INOSITOL PYROPHOSPHATE AND -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

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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

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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 , 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 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.

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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 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

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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).

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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

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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 damage to degrade certain proteins to facilitate DNA repair. Interestingly, IP6K1 regulates CRL4-mediated ubiquitination. Under basal conditions, IP6K1 binds and inhibits CRL4. UV radiation, which induces DNA damage, disrupts the IP6K1-CRL4 binding. This disruption then activates CRL4. Thus, IP6K1 is a novel CRL4 subunit that regulates nucleotide excision repair and cell death (Rao F, 2014).

Based on this information, it is conceivable that IP6K1 also regulates protein ubiquitination via modulation of Ube4A. Conversely, Ube4A may regulate insulin signaling and energy homeostasis by modulating IP6K1 stability. Moreover, Ube4A may also regulate cell metabolism by regulating other unknown target proteins. Therefore, the current project aims to; i) investigate role of Ube4A in cell metabolism; ii)) identify the co-factors and targets of Ube4A and; iii) characterize its interaction with IP6K1.

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Part One: Identification of Ube4A co-factor and target proteins

Results

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Figure 1: Ube4A enhances global protein ubiquitination

A B Ube4A

Active Inactive Active Inactive - - + + MG132 GST-Ube4A

Total ubiquitination

β Actin HEK293

Figure 1A: Immunoblot analysis reveals that overexpression of active, but not inactive, Ube4A enhances total protein ubiquitination following MG132 treatment. Figure 1B: Quantification of 1A demonstrates that total protein ubiquitination is increased ~2-fold in MG132 treated active Ube4A overexpressed samples compared to control. Inactive Ube4A overexpression although displays slightly increased total protein ubiquitination under basal conditions (black bars), it is not further enhanced following MG132 treatment.

First, we checked whether Ube4A overexpression enhances protein ubiquitination in HEK293 cells. To investigate this, we cloned both active and inactive version of

Ube4A (Naudin et al., 2014) in a mammalian GST-expression vector. Under basal condition, overexpression of active or inactive Ube4A does not display a major difference in total protein ubiquitination (Figure 1A; lanes 1 and 2). The proteasome inhibitor, MG132, blocks the degradation of the ubiquitinated proteins, thus enhancing levels of ubiquitinated proteins in the cell (Chakraborty A, 2011b; Han et al., 2009).

Active-Ube4A overexpressed cells display higher levels of total protein ubiquitination

(Figure 1A; lane 3 and lane 1) following MG132 treatment compared to the inactive-

Ube4A (Figure 1A; lane 4 and lane 2). Thus, the fold-increase in MG132 induced global

X

ubiquitination is higher in active-Ube4A compared to inactive samples (Figure 1B).

Thus, Ube4A overexpression enhances global protein ubiquitination.

Figure 2: Ube4A depletion reduces global protein ubiquitination and enhances Akt level

A B

C Sc 3 siRNA mTOR IRS1

GLUT4 HEK293

Figure 2A: Comparing lanes 1 and 4, total protein ubiquitination decreases nearly ~2 fold upon siRNA #3- mediated Ube4A depletion. siRNA #3 was determined to be the most effective in depleting endogenous Ube4A and thus was selected for subsequent experimentation. siRNA #3 decreased endogenous Ube4A expression nearly ~4 fold. (3) pAKT (S473) level increases nearly ~6 fold upon depleting Ube4A with siRNA #3. (4) However, this increase in phosphorylation of Akt is likely due to an increase in total Akt level, which showed an increase for all siRNA-treated samples compared to the control. Figure 2B: Quantification of 2A comparing relative protein levels between control and siRNA #3 for various antibodies. Figure 2C: Immunoblot showing no significant change upon Ube4A depletion in other proteins of the insulin signaling pathway.

