Glucose Regulation of Novel Protein Complexes with Potential Roles in the Metabolic Reprogramming of Human Leukemia Cells

Transcription

1 Wesleyan University The Honors College Glucose Regulation of Novel Protein Complexes with Potential Roles in the Metabolic Reprogramming of Human Leukemia Cells by Ryan Graff Class of 2014 A thesis submitted to the faculty of Wesleyan University in partial fulfillment of the requirements for the Degree of Bachelor of Arts with Departmental Honors in Molecular Biology & Biochemistry Middletown, Connecticut April, 2014

2 Acknowledgements First and foremost, I have to thank my absolutely incredible and dedicated research adviser and principal investigator, Ameeta Kelekar. Without you I absolutely could not have accomplished what I have today. Thank you for accepting and welcoming me in to your lab family with open arms. Thank you for encouraging me, for believing in me, for putting up with my frequently crazy lab hours, and for fighting to allow me to return for a second summer at the U of M despite several obstacles. By the end of the two summers I spent in your lab I truly felt I had found a second family and home in Minnesota. And with that, I would like to thank all of the members of the Kelekar lab. It was an absolute joy to work with and learn from you all. A huge thank you to Xazmin, Eric, and Jenna for teaching me essentially all that I know about working in a lab and running all of the experiments I did to make this thesis possible. It is only fitting that I give an enormous thank you as well to my thesis adviser (and general life adviser), Manju Hingorani. Thank you for accepting the challenge of working with me and my inevitable bouts of procrastination. Thank you for your willingness to be my adviser and for proposing my thesis to the department despite its foreign origins. Perhaps most importantly, thank you for always being willing to talk and having stockpiles of chocolate ready in your office for when I need them most. Last but definitely not least, I have to thank my beautiful roommates and friends for making my last year at Wesleyan one of the best years of my life. I would especially like to thank Brianne Wiemann and Johnny LaZebnik, who both constantly nagged me to continue working on this thesis and provided me with constant support and encouragement throughout the past year. ii

3 Abstract One of the hallmarks of cancer is the altered metabolic profile observed in cancer cells marked by increased glucose consumption and lactate production. Although these characteristics are largely understood, how cells undergo metabolic reprogramming to achieve this altered metabolic state is unknown. Here, we present a pair of novel of protein complexes as putative candidates with regulatory roles in the metabolic reprogramming of human leukemia cells. Each of these complexes contains an identical core of three proteins: the metabolic enzyme GAPDH and two members of the apoptosis regulating Bcl-2 protein family, as well as two to three additional peripheral proteins that vary between the two complexes. We explored the response of these complexes to various lengths of glucose deprivation in order to characterize the complexes and elucidate potentially functionally important information. Complexes were isolated by gel filtration chromatography and immunoprecipitation and run through a variety of in vitro assays involving Western blotting and 2D gel electrophoresis. One of the complexes was found to have an inherent dependence on the presence of glucose for its integrity and, possibly, its function, suggesting a role for it in promoting glycolysis as observed in the Warburg effect. A potential role for EF-1ϒ (a protein in this complex) in the protective glutathionylation of GAPDH in response to glucose deprivation is suggested. The second complex was found to be less responsive to glucose deprivation, possibly indicating an alternative role for this complex in promoting metabolism necessary for cell survival. These results provide evidence for the roles of these complexes in the regulation of cancer cell metabolism, potentially highlighting the complexes as novel targets for cancer therapeutics. iii

4 Table of Contents Page Acknowledgements Abstract List of Figures ii iii vi Chapter 1: Introduction 1.1 Apoptosis and the Intrinsic Pathway The Bcl-2 Superfamily as Regulators of Apoptosis Apoptotic Misregulation and Cancer Cancer Cell Metabolism and the Connection to Apoptosis The Dual Functionality of the Bcl-2 Superfamily The Novel Noxa/Mcl-1 Protein Complexes Specific Aims and Proposed Studies 37 Chapter 2: Materials and Methods 2.1 Cell lines, cell culture, and cell lysis Glucose Deprivation Immunoprecipitation and Antibodies Western Blotting Gel Exclusion Chromatography Two-Dimensional Gel Electrophoresis Gel Staining 43 Chapter 3: Results 3.1 Dynamic interplay suggested to occur between complexes Shifts in complex expression shown with glucose deprivation Primary T cells show similar complex expression patterns Modifications to cellular components of the complexes Modifications to direct complex components 68 iv

