XBP1-NLuc cell line, the IRE1-GFP cell line demonstrated an increased oligomerization of IRE1 upon ER stress induction. The KIRA6 inhibitor of IRE1, which prevents IRE1 oligomerization, decreased the formation of IRE1-GFP clusters. Additionally, the IRE1-endonuclease-activity inhibitor 4μ8c induced the formation of IRE1-GFP clusters. Curiously, the distribution of the intensity of IRE1-GFP clusters was bimodal and could point to two manners of IRE1 clustering and/or activation. Together, the experiments done with cells transfected with CDNF, MANF or MANF mutants, suggested that the tested neurotrophic factors decreased IRE1 oligomerization and its activation. However, there were substantial problems in the quantification of viable cells, which should be considered in the interpretation of these results. No significant difference among the tested neurotrophic factors was observed. In conclusion, the XBP1-NLuc reporter cell line provided a reliable reporter of IRE1 endonuclease activity, whose expression is increased during the ER stress. Together with IRE1-GFP cell line, it revealed the amount of IRE1 oligomerization and activation under various treatments and at different time points relative to treatments. Due to the effectiveness and accuracy, the XBP1-NLuc cell line can be further used in studying the regulation and activation of IRE1, as well as for the identification of ER-stress modulating molecules, which can be used for development of novel treatments for ER stress associated diseases, such as Parkinson’s disease.
  • Khan, Nahid A.; Auranen, Mari; Paetau, Ilse; Pirinen, Eija; Euro, Liliya; Forsström, Saara; Pasila, Lotta; Velagapudi, Vidya; Carroll, Christopher J.; Auwerx, Johan; Suomalainen, Anu (2014)
  • Danilova, Tatiana; Lindahl, Maria (2018)
    Mesencephalic astrocyte-derived neurotrophic factor (MANF) was originally identified as a secreted trophic factor for dopamine neurons in vitro. It protects and restores damaged cells in rodent models of Parkinson's disease, brain and heart ischemia, spinocerebellar ataxia and retina in vivo. However, its exact mechanism of action is not known. MANF is widely expressed in most human and mouse organs with high levels in secretory tissues. Intracellularly, MANF localizes to the endoplasmic reticulum (ER) and ER stress increases it's expression in cells and tissues. Furthermore, increased MANF levels has been detected in the sera of young children with newly diagnosed Type 1 (T1D) diabetes and Type 2 (T2D) diabetic patients. ER stress is caused by the accumulation of misfolded and aggregated proteins in the ER. It activates a cellular defense mechanism, the unfolded protein response (UPR), a signaling cascade trying to restore ER homeostasis. However, if prolonged, unresolved ER stress leads to apoptosis. Unresolved ER stress contributes to the progressive death of pancreatic insulin-producing beta cells in both T1D and T2D. Diabetes mellitus is characterized by hyperglycemia, caused by the inability of the beta cells to maintain sufficient levels of circulating insulin. The current medications, insulin and antidiabetic drugs, alleviate diabetic symptoms but cannot reconstitute physiological insulin secretion which increases the risk of devastating vascular complications of the disease. Thus, one of the main strategies in improving current diabetes therapy is to define and validate novel approaches to protect beta cells from stress as well as activate their regeneration. Embryonic deletion of the Manf gene in mice led to gradual postnatal development of insulin-deficient diabetes caused by reduced beta cell proliferation and increased beta cell death due to increased and sustained ER stress. In vitro, recombinant MANF partly protected mouse and human beta cells from ER stress-induced beta cell death and potentiated mouse and human beta cell proliferation. Importantly, in vivo overexpression of MANF in the pancreas of T1D mice led to increased beta cell proliferation and decreased beta cell death, suggesting that MANF could be a new therapeutic candidate for beta cell protection and regeneration in diabetes.