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Based on the previous experiment, we presume that Ube4A depletion should reduce total protein ubiquitination. In order to test this hypothesis, we depleted Ube4A protein level using small interference RNA (siRNA). A number of siRNAs were tested of which siRNA#3 worked best and thus, was used in subsequent experiments (Figure 2A).

Under basal conditions, siRNA-mediated Ube4A depletion decreases total protein ubiquitination to ~half (Figure 2A: lanes 4 and 1; Figure 2B: panels 1 and 2).

Ube4A depletion is associated with an increase in stimulatory phosphorylation of

Akt (serine 473) without altering its protein level in the colon cancer cell line HT29

(Naudin C, 2014). Thus, we monitored Akt phosphorylation in Ube4A depleted HEK293 cells. Indeed, Akt phosphorylation is higher in Ube4A depleted samples (Figures 2A;

Figure 2B; panel 3). This indicates that Ube4A depletion enhances Akt activity in

HEK293 cells. However, we observed that enhanced Akt phosphorylation is due to an enhancement in its protein level in Ube4A depleted samples (Figure 2A; Figure 2B; panel

4). This result indicates that Akt is a target for Ube4A.

Next, we checked effects of Ube4A depletion on other proteins involved in insulin signaling pathway (Figure 2C). Ube4A depletion does not seem to affect other proteins such as mTOR, insulin receptor substrate-1 (IRS1), or glucose transporter-4

(GLUT4) (Figure 2C). Thus, Ube4A specifically regulates Akt protein level.

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Figure 3: Energy starvation enhances protein ubiquitination, Ube4A, and BiP protein level

A B 0 1 2 4 6 20 Glucose starvation (h) 15 Ube4A ~12

10 ~7

Total 5 ubiquitination (20 hr/ (20 0 hr starvation)

Fold Increase in Protein Level Protein in Increase Fold 0 Ubiquitin Ube4a C BiP 2.0 β Actin BiP Ube4A HEK293 1.5 actin (AU) actin β 1.0

0.5 Protein level/ Protein 0.0 0.0 0.5 1.0 1.5 2.0 Glucose Starvation (h) Figure 3A: (1) Total protein ubiquitination increases ~7 fold following overnight starvation. (2) Ube4A level increases ~12 fold following overnight starvation. (3) BiP level decreases initially and its level is restored following overnight starvation. Figure 3B: Quantification of 3A showing fold-increase of total protein ubiquitination and Ube4a protein level after starvation. Figure 3C: Line graph showing initial inverse relationship between Ube4a and BiP level from acute glucose starvation.

Glucose starvation induces protein ubiquitination and autophagy in cells, which generates amino acids. Amino acids, via gluconeogenesis, provide necessary energy for cell survival under these conditions (Choi et al., 2015). We tested whether Ube4A is involved in glucose deprivation-mediated protein ubiquitination. As expected, total protein ubiquitination is augmented when HEK293 cells were deprived of glucose and pyruvate (Figure 3A; lanes 6 and 1). Interestingly, Ube4A protein level is also dramatically enhanced under this condition (Figure 3A; lanes 6 and 1). Glucose

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starvation also induces endoplasmic reticulum (ER) stress. Therefore, we monitored level of an ER stress marker chaperone, BiP (Kohno et al., 1993). Interestingly, BiP protein level is decreased during the initial periods of starvation (1-4h) but subsequently recovered (6-20h) (Figure 3A; lanes 6 and 1). Thus, Ube4A up-regulation is inversely correlated with BiP protein level during acute starvation (Figure 3C), but not after prolonged starvation (Figure 3A; lanes 6 and 1). It is conceivable that Ube4A destabilizes

BiP during acute energy stress. Thus, Ube4A seems to be involved in energy starvation- mediated protein ubiquitination and degradation.

