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Inositol Pyrophosphate and Ubiquitin-Proteasomal Pathways

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Inositol Pyrophosphate and Ubiquitin-Proteasomal Pathways INOSITOL PYROPHOSPHATE AND UBIQUITIN-PROTEASOMAL PATHWAYS REGULATE CELL SIGNALING AND METABOLISM by Hector Mora A Thesis Submitted to the Faculty of The Wilkes Honors College in Partial Fulfillment of the Requirements for the Degree of Bachelor of Arts in Liberal Arts and Sciences with a Concentration in Biological Chemistry Wilkes Honors College of Florida Atlantic University Jupiter, Florida July 2015 INOSITOL PYROPHOSPHATE AND UBIQUITIN-PROTEASOMAL PATHWAYS REGULATE CELL SIGNALING AND METABOLISM by Hector Mora This thesis was prepared under the direction of the candidate’s thesis advisor, Dr. Anutosh Chakraborty, and has been approved by the members of her/his supervisory committee. It was submitted to the faculty of The Honors College and was accepted in partial fulfillment of the requirements for the degree of Bachelor of Arts in Liberal Arts and Sciences. SUPERVISORY COMMITTEE: ____________________________ Dr. Anutosh Chakraborty, PI ____________________________ Dr. Chitra Chandrasekhar, 1st Reader ____________________________ Dr. Shree Kundalkar, 2nd Reader ______________________________ Dean Jeffrey Buller, Wilkes Honors College ____________ Date II Table of Contents Item Page # Abstract……………………………………………………………………………..……IV Introduction……………………………………………………….………………............V Part One Results …………….………………………….……...………………………..IX Part Two Results …………….………………………….……...…………………...…XIX Discussion……………………………………….…………...…………………...…..XXV Materials …………………….…………………………………………………..…..XXVI Methods …….…………………………………………………………………..……XXVI Pharmacological Treatments………………………………...………………...……..XXXI Bibliography…………………………………………………………...…......…..XXXVIII III Abstract Author: Hector Mora Title: Inositol Pyrophosphate And Ubiquitin-Proteasomal Pathways Regulate Cell Signaling And Metabolism Institution: Wilkes Honors College of Florida Atlantic University Thesis Advisor: Dr. Anutosh Chakraborty Degree: Bachelor of Arts in Liberal Arts and Sciences Concentration: Biological Chemistry Year: 2015 Background: The prevalence of obesity and type-2 diabetes (T2D) related comorbidities in the US emphasize the need for their treatment. Enzymes implicated in these diseases are favorable targets due to their specific catalytic activity. The Chakraborty lab discovered that inositol hexakisphosphate kinase-1 (IP6K1), an enzyme that synthesizes the signaling inositol pyrophosphate IP7, promotes fat accumulation and insulin resistance. Thus, IP6K1 is a novel target in obesity/T2D. However, how IP6K1 expression, stability, and activity are modulated in vivo remains unknown. Recent experiments establish that IP6K1 interacts with a novel ubiquitin ligase, Ube4A. Studies are ongoing to determine the cellular role of Ube4A as well as the physiological consequences of IP6K1-Ube4A interaction. Conclusion: The current study indicates that Ube4A mediated protein ubiquitination regulates cell metabolism. Furthermore, Ube4A is a component of the DNA damage- induced ubiquitination complex. Thus, Ube4A may regulate age/stress-induced DNA damage as observed in aging related metabolic diseases and neurodegeneration. IV Introduction The predominance of obesity and type-2 diabetes (T2D) related comorbidities such as atherosclerosis, hepatic steatosis, sleep apnea, neurodegeneration and cancer, emphasize the urge for the treatment of these diseases (Must et al., 1999). Extensive research established that a combination of drug and lifestyle modification is essential to ameliorate obesity/T2D (Wadden et al., 2001). Unfortunately, current medications are only partly effective (Vervoort G, 2007) and thus, a safe and effective anti-obesity/anti- diabetic drug has a projected market of $3.7 billion (Rodgers RJ, 2012). Therefore, extensive research is ongoing to identify targetable proteins to develop pharmacotherapy against obesity/T2D (Whittle A, 2013). In particular, enzymes are considered favorable targets due to their specific catalytic activity. In this regard, the Chakraborty laboratory is focused on identifying novel protein targets in obesity/T2D. The group discovered inositol hexakisphosphate kinase-1 (IP6K1) as one such target (Chakraborty A, 2011a; Mackenzie RW, 2014). IP6K1 is the major enzyme that synthesizes the signaling inositol pyrophosphate IP7 (Chakraborty A, 2011a). Mice deleted of IP6K1 are protected against age and high-fat diet induced obesity/T2D and fatty liver (Chakraborty A, 2010). IP6K1-generated IP7 inhibits the protein kinase Akt. Akt is an insulin-sensitizing kinase and thus, its inhibition causes insulin resistance (Mackenzie RW, 2014; Manning BD, 2007). Accordingly, IP6K1-KO mice display increased Akt activity and are insulin hypersensitive. Thus, IP6K1 