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Browsing by Author "Roach, Peter"
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Item Glycogenin is Dispensable for Glycogen Synthesis in Human Muscle, and Glycogenin Deficiency Causes Polyglucosan Storage(Oxford University Press, 2020-02-01) Visuttijai, Kittichate; Hedberg-Oldfors, Carola; Thomsen, Christer; Glamuzina, Emma; Kornblum, Cornelia; Tasca, Giorgio; Hernandez-Lain, Aurelio; Sandstedt, Joakim; Dellgren, Göran; Roach, Peter; Oldfors, Anders; Biochemistry and Molecular Biology, School of MedicineGlycogenin is considered to be an essential primer for glycogen biosynthesis. Nevertheless, patients with glycogenin-1 deficiency due to biallelic GYG1 (NM_004130.3) mutations can store glycogen in muscle. Glycogenin-2 has been suggested as an alternative primer for glycogen synthesis in patients with glycogenin-1 deficiency. OBJECTIVE: The objective of this article is to investigate the importance of glycogenin-1 and glycogenin-2 for glycogen synthesis in skeletal and cardiac muscle. DESIGN, SETTING, AND PATIENTS: Glycogenin-1 and glycogenin-2 expression was analyzed by Western blot, mass spectrometry, and immunohistochemistry in liver, heart, and skeletal muscle from controls and in skeletal and cardiac muscle from patients with glycogenin-1 deficiency. RESULTS: Glycogenin-1 and glycogenin-2 both were found to be expressed in the liver, but only glycogenin-1 was identified in heart and skeletal muscle from controls. In patients with truncating GYG1 mutations, neither glycogenin-1 nor glycogenin-2 was expressed in skeletal muscle. However, nonfunctional glycogenin-1 but not glycogenin-2 was identified in cardiac muscle from patients with cardiomyopathy due to GYG1 missense mutations. By immunohistochemistry, the mutated glycogenin-1 colocalized with the storage of glycogen and polyglucosan in cardiomyocytes. CONCLUSIONS: Glycogen can be synthesized in the absence of glycogenin, and glycogenin-1 deficiency is not compensated for by upregulation of functional glycogenin-2. Absence of glycogenin-1 leads to the focal accumulation of glycogen and polyglucosan in skeletal muscle fibers. Expression of mutated glycogenin-1 in the heart is deleterious, and it leads to storage of abnormal glycogen and cardiomyopathy.Item Ssu72 and Rtr1 Serine 5 Phosphates and Their Role in NNS and CPF Transcription Termination(2020-05) Victorino, Jose Fabian; Mosley, Amber; Roach, Peter; Georgiadis, Millie; Liu, Yunlong; Arrizabalaga, GustavoPolyadenylation dependent transcription termination is dependent on the Cleavage and Polyadenylation Factor complex (CPF) which is essential for the termination and processing of mature RNA. Polyadenylation (PolyA) independent transcription termination is carried out by the NNS (Nrd1-Nab3-Sen1) termination pathway, which helps regulate termination and processing of non-coding RNA (ncRNA). The disruption of these pathways can impact expression of nearby genes, both protein coding and noncoding. Recruitment of termination pathway components is achieved through a domain unique to the largest subunit of RNA Polymerase II (RNAPII) referred to as the Cterminal domain (CTD), which contains a repeating heptad sequence, Y1S2P3T4S5P6S7, and acts as a docking site for transcription regulatory proteins. Ssu72 is a serine 5 phosphatase and an essential member of the CPF complex. Rtr1 is also a serine 5 phosphatase, but its mechanism of action is less well characterized. Both Rtr1 and Ssu72 regulate transcription machinery recruitment through control of the phosphorylation status of the CTD. My studies have focused on Rtr1 and Ssu72 mutants in yeast which show evidence of transcription termination related phenotypes. Chromatin immunoprecipitation of RNAPII followed by exonuclease treatment (ChIP-exo) studies provide evidence of RNAPII transcription continuing through termination sites at ncRNA genes as a result of a hyperactive Ssu72-L84F mutant, while an RTR1 knockout results in increased premature RNAPII transcription termination. Northern blots and RNA sequencing confirm premature transcription termination and decreased total RNA expression in the RTR1 knockout and increased length of ncRNA transcripts as well as total RNA expression in the Ssu72-L84F mutant. Mass spectrometry analysis has identified changes in the protein-protein interactions (PPI) within the CPF complex in the Ssu72-L84F mutant and decreased PPIs between different transcription machinery in RTR1 knockout cells. My results show that the CTD phosphatases Rtr1 and Ssu72 play unique roles in the regulation of RNAPII termination in eukaryotes.Item Sterol regulatory element-binding protein-1 (SREBP-1) is required to regulate glycogen synthesis and gluconeogenic gene expression in mouse liver(ASBMB, 2014-01-07) Ruiz, Rafaela; Jideonwo, Victoria; Ahn, Miwon; Surendran, Sneha; Tagliabracci, Vincent S.; Hou, Yongyong; Gamble, Aisha; Kerner, Janos; Irimia-Dominguez, Jose M.; Puchowicz, Michelle A.; Hoppel, Charles; Roach, Peter; Morral, Nuria; Department of Medical & Molecular Genetics, IU School of MedicineSterol regulatory element-binding protein-1 (SREBP-1) is a key transcription factor that regulates genes in the de novo lipogenesis and glycolysis pathways. The levels of SREBP-1 are significantly elevated in obese patients and in animal models of obesity and type 2 diabetes, and a vast number of studies have implicated this transcription factor as a contributor to hepatic lipid accumulation and insulin resistance. However, its role in regulating carbohydrate metabolism is poorly understood. Here we have addressed whether SREBP-1 is needed for regulating glucose homeostasis. Using RNAi and a new generation of adenoviral vector, we have silenced hepatic SREBP-1 in normal and obese mice. In normal animals, SREBP-1 deficiency increased Pck1 and reduced glycogen deposition during fed conditions, providing evidence that SREBP-1 is necessary to regulate carbohydrate metabolism during the fed state. Knocking SREBP-1 down in db/db mice resulted in a significant reduction in triglyceride accumulation, as anticipated. However, mice remained hyperglycemic, which was associated with up-regulation of gluconeogenesis gene expression as well as decreased glycolysis and glycogen synthesis gene expression. Furthermore, glycogen synthase activity and glycogen accumulation were significantly reduced. In conclusion, silencing both isoforms of SREBP-1 leads to significant changes in carbohydrate metabolism and does not improve insulin resistance despite reducing steatosis in an animal model of obesity and type 2 diabetes.