Figure 4: Ube4A overexpression reduces protein levels of BiP, LC3B and β-catenin following ER stress under energy-starved condition

A B

1.0 Control Thapsigargin 0.8

0.6 actin (AU) actin β 0.4

Protein/ 0.2

0.0 - + - + - + Myc-Ube4a BiP β-Catenin Myc-Ube4a

Figure 4: (1) Thapsigargin treatment increases BiP protein level nearly ~5 fold in absence of Ube4A overexpression. There is not much change in BiP level upon ER stress stimulation when Ube4A is overexpressed. (2) β-catenin protein level spikes nearly ~2 fold in absence of Ube4A under ER stress. Meanwhile, there is not much change in β-catenin level upon ER stress stimulation when Ube4A is overexpressed. (3) Ube4A decreases nearly ~3 fold upon Thapsigargin-induced ER stress. Figure 4B: Quantification of 4A showing various changes to protein levels following Thapsigargin treatment.

Next, we monitored effects of Ube4A overexpression on BiP induction of ER stress. ER stress was induced in glucose-starved cells by addition of the inhibitor of endoplasmic reticulum calcium-ATPase called thapsigargin (Cox et al., 2011). ER stress

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reduces Ube4A protein level (Figure 4A: lanes 2 and 4; Figure 4B; panel 3), which indicates that Ube4A may also undergo proteasomal degradation. Ube4A overexpression does not influence BiP protein level under glucose-starved condition (Figure 4A: lanes 1 and 2; Figure 4B: panel 1). Interestingly, Ube4A reduces BiP level following ER stress condition (Figure 4A: lanes 3 and 4; Figure 4B: panel 1). BiP facilitates protein folding, which protects them from misfolding and degradation during ER stress (Kohno et al.,

1993). Conversely, Ube4A degrades proteins and thus, Ube4A-mediated degradation of

BiP may facilitate ER stress induced protein degradation.

β-catenin is a transcriptional activator which promotes cell survival (Petherick et al., 2013). Accordingly, β-catenin is degraded following glucose starvation (Choi et al.,

2015). We observe that Ube4A overexpression reduces β-catenin protein level under glucose-starved condition (Figure 4A: lanes 1 and 2). ER stress enhances β-catenin level in control cells (Figure 4A: lanes 1 and 3). However, Ube4A overexpression dramatically reduces β-catenin protein level (Figure 4A: lanes 3 and 4). Thus, Ube4A overexpression regulates protein levels of BiP and β-catenin, which may modulate cell survival.

Figure 5: Ube4A is degraded via the ubiquitin-proteasomal pathway

A B

- + MG132 1.0

Sc 2+3 3 Sc 2+3 3 siRN A actin (AU) 0.8 β Ube4A 0.6

0.4 β Actin 0.2

HEK293 0.0 Endogenous Ube4a level/Endogenous Control MG132 siRNA Scrambled

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Figure 5A: Pharmacologic inhibition of proteasomal pathway augments endogenous Ube4A protein level. Figure 5B: Graph showing fold-increase in Ube4A protein level following MG132 treatment.

The previous experiment indicates that Ube4A may also undergo degradation.

Therefore, we monitored Ube4A protein level following treatment of the proteasomal

inhibitor MG132. Indeed, MG132 enhances Ube4A protein level (Figure 5; lanes 1 and

4). Thus, this experiment indicates that the UPS pathway degrades Ube4A.

Figure 6: Identification of Ubed4a interacting proteins by mass spectrometry

GST Ube4A P1030A Figure 6: Coomassie stain to demonstrate various proteins that co-purified with GST-control, active GST- Ube4A and inactive GST-Ube4A proteins. The proteins 170 were subsequently identified by liquid chromatography 130 and mass spectrometry (LC-MS/MS).

95 Thus far, we identified Akt, BiP and β-

72 catenin as putative targets of Ube4A. To

56 obtain a complete list of Ube4A target

43 proteins, we employed mass spectrometry.