5 Chapter 4: Discussion 4.1 Overview Stability of the complexes is dependent on glucose A role for EF-1ϒ as a putative glutathione transferase Roles for the described complexes in metabolic regulation Alterations to cellular complex components with deprivation 80 Chapter 5: Conclusions and Future Directions 83 References 85 v

6 List of Figures Chapter 1: Introduction Page Figure 1.1: The Apoptotic Pathways and Their Regulation 3 Figure 1.2: BH Domain Structures of the Bcl-2 Protein Superfamily Figure 1.3: Metabolic Pathways Associated with Rapid Cell Proliferation and their Regulation by Oncogenes and Tumor Suppressor Genes Figure 1.4: Western Blots of Column Fractionated Lysates Reveal Noxa Complexes Figure 1.5: A Graphic Model and Chart Representation of the Described Complexes Chapter 3: Results Figure 3.1: Native Gel Electrophoresis of Mcl-1 Immunoprecipitates of Cytosolic Lysates from Unperturbed and Glucose Deprived Jurkat Cells Figure 3.2: Western Blotting of Mcl-1 Immunoprecipitates of Cytosolic Lysates from Unperturbed and Glucose Deprived Jurkat Cells Resolved by SDS PAGE Figure 3.3: Western Blotted Fractions of Cytosolic Lysates of Unperturbed and Glucose Deprived Jurkat Cells Run by Column Chromatography Figure 3.4: Western Blotted Fractions of Cytosolic Lysates of Unstimulated and Stimulated Primary T Cells Run by Column Chromatography Figure 3.5: 2D Gel Electrophoresis of Cytosolic Lysates of Unperturbed and Glucose Deprived Jurkat Cells Figure 3.6: Western Blotted 2D Gel Electrophoresis of Cytosolic Lysates of Unperturbed and Glucose Deprived Jurkat Cells Figure 3.7: 2D Gel Electrophoresis of Mcl-1 IPs of Pooled Gel Filtrated Lysate Fractions Shown to Contain Mcl-1 from Lysate of Unperturbed and Glucose Deprived Jurkat Cells vi

7 Chapter One: Introduction 1.1 Apoptosis and the Intrinsic Pathway The term apoptosis was first coined by John Kerr as a way to describe controlled cell deletion (Kerr et al., 1972). This process is now known to have implications in tissue homeostasis, growth and development, and bodily defense mechanisms in response to damaging agents and disease. When this process is not properly regulated, diseases such as cancer, autoimmune disorders, and autophagy can occur. When considering apoptosis, it is important to distinguish it from other forms of cell death. While apoptosis is an active and controlled mechanism of cell death, oncosis (also known as ischemic cell death) is the passive or accidental death of a cell (Manjo and Joris 1995). Necrosis, on the other hand, is the term used to describe the secondary morphological changes that occur in cells as a result of either apoptosis or oncosis and is not, in fact, a term for a form of cell death (Manjo and Joris 1995). The necrosis that results from apoptosis is also very distinct to the necrosis that results from oncosis. Apoptosis results in cell shrinkage, followed by a blebbing of the cell membrane, before budding occurs to form apoptotic bodies. Neighboring cells then engulf these apoptotic bodies via phagocytosis to prevent the release of cellular contents to the environment (Elmore 2007; Manjo and Joris 1995). Oncosis, on the other hand, results in cell swelling and a loss of integrity of the cell membrane that eventually results in rupturing of the membrane to release the contents of the cell into the environment (Elmore 2007; Manjo and Joris 1995). 1

8 Whether a cell commits to the apoptotic or oncotic cell death pathway depends on the cell death signal, the developmental stage of the cell, and the tissue type (Zeiss 2003). Within the apoptotic pathway, there are two established routes of cell death: the intrinsic or mitochondrial pathway and the extrinsic or death receptor pathway. The pathway that is important with respect to the research described in this thesis is the intrinsic, non-receptor mediated pathway. An outline of this process and the proteins involved in its regulation is shown in the figure below. Details of this process will be described in the following sections of this introduction as the complexes being analyzed in subsequent chapters were initially discovered through studies on the proteins involved in regulation of these pathways. Thus, an understanding of the pathways and the related proteins are essential to a fundamental understanding of the substituents of the complexes at hand. 2