is a novel target in obesity and diabetes. IP7 is a signaling molecule and thus, it regulates diverse cellular processes (Chakraborty A, 2011a). IP7 regulates its protein targets by i) binding or ii) by V phosphorylating serine or threonine residues which are already phosphorylated by a priming kinase (Chakraborty A, 2011a). In addition, IP6K1 modulates protein functions by direct protein-protein interaction which may (Koldobskiy MA, 2010) or may not require its catalytic activity (Chakraborty A, 2014). Thus, IP6K1 exerts pleiotropic effects on diverse protein targets to regulate cell metabolism and survival. Yet, all the physiological cellular protein targets of IP6K1 have not been identified. Moreover, how IP6K1 expression, stability and activity are modulated in vivo also remains elusive. Therefore, research is ongoing in the laboratory to identify targets and regulators of IP6K1. In this direction, the laboratory recently discovered that IP6K1 interacts with the ubiquitin ligase, Ube4A. The ubiquitin-proteasome system (UPS) is the major proteolytic pathway that regulates cellular protein homeostasis. Protein ubiquitination requires three distinct families of ubiquitin ligase enzymes. First, an E1 ligase stimulates ubiquitin in an ATP- dependent manner and forms a covalent bond between its active site and the C-terminal end of the ubiquitin protein. Thereafter, the ubiquitin moves from the active site of the E1 ligase to an E2 ligase, the ubiquitin-conjugating enzyme. Finally, an E3 ligase binds to both the E2-bound ubiquitin and the protein substrate and stimulates the transfer of the ubiquitin molecule onto the substrate (Chernorudskiy AL, 2013 ; Kleiger G, 2014; Komander D, 2012). Two major kinds of E3 ligases can be found in eukaryotes. These are defined by the presence of either a HECT or a RING domain (Deshaies RJ, 2009). In addition, a domain similar to the RING finger has been recognized and implicated in degradation via the UPS. This domain has been termed a ‘U box’ due to its occurrence in the UFD2 ubiquitination factor found in yeast Ub fusion degradation protein (C., 2002). VI UFD are also known as E4 ligases (Koegl M, 1999) since they poly-ubiquitinate already ubiquitinated proteins in order to ensure their degradation (Koegl M, 1999; Liu C, 2011). E4 ligases are being characterized in mammals. In humans, UFD homologs Ube4A and Ube4B, are located in common neuroblastoma deletion regions and are subject to mutations in tumors (Carén H, 2006). Moreover, Ube4A facilitates the degradation of a receptor tyrosine kinase EPHA2 in colon cancer cells via the Src-like adaptor protein (SLAP) (Naudin C, 2014). Evidently, the UPS modulates diverse cellular processes and thus, its aberration leads to various diseases (S., 2008). Therefore, the UPS is being targeted in diseases such as cancer and neurodegeneration (Kar G, 2013). While less explored, some reports suggest that the UPS plays important roles in metabolic tissues, such as the liver. For example, insulin resistance was observed upon the degradation of the insulin receptor substrate-1 (IRS1) by the E3 ligase CRL7 (Wing, 2008). Meanwhile, the E3 ligase, Casitas B-lineage lymphoma (Cbl)-b, promotes obesity-induced insulin resistance by regulating the production of macrophages in adipose tissue (Abe T, 2014). On the other hand, another E3 ligase, Pellino3, demonstrates a protective role in adipose tissue by reducing obesity-induced inflammation and insulin resistance (Yang S, 2014). Thus, the UPS may enhance or reduce insulin sensitivity, and thus regulate obesity/T2D, depending on the E3 ligase and the tissue specific protein target on which it acts. Literature on the role of the UPS in adipose tissue metabolism is limited. The Fbxw7 ligase inhibits adipocyte differentiation via degradation of the adipogenic transcription factor C/EBPα (Bengoechea-Alonso MT, 2010). Furthermore, the E3 ligase, COP1, along with the pseudokinase Tribbles 3 (TRB3) activates the degradation of acetyl VII CoA carboxylase (ACC) in adipose tissue (Qi L, 2006). Finally, PPARγ, the metabolic master regulator of adipose tissue, also undergoes proteasomal degradation (Floyd ZE, 2012). Yet, functions of E4 ligases such as Ube4A in adipose tissue metabolism have not been explored. Accumulation of DNA damage is one of the causes of aging related diseases including obesity/T2D, cancer and neurodegeneration (Shimizu et al., 2014). DNA damage causes senescence or apoptosis, which impairs cell/tissue functions. In addition, damaged DNA induces tissue inflammation that disrupts the homeostasis of systemic metabolism. Therefore, proteins/molecules that modulate DNA repair are being targeted in metabolic and other diseases (Shimizu I, 2014). DNA damage induces a specific UPS pathway. An E3 ubiquitin ligase called the Cullin-RING ubiquitin ligase 4 (CRL4) is induced by DNA
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