34 GST-control and active and inactive versions 26 of Ube4A were overexpressed in HEK293 17 cells. GST-Ube4A and the controls were

purified by GST-pull down assay. Purified GST pull-down: HEK293 proteins were run on SDS-PAGE and the purified proteins were visualized by Coomassie

stain (Figure 6). We also observed several co-purified proteins. We sequenced all the

proteins by LC-MS/MS. Proteins that were bound to active and/or inactive Ube4A, but

not GST-control, are regarded as Ube4A interacting proteins.

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Table 1: Mass spectrometry results showing protein binding-partners for Ube4A

Table 1: Table of proteins found binding to GST-control, GST-Ube4A, and GST-Inactive Ube4A following mass spectrometry analysis.

Table 1 represents a list of Ube4A interacting proteins. The numbers associated with each protein are the number of peptides that were identified. Ube4A was detected as the major protein in active and inactive Ube4A samples as it was overexpressed (Sl. 1).

Detailed analysis of the interactors reveals that Ube4A primarily binds to proteins of three distinct pathways; i) DNA damage induced UPS (Sl. 2-4); ii) protein translation (Sl no. 5-9) and; iii) fatty acid metabolism (Sl. No. 10-12). IP6K1 is identified as the major

Ube4A interacting protein, which further validates our previous discovery that IP6K1 binds Ube4A in the adipocytes (Sl. 2). Interestingly, IP6K1 is also involved in regulation of all of the above pathways (Chakraborty A, 2010; Chakraborty A, 2011a; Rao F, 2014).

For example, Ube4A binds the DNA damage binding protein DDB1 and DDB1-Cul4 associated factor 11 (DCAF11; Sl. 3 and 4). As mentioned earlier, IP6K1 binds

CUL4/CRL4, DDB1 and DCAF1 (Rao F, 2014). Thus, Ube4A is a novel component of the DNA damage induced ubiquitin pathway, which acts in concert with the E3 ligase

CRL4 and its regulator, IP6K1. Moreover, IP6K1 is a major regulator of fatty acid

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metabolism (Chakraborty A, 2011a). Thus, our present research strongly indicates that

Ube4A modulates Akt in the insulin/growth factor signaling pathway as well as BiP in the ER stress pathway. In addition, IP6K1 and Ube4A may act in concert to regulate

DNA damage induced protein ubiquitination and fatty acid metabolism.

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Part 2: Characterization of IP6K1-Ube4A interaction

Results

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Figure 7: IP6K1 binds and enhances Ube4A protein level

Figure 7: GST-versions of both active and inactive - Active Inactive IP6K1 IP6K1 bind to enhance Ube4A protein level. GST- tagged IP6K1 was purified by GST-pull down assay Load: Ube4A following its overexpression in HEK293 cells. GST- vector (lane 1) was used as a control. Endogenous Bound: Ube4A Ube4A is co-purified with both active and inactive IP6K1 (lanes 2 and 3; bound Ube4A). Ube4A protein Pull-down: level is higher in IP6K1 overexpressed samples GST-IP6K1 compared to GST-control (lanes 2 and 3; load: Ube4A). Details are in the methods section.

Pull-down: GST HEK293 In this part, we characterized IP6K1-Ube4A interaction in detail. First, we monitored whether Ube4A is co-purified with overexpressed IP6K1. To investigate this, we conducted an experiment in HEK293 cells. Catalytically active and inactive versions of GST-tagged IP6K1 were overexpressed in cells. GST-pull down assay demonstrates that both active and inactive IP6K1 bind endogenous Ube4A (Figure 7: lanes 3 and 2) whereas the GST-control protein does not (Figure 7: lane 1). Moreover, IP6K1 overexpression substantially enhances Ube4A protein level (Figure 7: lanes 3 and 2).

This experiments indicates that i) IP6K1 activity is not critical in its binding with Ube4A and ii) IP6K1 enhances Ube4A protein level.

