Browsing by Subject "Proteasome"

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  • Zhang, Yuemei; Ertbjerg, Per (2018)
    This study aimed to determine the effect of frozen-then-chilled storage on free Ca2+, proteolytic enzyme activity of calpains and the proteasome, water-holding capacity and shear force of porcine longissimus thoracis et lumborum muscle. Pork loins were subjected to either chilled storage at 2 +/- 1 degrees C for 1, 2, 4, 6 and 9 days, or frozen then chilled storage (20 +/- 1 degrees C for 1 week followed by thawing overnight). Free Ca2+ increased with chilled storage in the non-frozen group. Frozen-then-chilled storage increased free Ca2+ concentration, followed by a faster decrease of calpain-1 activity and activation of around 50% of calpain-2. Proteasome activity was reduced by around 40% following freezing-thawing. Purge loss increased and water-holding capacity of myofibrils decreased in the frozen-thawed group, suggesting considerable denaturation of myofibrillar proteins. Shear force was not affected by freezing-thawing, and we speculate that the tenderizing effect of calpain activation was counteracted by loss of proteasome activity and substantial exudate loss.
  • Dixit, Pragyesh; Kokate, Shrikant B.; Poirah, Indrajit; Chakraborty, Debashish; Smoot, Duane T.; Ashktorab, Hassan; Rout, Niranjan; Singh, Shivaram P.; Bhattacharyya, Asima (2021)
    Background Helicobacter pylori-mediated gastric carcinogenesis is initiated by a plethora of signaling events in the infected gastric epithelial cells (GECs). The E3 ubiquitin ligase seven in absentia homolog 2 (Siah2) is induced in GECs in response to H. pylori infection. Posttranslational modifications of Siah2 orchestrate its function as well as stability. The aim of this study was to evaluate Siah2 phosphorylation status under the influence of H. pylori infection and its impact in gastric cancer progression. Methods H. pylori-infected various GECs, gastric tissues from H. pylori-infected GC patients and H. felis-infected C57BL/6 mice were evaluated for Siah2 phosphorylation by western blotting or immunofluorescence microscopy. Coimmunoprecipitation assay followed by mass spectrometry were performed to identify the kinases interacting with Siah2. Phosphorylation sites of Siah2 were identified by using various plasmid constructs generated by site-directed mutagenesis. Proteasome inhibitor MG132 was used to investigate proteasome degradation events. The importance of Siah2 phosphorylation on tumorigenicity of infected cells were detected by using phosphorylation-null mutant and wild type Siah2 stably-transfected cells followed by clonogenicity assay, cell proliferation assay, anchorage-independent growth and transwell invasion assay. Results Siah2 was phosphorylated in H. pylori-infected GECs as well as in metastatic GC tissues at residues serine(6) (Ser(6)) and threonine(279) (Thr(279)). Phosphorylation of Siah2 was mediated by MRCK beta, a Ser/Thr protein kinase. MRCK beta was consistently expressed in uninfected GECs and noncancer gastric tissues but its level decreased in infected GECs as well as in metastatic tissues which had enhanced Siah2 expression. Infected murine gastric tissues showed similar results. MRCK beta could phosphorylate Siah2 but itself got ubiquitinated from this interaction leading to the proteasomal degradation of MRCK beta and use of proteasomal inhibitor MG132 could rescue MRCK beta from Siah2-mediated degradation. Ser(6) and Thr(279) phosphorylated-Siah2 was more stable and tumorigenic than its non-phosphorylated counterpart as revealed by the proliferation, invasion, migration abilities and anchorage-independent growth of stable-transfected cells. Conclusions Increased level of Ser(6) and Thr(279)-phosphorylated-Siah2 and downregulated MRCK beta were prominent histological characteristics of Helicobacter-infected gastric epithelium and metastatic human GC. MRCK beta-dependent Siah2 phosphorylation stabilized Siah2 which promoted anchorage-independent survival and proliferative potential of GECs. Phospho-null mutants of Siah2 (S6A and T279A) showed abated tumorigenicity.