9 Figure 1.1: The Apoptotic Pathways and Their Regulation Figure 1.1: The Apoptotic Pathways and Their Regulation: A model of the intrinsic (1) and extrinsic (2) pathways of apoptosis with the regulatory Bcl-2 proteins shown (Kang and Reynolds 2009). The intrinsic pathway is the major form of apoptosis that occurs in vertebrate cells (Green and Kroemer 2004). It is induced in response to various cell stresses including DNA damage, redox stress, cytoskeletal damage, endoplasmic reticulum stress, loss of adhesion, growth factor withdrawal, and viral infections, among others 3

10 (Chipuk et al. 2006; Cotter 2009; Elmore 2007). It is known as the mitochondrial pathway because it is primarily dependent on a disruption of the integrity of the outer mitochondrial membrane (Fulda and Debatin 2006). This permeabilization of the membrane is lethal to cells because it results in either a loss of the metabolic function of the mitochondria, a release of death inducing molecules both caspase dependent and independent from the mitochondrial intermembrane space, or both (Green and Kroemer 2004). Caspases are cysteine aspartases that cleave proteins at aspartic acid peptide bonds in order to initiate apoptosis. Two types of caspases exist in cells: initiator and effector caspases. Initiator caspases (e.g. caspase-9) cleave and activate effector caspases (e.g. caspase-3), which subsequently cleave further downstream targets to induce apoptosis (Chang and Yang 2000). Upon permeabilization of the mitochondrial membrane, pro-apoptotic factors are released into the cytosol from the outer mitochondrial membrane in two groups: the initial caspase dependent group (consisting of cytochrome c, Smac/DIABLO, and the serine protease HtrA2/Omi) and a later caspase independent group (consisting of AIF, endonuclease G, and CAD) (Chipuk and Green 2008; Elmore 2007). Upon release, cytochrome c associates with and activates Apaf-1 and procaspase-9 to form the apoptosome (Elmore 2007). This leads to activation of initiator caspase-9, followed by effector caspases, such as caspase-3 and -7, and culminates in cell death typical of apoptosis (Chipuk et al. 2006; Elmore 2007). Smac/DIABLO and HtrA2/Omi function by inhibiting the activity of IAPs (inhibitors of apoptosis proteins) (Elmore 2007). All of the later released, caspase independent pro-apoptotic factors translocate to the nucleus to facilitate DNA fragmentation and chromatin 4

11 condensation once the cell is completely committed to the apoptotic pathway (Elmore 2007). In the intrinsic pathway the permeabilization of the outer mitochondrial membrane, as well as the release of cytochrome c and other pro-apoptotic agents, is considered to be the point of no return for the cell (Chipuk et al. 2006; Kelekar and Thompson 1998). This final step that commits the cell to apoptosis must therefore be tightly regulated. 1.2 The Bcl-2 Superfamily as Regulators of Apoptosis The release of death inducing agents from the mitochondrial intermembrane space is regulated by the B cell lymphoma 2 (Bcl-2) protein superfamily (Cory and Adams 2002). This class of proteins consists of both pro- and anti-apoptotic proteins that all share at least one of the four conserved alpha helical Bcl-2 homology (BH) domains (Kelekar and Thompson 1998). Most of the Bcl-2 proteins also contain a C- terminal signal sequence and transmembrane region that localizes the proteins to the outer mitochondrial membrane, endoplasmic reticular membrane, and outer nuclear envelope (Krajewski et al. 1993; Akao et al. 1994). This protein family is unique due to the ability of its members to form both homo and heterodimers with one another (Kelekar and Thompson 1998; Oltval et al. 1993; Yang et al. 1995; Sedlack et al. 1995). This dimerization has been shown, at least in some instances, to occur as a result of shared BH domains between dimer members (Sedlack et al. 1995). These interactions between members of the Bcl-2 family are believed to be responsible for regulating the apoptotic pathway (Cory and Adams 2002; Kelly and Strasser 2011). 5

12 Within the Bcl-2 superfamily, there are five anti-apoptotic proteins: Bcl-2, Bcl-xL, Bcl-w, A1, and Mcl-1 (myeloid cell leukemia sequence 1), along with two families of pro-apoptotic proteins: the multi-bh domain family and the BH3-only family (Cory and Adams 2002; Kelly and Strasser 2011). The anti-apoptotic Bcl-2 proteins contain all four of the BH domains as well as a transmembrane region (Yip and Reed 2008). The only commonality between the pro-apoptotic family members is the presence of the BH3 domain (Kelekar and Thompson 1998). The BH4 domain has been described as imparting an anti-apoptotic function to the majority of the antiapoptotic proteins (Cheng et al. 1997; Hunter et al. 1996; Sugioka et al. 2003). Cleavage of this domain from anti-apoptotic Bcl-2 proteins has been shown to either partially or fully negate their anti-apoptotic function or impart a pro-apoptotic function to the deletion mutants (Cheng et al. 1997; Hunter et al. 1996). An illustration of the domain structures of each type of Bcl-2 protein is shown in the figure below. 6