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Figure 8: IP6K1 overexpression enhances Ube4A protein level upon differentiation in 3T3L1 adipocytes

A 0 1 3 4 5 6 Days di fferentiation - + - + - + - + - + - + Myc-IP6K1 Myc-IP6K1

β Actin 3T3L1 adipocytes

B C Myc Myc-IP6K1 1.0 0 5 8 0 5 8 Days Myc 0.8 Myc-IP6K1 Ube4A 0.6 actin (AU) actin

β Actin β 0.4 3T3L1 differentiation

Ube4a/ 0.2

0.0 0 5 8 Days

Figure 8A: Immunoblot showing increased expression of Myc-IP6K1 following IP6K1 overexpression in conjunction with increased adipocyte differentiation. Figure 8B: Myc-IP6K1 was overexpressed in pre- adipocytes, which showed enhanced Ube4A binding following differentiation. Figure 8C: Quantification of 10A showing a ~2-fold increase in Ube4A level for Myc-IP6K1 overexpressed cells.

Next, we assessed whether IP6K1 stabilizes Ube4A in the adipocytes. To perform this experiment, we utilized Myc-tagged control vector or Myc-IP6K1 overexpressed

3T3L1 preadipocytes, which were previously generated in the laboratory. The cells were differentiated to adipocytes following standard procedure (Chakraborty A, 2010). The laboratory previously observed that IP6K1 protein level is dramatically augmented during differentiation of preadipocytes to adipocytes (Figure 8A; Ghoshal et al. unpublished data). Accordingly, Ube4A protein level is higher in Myc-IP6K1- compared to Myc- control- overexpressed adipocytes (Figure 8B and 8C). This result suggests that IP6K1-

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Ube4A interaction may regulate adipocyte differentiation and metabolism, which is critical in maintaining insulin and energy homeostasis.

Figure 9: Ube4A protein level is reduced in high-fat fed IP6K1-KO mice

A B WT KO IP6K1

Ube4A

β Actin EWAT: High fat diet

Figure 9A: Ube4A protein level was analyzed in vivo from mice lysate samples obtained from the lab. IP6K1-KO mice displayed significantly reduced Ube4A protein levels compared to wild type mice. Figure 9B: Quantification of Ube4A level showing a ~4-fold decrease in IP6K1-KO mice compared to wild type mice.

Is IP6K1 mediated stabilization of Ube4A relevant in vivo? To investigate this, we employed IP6K1-KO mice which are protected against high fat diet induced weight gain and insulin resistance (Chakraborty A, 2010). We compared Ube4A protein level in the epididymal white adipose tissue (EWAT) of WT and IP6K1-KO mice. Chow

(regular) diet-fed mice do not display any significant alteration in the Ube4A protein level (data not shown). However, high fat-fed (60% Kcal from fat) IP6K1-KO exhibit significantly less Ube4A protein level compared to their WT littermates (Figures 9A and

9B). Thus, IP6K1 stabilizes Ube4A protein in the adipose tissue especially under high fat diet conditions. As demonstrated in part 1, Ube4A degrades Akt. Thus, IP6K1 mediated stabilization of Ube4A in high fat fed mice may enhance degradation of Akt which can cause insulin resistance.

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Figure 10: IP6K1 protein is a degradable protein

Figure 10: Immunoblot showing result of inhibition of protein synthesis on IP6K1 level. IP6K1 seems to be fully depleted after 8 hours of CHX treatment.

By far, we demonstrated that

IP6K1 stabilizes Ube4A protein. Does

Ube4A regulate IP6K1 protein level? Prior to answering this question, we wanted to determine whether IP6K1 is a degradable protein in 3T3L1 adipocytes. To monitor this, we treated Myc-IP6K1 overexpressed 3T3L1 adipocytes with Cycloheximide.