  • Dixit, Pragyesh; Kokate, Shrikant B; Poirah, Indrajit; Chakraborty, Debashish; Smoot, Duane T; Ashktorab, Hassan; Rout, Niranjan; Singh, Shivaram P; Bhattacharyya, Asima (BioMed Central, 2021)
    Abstract Background Helicobacter pylori-mediated gastric carcinogenesis is initiated by a plethora of signaling events in the infected gastric epithelial cells (GECs). The E3 ubiquitin ligase seven in absentia homolog 2 (Siah2) is induced in GECs in response to H. pylori infection. Posttranslational modifications of Siah2 orchestrate its function as well as stability. The aim of this study was to evaluate Siah2 phosphorylation status under the influence of H. pylori infection and its impact in gastric cancer progression. Methods H. pylori-infected various GECs, gastric tissues from H. pylori-infected GC patients and H. felis-infected C57BL/6 mice were evaluated for Siah2 phosphorylation by western blotting or immunofluorescence microscopy. Coimmunoprecipitation assay followed by mass spectrometry were performed to identify the kinases interacting with Siah2. Phosphorylation sites of Siah2 were identified by using various plasmid constructs generated by site-directed mutagenesis. Proteasome inhibitor MG132 was used to investigate proteasome degradation events. The importance of Siah2 phosphorylation on tumorigenicity of infected cells were detected by using phosphorylation-null mutant and wild type Siah2 stably-transfected cells followed by clonogenicity assay, cell proliferation assay, anchorage-independent growth and transwell invasion assay. Results Siah2 was phosphorylated in H. pylori-infected GECs as well as in metastatic GC tissues at residues serine6 (Ser6) and threonine279 (Thr279). Phosphorylation of Siah2 was mediated by MRCKβ, a Ser/Thr protein kinase. MRCKβ was consistently expressed in uninfected GECs and noncancer gastric tissues but its level decreased in infected GECs as well as in metastatic tissues which had enhanced Siah2 expression. Infected murine gastric tissues showed similar results. MRCKβ could phosphorylate Siah2 but itself got ubiquitinated from this interaction leading to the proteasomal degradation of MRCKβ and use of proteasomal inhibitor MG132 could rescue MRCKβ from Siah2-mediated degradation. Ser6 and Thr279 phosphorylated-Siah2 was more stable and tumorigenic than its non-phosphorylated counterpart as revealed by the proliferation, invasion, migration abilities and anchorage-independent growth of stable-transfected cells. Conclusions Increased level of Ser6 and Thr279-phosphorylated-Siah2 and downregulated MRCKβ were prominent histological characteristics of Helicobacter-infected gastric epithelium and metastatic human GC. MRCKβ-dependent Siah2 phosphorylation stabilized Siah2 which promoted anchorage-independent survival and proliferative potential of GECs. Phospho-null mutants of Siah2 (S6A and T279A) showed abated tumorigenicity.
  • Felszeghy, Szabolcs; Viiri, Johanna; Paterno, Jussi J.; Hyttinen, Juha M. T.; Koskela, Ali; Chen, Mei; Leinonen, Henri; Tanila, Heikki; Kivinen, Niko; Koistinen, Arto; Toropainen, Elisa; Amadio, Marialaura; Smedowski, Adrian; Reinisalo, Mika; Winiarczyk, Mateusz; Mackiewicz, Jerzy; Mutikainen, Maija; Ruotsalainen, Anna-Kaisa; Kettunen, Mikko; Jokivarsi, Kimmo; Sinha, Debasish; Kinnunen, Kati; Petrovski, Goran; Blasiak, Janusz; Bjorkoy, Geir; Koskelainen, Ari; Skottman, Heli; Urtti, Arto; Salminen, Antero; Kannan, Ram; Ferrington, Deborah A.; Xu, Heping; Levonen, Anna-Liisa; Tavi, Pasi; Kauppinen, Anu; Kaarniranta, Kai (2019)
    Age-related macular degeneration (AMD) is a multi-factorial disease that is the leading cause of irreversible and severe vision loss in the developed countries. It has been suggested that the pathogenesis of dry AMD involves impaired protein degradation in retinal pigment epithelial cells (RPE). RPE cells are constantly exposed to oxidative stress that may lead to the accumulation of damaged cellular proteins, DNA and lipids and evoke tissue deterioration during the aging process. The ubiquitin-proteasome pathway and the lysosomal/autophagosomal pathway are the two major proteolytic systems in eukaryotic cells. NRF-2 (nuclear factor-erythroid 2-related factor-2) and PGC-1 alpha (peroxisome proliferator-activated receptor gamma coactivator-1 alpha) are master transcription factors in the regulation of cellular detoxification. We investigated the role of NRF-2 and PGC-1 alpha in the regulation of RPE cell structure and function by using global double knockout (dKO) mice. The NRF-2/PGC-1 alpha dKO mice exhibited significant age-dependent RPE degeneration, accumulation of the oxidative stress marker, 4-HNE (4-hydroxynonenal), the endoplasmic reticulum stress markers GRP78 (glucose-regulated protein 78) and ATF4 (activating transcription factor 4), and damaged mitochondria. Moreover, levels of protein ubiquitination and autophagy markers p62/SQSTM1 (sequestosome 1), Beclin-1 and LC3B (microtubule associated protein 1 light chain 3 beta) were significantly increased together with the Iba-1 (ionized calcium binding adaptor molecule 1) mononuclear phagocyte marker and an enlargement of RPE size. These histopathological changes of RPE were accompanied by photoreceptor dysmorphology and vision loss as revealed by electroretinography. Consequently, these novel findings suggest that the NRF-2/PGC-1 alpha dKO mouse is a valuable model for investigating the role of proteasomal and autophagy clearance in the RPE and in the development of dry AMD.