  • Kovaleva, Vera; Saarma, Mart (2021)
    Parkinson's disease (PD) pathology involves progressive degeneration and death of vulnerable dopamine neurons in the substantia nigra. Extensive axonal arborization and distinct functions make this type of neurons particularly sensitive to homeostatic perturbations, such as protein misfolding and Ca2+ dysregulation. Endoplasmic reticulum (ER) is a cell compartment orchestrating protein synthesis and folding, as well as synthesis of lipids and maintenance of Ca2+ homeostasis in eukaryotic cells. When misfolded proteins start to accumulate in ER lumen the unfolded protein response (UPR) is activated. UPR is an adaptive signaling machinery aimed at relieving of protein folding load in the ER. When UPR is chronic, it can either boost neurodegeneration and apoptosis or cause neuronal dysfunctions. We have recently discovered that mesencephalic astrocyte-derived neurotrophic factor (MANF) exerts its prosurvival action in dopamine neurons and in an animal model of PD through the direct binding to UPR sensor inositol-requiring protein 1 alpha (IRE1) and attenuation of UPR. In line with this, UPR targeting resulted in neuroprotection and neurorestoration in various preclinical animal models of PD. Therefore, growth factors (GFs), possessing both neurorestorative activity and restoration of protein folding capacity are attractive as drug candidates for PD treatment especially their blood-brain barrier penetrating analogs and small molecule mimetics. In this review, we discuss ER stress as a therapeutic target to treat PD; we summarize the existing preclinical data on the regulation of ER stress for PD treatment. In addition, we point out the crucial aspects for successful clinical translation of UPR-regulating GFs and new prospective in GFs-based treatments of PD, focusing on ER stress regulation.
  • Pakarinen, Emmi; Danilova, Tatiana; Voikar, Vootele; Chmielarz, Piotr; Piepponen, Petteri; Airavaara, Mikko; Saarma, Mart; Lindahl, Maria (2020)
    Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an endoplasmic reticulum (ER) localized protein that regulates ER homeostasis and unfolded protein response (UPR). The biology of endogenous MANF in the mammalian brain is unknown and therefore we studied the brain phenotype of MANF-deficient female and male mice at different ages focusing on the midbrain dopamine system and cortical neurons. We show that a lack of MANF from the brain led to the chronic activation of UPR by upregulation of the endoribonuclease activity of the inositol-requiring enzyme 1 alpha (IRE1 alpha) pathway. Furthermore, in the aged MANF-deficient mouse brain in addition the protein kinase-like ER kinase (PERK) and activating transcription factor 6 (ATF6) branches of the UPR pathways were activated. Neuronal loss in neurodegenerative diseases has been associated with chronic ER stress. In our mouse model, increased UPR activation did not lead to neuronal cell loss in the substantia nigra (SN), decrease of striatal dopamine or behavioral changes of MANF-deficient mice. However, cortical neurons lacking MANF were more vulnerable to chemical induction of additional ER stress in vitro. We conclude that embryonic neuronal deletion of MANF does not cause the loss of midbrain dopamine neurons in mice. However, endogenous MANF is needed for maintenance of neuronal ER homeostasis both in vivo and in vitro.
  • Siyo, Vuyolwethu; Schaefer, Georgia; Hunter, Roger; Grafov, Andriy; Grafova, Iryna; Nieger, Martin; Katz, Arieh A.; Parker, M. Iqbal; Kaschula, Catherine H. (2017)
    Garlic is a food and medicinal plant that has been used in folk medicine since ancient times for its beneficial health effects, which include protection against cancer. Crushed garlic cloves contain an array of small sulfur-rich compounds such as ajoene. Ajoene is able to interfere with biological processes and is cytotoxic to cancer cells in the low micromolar range. BisPMB is a synthetic ajoene analogue that has been shown in our laboratory to have superior cytotoxicity to ajoene. In the current study we have performed a DNA microarray analysis of bisPMB-treated WHCO1 oesophageal cancer cells to identify pathways and processes that are affected by bisPMB. The most significantly enriched biological pathways as assessed by gene ontology, KEGG and ingenuity pathway analysis were those involving protein processing in the endoplasmic reticulum (ER) and the unfolded protein response. In support of these pathways, bisPMB was found to inhibit global protein synthesis and lead to increased levels of ubiquitinated proteins. BisPMB also induced alternate splicing of the transcription factor XBP-1; increased the expression of the ER stress sensor GRP78 and induced expression of the ER stress marker CHOP/GADD153. CHOP expression was found to be central to the cytotoxicity of bisPMB as its silencing with siRNA rendered the cells resistant to bisPMB. The MAPK proteins, JNK and ERK1/2 were activated following bisPMB treatment. However JNK activation was not critical in the cytotoxicity of bisPMB, and ERK1/2 activation was found to play a pro-survival role. Overall the ajoene analogue bisPMB appears to induce cytotoxicity in WHCO1 cells by activating the unfolded protein response through CHOP/GADD153.