Cycloheximide blocks protein synthesis and thus, the level of already synthesized protein

(in this case IP6K1) will be gradually depleted by degradation. This experiment helps in determining the half-life of proteins in cells (Chakraborty A, 2011b). We observed that

IP6K1 protein level is drastically diminished by 8h of Cycloheximide treatment which indicates that the protein has a half-life of ~4h (Figure 10). Thus, IP6K1 is degraded at a moderate rate. The UPS is the major pathway that degrades proteins. Moreover, Ube4A binds IP6K1. Thus, we presume that Ube4A is the ubiquitin ligase that degrades IP6K1.

However, we do not have evidence for this yet. Current experiments are ongoing in this direction.

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Figure 11: Known phosphorylation sites of IP6K1

Figure 11: Putative ubiquitination and degradation motifs in IP6K1.

How might Ube4A degrade IP6K1? Although the precise mechanism is not clear, it is conceivable that Ube4A ubiquitinates IP6K1 which leads to its degradation.

Bioinformatic analyses reveal that IP6K1 contains several lysine residues that are potential ubiquitination sites (red: highly likely; blue: less likely) (Figure 11). Moreover,

IP6K1 contains a highly stringent degradation-specific PEST (proline-glutamte-serine- threonine) consensus motif, which is phosphorylated in cells (Chakraborty et al. unpublished observation). IP6K2, the other IP6K isoform possesses a similar motif which leads to its degradation (Chakraborty A, 2011b). Thus, we presume that Ube4A regulates

IP6K1 stability by ubiquitinating one or more of the lysine residues. Furthermore, the ubiquitination may be influenced by phosphorylation by various protein kinases that are yet to be identified.

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Discussion

The current study reveals

Ube4A as a novel regulator of cell metabolism and stress. Ube4A regulates several important proteins in the cell such as Akt, BiP, β-catenin and IP6K1 (Figure 12). These proteins regulate various cellular pathways, Figure 12: Summary of IP6K1 and Ube4a Interactions including cell survival and metabolism. Moreover, aberrations in these proteins lead to diverse diseases like obesity/T2D, cancer and neurodegeneration. Thus, our study strongly indicates that

Ube4A may also be targeted in these diseases. Current studies are ongoing in the laboratory to determine Ube4A’s role in obesity/T2D.

The discovery that IP6K1 stabilizes Ube4A is interesting and adds another dimension to the complexity. In general, ubiquitin ligases regulate protein targets.

However, we demonstrate that IP6K1 regulates the regulator of protein homeostasis.

Therefore, our study unravels a hitherto unknown function of this unique class of enzyme. Further studies are needed to decipher the molecular mechanism of these interactions.

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Materials

Auxotrophic HEK-293 cells, requiring glucose and serum containing media to

survive

• 500 mL DMEM Phenol-Red Media, GIBCO/Invitrogen, Cat no: 11330-057

• Heat Inactivated Fetal Bovine Serum

• Various vectors containing the for Ube4A

• Jetprime Transfection Kit

• Cell Culture Incubator

• Gel Electrophoresis Equipment

• Gel Transfer Equipment

• Various Antibodies, noted beside Blots

• Film Developer equipment

• Chemiluminescent Film

• Antibodies: Endogenous Ube4A, BiP, Ubiquitin, β-catenin, LC3B,

Methods

Cell Culture: HEK293 cells were obtained to be transfected. Media contained ~10%

FBS, ~1% PenStrep, and was filtered. HEK293 cells were grown to 80-90% confluency.

Before splitting cells, the cell count was measured by using a cell counter. Cell count was around ~2 * 10^5. Cells were split evenly into one 6-well plate for experimentation and one 10 cm plate was left for maintenance.

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Transfection and Treatment: HEK293 cells in the 6-well plate were transfected when they reached 60-80% confluency. Generally, two micrograms of DNA or 50nM siRNA was gathered depending on the experiment. The transfection was performed according to the JetPrime protocol. Transfections proceeded for 24-48 hours. Cells were checked periodically throughout the transfection in order to estimate the toxicity of the proteins being used as well as to ensure that there will be enough live cells left for analysis.