  • Arpalahti, Leena; Haglund, Caj; Holmberg, Carina I (Springer, 2020)
    Advances in Experimental Medicine and Biology
    The most common form of pancreatic cancer, pancreatic ductal adenocarcinoma (PDAC), has a dismal 5-year survival rate of less than 5%. Radical surgical resection, in combination with adjuvant chemotherapy, provides the best option for long-term patient survival. However, only approximately 20% of patients are resectable at the time of diagnosis, due to locally advanced or metastatic disease. There is an urgent need for the identification of new, specific, and more sensitive biomarkers for diagnosis, prognosis, and prediction to improve the treatment options for pancreatic cancer patients. Dysregulation of proteostasis is linked to many pathophysiological conditions, including various types of cancer. In this review, we report on findings relating to the main cellular protein degradation systems, the ubiquitin-proteasome system (UPS) and autophagy, in pancreatic cancer. The expression of several components of the proteolytic network, including E3 ubiquitinligases and deubiquitinating enzymes, are dysregulated in PDAC, which accounts for approximately 90% of all pancreatic malignancies. In the future, a deeper understanding of the emerging role of proteostasis in pancreatic cancer has the potential to provide clinically relevant biomarkers and new strategies for combinatorial therapeutic options to better help treat the patients.
  • Zeng, Zhen; Li, Cheng; Ertbjerg, Per (2017)
    The purpose of this study was to increase the knowledge on the relationship between proteolysis of myofibrillar proteins and the water-holding of meat. Myofibrils isolated from porcine longissimus thoracic et lumborum muscle were used as a model system. Myofibrils were incubated with either calpain-2, the proteasome or a lysosomal extract at 25 degrees C for 2 h. All three proteolytic systems improved the relative water-holding and generally there was a larger effect with increasing amount of enzymes in the incubation. The improved water-holding occurred in parallel to degradation of myofibrillar proteins. Desmin was degraded by calpain-2 as well as by lysosomal enzymes and a-actinin was released by the proteasome. We here propose a model in which degradation of proteins in and around the Z-disk allows overall swelling of the filament lattice and more specifically in the I band area. In conclusion, proteolytic degradation of myofibrillar proteins by calpain-2, the proteasome or lysosomal enzymes improves the water-holding of myofibrils.
  • Ilomäki, Miika Valtteri (Helsingfors universitet, 2016)
    Ubiquitin is an important modifier in eukaryotic cells through many effects on the targeted protein. Ubiquitination is a reaction cascade, catalyzed by E1 – E2 and finally E3 enzyme which completes the ubiquitination. In this study preselected 61 RING type ubiquitin E3 proteins of Arabidopsis thaliana were classified, grouped and analysed to characterize what kind of domains and groups were included. Proteins which contain a RING domain, can either ubiquitinate substrates independently or function as part of a multi-subunit complex. RING E3s are known to act as a molecular adaptors for the E2s and the substrates. It is the E3 ligase that is responsible for selecting the target protein for ubiquitination and later for degradation in proteasome in to peptides. The same preselected 61 genes were also researched from the Betula pendula Roth genome. Web Apollo database was used to annotate genes from the recently sequenced silver birch genome. As a result 32 gene homologs that included RING domain were identified in silver birch.