Transfections were terminated by aspirating the media and washing with ice cold 1x PBS.

6-well plates were then store in -80 C until ready for lysate preparation.

Cell Lysate Preparation: After the transfection, the cell lysates were prepared. Each well was prepared for protein extraction. The 6-well plate was removed from -80 °C and put on ice. While on ice, ~100 µL of fresh cell lysis buffer with protease and phosphatase inhibitors was added to each well. The protease and phosphatase inhibitors are essential to prevent the degradation of proteins after the cells undergo apoptosis and lyse. The plate was kept on ice for 15 minutes, until it thawed and the lysis buffer acted on the entire well surface. The well contents were scratched and pipeted into 1.5 mL Eppendorf tubes. The tubes containing the cell lysates were centrifuged at 12,000 rpm for 10 minutes at 4 °C. At the end of the spin down, there was a white pellet in each Eppendorf tube containing the cell membrane and other large structural proteins from the cells. Only the supernatant was kept and pipeted it into new Eppendorf tubes. The tubes containing the pellet were thrown away. Optionally, the supernatants could be stored at -80 °C until we were ready to measure the protein concentration.

Protein Concentration Determination: In order to measure the protein content of each cell lysate, a BCA kit was be used. First, the supernatants were diluted in a 1:10 ration

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with water. The BCA kit contains BSA (albumin) standards including 0.125mg/mL,

0.25mg/mL, 0.5mg/mL, 1.0mg/mL, and 2.0mg/mL. These standards were used to compare the protein concentrations of the obtained samples using a spectrophotometer.

The reagents of the BCA kit were diluted in a 1:50 ratio. For example, take 200 uL of the blue reagent and 9,800 uL of clear reagent. 10 uL of water, the standards, and the samples were added in duplicate to a 96-well plate for photospectrometric analysis. The phospectrometer was set to a wavelength of 562 nm and each well in duplicate was analyzed and the absorbances were recorded. Because the samples and standards were added to the 96-well plate in duplicate, the mean value of the absorbance for each pair was considered, as it is closest to the true value for each sample. A standard curve was derived and the samples were compared to the standards of known concentration.

Sample preparation for SDS-PAGE: By using Excel software, the samples were prepared to contain equal amounts of protein per microliter. Samples consist of the supernatant, water, and 4x LDS. The 4x LDS includes a 1:10 ratio of beta-mercapto ethanol. If using a 15-well gel, which can hold approximately 20 uL of sample, prepare samples in such a way so that there is 1ug/uL to load in the well. Thus, 20 uL of sample will load 20 ug of proteins that can be analyzed. A 10-well gel can hold approximately 40 uL, so therefore samples should be prepared in such a way that 40 uL will contain 40 ug of proteins.

SDS-PAGE: Before running the gel, the samples were boiled for 5 minutes and spun down for a few seconds to collect everything at the bottom of the tube. SDS running buffer was prepared. ~6.5 uL of pre-stained molecular markers were loaded into the first

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lane of the gel and then the rest of the samples. The gels were run at a fixed voltage of

200 volts for 50 minutes.

Transfer of proteins in a nitrocellulose membrane: The proteins in the gel were transferred onto a nitrocellulose membrane by carefully removing the gel and placing it in 1x transfer buffer. An appropriately-sized membrane was then placed over the gel. The transfer was run at a fixed current of 0.72 amps for 45 minutes.

Blocking: In order to tag particular proteins of interest with antibodies, the membrane was first blocked with 5% milk in 1x TBST solution for 2 hours. Blocking prevents the antibodies from binding to a part of the membrane where there is no protein. After blocking, the membrane was washed three times with 1x TBST, for 5 minutes each rinse.

This removed excess milk and left the membrane ready for incubation with a primary antibody.

Primary Antibody: The 1x TBST was removed and the blocked membrane was incubated with primary antibody in 5% BSA in 1x TBST. Generally, the primary antibody dilution should be ~1:1000. However, for any tagged antibody (Myc, GST,

GFP, etc.) the dilution should be at least 1:5000 and diluted in 5% milk in 1x TBST instead of 5% BSA. Once the primary antibody was added, the membrane was stored in a

4 °C cold room on a shaker overnight.

Secondary Antibody: The next day, the primary antibody was removed and stored for future use. The membrane was then washed three times with 1x TBST for 10 minutes each wash. Next, the membrane was incubated with a secondary antibody that was HRP- linked. HRP stands for horse radish peroxidase, and reacts with a special substrate to give off light. The membrane gave off light and imprinted blots of varying intensity on a light-

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sensitive film. Secondary antibody incubation was done for at least one hour. It was essential to check the compatibility of the primary and secondary antibody (i.e. if the primary antibody came from a mouse, the secondary antibody must also be mouse).

Secondary antibodies had a dilution of 1:5000 in 5% milk (in 1x TBST).

Chemiluminescence: Following incubation with secondary antibody, the membrane was washed three times with 1x TBST for 10 minutes each. A reactive substrate was added to the membrane and allowed to react for 5 minutes. Afterwards, the membrane was placed in a Hyper-cassette and taken to a dark room to be developed. The Chemiluminescent films were opened only under red light. The films were exposed to the membrane for varying periods of time, from seconds to several minutes. After exposure, the film was inserted into the film developer were it was fixed and ready to be analyzed.

Analyses of films: Before removing the membrane from the Hyper-cassette, the location of the molecular markers was marked on the film by overlaying the film on the membrane. This was essential in order to ensure that the correct protein was being identified and analyzed.

Stripping of the Membrane: In the case that we wanted to check the binding of another antibody in at a similar molecular weight to a previous antibody, the membrane was stripped of antibodies by using OneMinute Strip. The membrane was bathed in solution for one minute on a shaker. Afterwards, the solution was recovered for future use. The membrane was reblocked with milk and washed with 1x TBST. Following that, the membrane was ready to be re-incubated with a new primary antibody following the steps above.

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Pharmacological Treatments

MG132: MG132 (carbobenzoxy-Leu-Leu-leucinal) is a peptide aldehyde, which effectively blocks the proteolytic activity of the 26S proteasome complex (Han et al.,

2009). MG132 treatment consisted of 20uM concentration in 6-well plates. Treatment lasted 3 hours, after which the cells were lysed and the proteins were analyzed.

Thapsigargin: During the unfolded protein response, Ca2+ ionophores such as ionomycin induce the UPR by depleting ER luminal Ca2+in mammalian cells (Cox et al., 2011).

Many ER-resident chaperones bind Ca2+ with high capacity and may require Ca2+ to function (Cox et al., 2011). Thus, Thapsigargin induces the UPR by depleting ER Ca2+ by irreversibly inhibiting SERCA Ca2+ ATPases in the ER membrane.

Cycloheximide: Cycloheximide was found to have a potent anti-proliferative effect on tumor cell lines. It selectively inhibits protein translation by blocking the translocation step in elongation (Schneider-Poetsch et al., 2010).

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Acknowledgements

I would like to sincerely thank my PI, Dr. Anutosh Chakraborty, for all of his help, guidance, and patience during this learning process. He has been instrumental in my learning and has inspired me to pursue further research. Additionally, I would like to thank my post-Doctorate mentors: Sarbani Ghoshal, Ph.D, Qingzhang Zhu, Ph.D, and

Ugander Gajjalaiahvari, Ph.D, for their guidance and time. I would like to thank Dr.

Chandrasekhar for her advising and superior teaching as well as Dr. Kundalkar for the opportunity of TAing in three different science labs this past year. I would also like to thank my parents and brother for supporting me and sacrificing so much so that I could have opportunities for a better life. I would not have made it this far without them.

Finally, I would like to thank Margaux Ehrlich for all her love and support during this past year.

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