13 Figure 1.2: BH Domain Structures of the Bcl-2 Protein Superfamily Figure 1.2: BH Domain Structures of the Bcl-2 Protein Superfamily: Comparisons of the presence of Bcl-2 homology (BH) domains between the three Bcl-2 protein families (Yip and Reed 2008). Three of the Bcl-2 members, the anti-apoptotic Bcl-2 and Bcl-xL and the proapoptotic multi-bh domain Bax, have been shown to have a structural similarity to that of the bacterial ion-pore-forming toxins diphtheria toxin and the colicins (Muchmore et al. 1996; Schendel et al. 1997; Minn et al. 1997; Schlesinger et al. 1997). The BH1 and BH2 domains of these proteins flank two hydrophobic helices in the protein core that are believed to insert themselves into membranes in order to allow for the formation of a pore (Kelekar and Thompson 1998; Muchmore et al. 1996). The fact that proteins known to regulate apoptosis at the mitochondrial membrane surface were found to regulate ion flux across membranes was no surprise, given that subcellular distribution of ions and small molecules, as well as a collapse 7

14 of the mitochondrial proton gradient, are known to regulate the drive toward apoptosis in cells (Kelekar and Thompson 1998). Although Bax differs from Bcl-2 and Bcl-xL in that it is a pro-apoptotic as opposed to a pro-survival protein, it was found that the pores formed by Bax differ in their ion selectivity, conductance, and voltage dependence as compared to the pores formed by its anti-apoptotic relatives (Schlesinger et al. 1997). Of the anti-apoptotic proteins, the structures of Bcl-2, Bcl-xL, and Bcl-w all share a hydrophobic groove formed by the BH1, BH2, and BH3 domains that allows for binding to the BH3 domain of pro-apoptotic BH3-only proteins (Cory and Adams 2002; Sattler et al. 1997). The anti-apoptotic proteins A1 and Mcl-1 have more divergent sequences as compared to Bcl-2, Bcl-xL, and Bcl-w and are known to have weaker survival activity, possibly indicating that these two proteins have additional functions that have yet to be fully elucidated (Cory and Adams 2002). Before discussing the pro-apoptotic members of the Bcl-2 family, an introduction to the domain that gives these proteins their cytotoxic potential must be made. The BH3 domain was first discovered as a stretch of sixteen amino acids in the multi-bh domain pro-apoptotic protein Bak and was described as the region of Bak that is necessary and sufficient for its cytotoxic activity and binding to Bcl-xL (Chittenden 1995; Kelekar and Thompson 1998). This domain was later found to form an amphipathic alpha helix that bound strongly to the hydrophobic pocket formed by the BH1, BH2, and BH3 domains of some of the anti-apoptotic proteins (Cory and Adams 2002; Sattler et al. 1997). Although it is contained in every member 8

15 of the Bcl-2 superfamily, the BH3 domain is known as a functionally important domain for the death promoting activity of the pro-apoptotic members of the family. The majority of the pro-apoptotic Bcl-2 proteins belong to the BH3-only family. This subgroup of proteins is comprised of Bim, Puma, Bid, Noxa, Bad, Hrk, Bmf, and Bik (Kelly and Strasser 2011). These proteins appear to promote death via inhibition of anti-apoptotic Bcl-2 members or activation of other pro-apoptotic Bcl-2 members (namely Bax and Bak) (Huang and Strasser 2000). Although all members of the Bcl-2 family contain the BH3 domain, the BH3-only family members are the only proteins in the Bcl-2 family that share no sequence similarity to the anti-apoptotic proteins other than their shared BH3 domain (Kelly and Strasser 2011). They also lack the ability to homodimerize and can only form heterodimers with anti-apoptotic Bcl-2 members (Kelly and Strasser 2011). Some of the BH3-only proteins namely Bim, Puma, and tbid bind all anti-apoptotic proteins with high affinity (Kuwana et al. 2005). Others such as Noxa, which binds specifically to Mcl-1 and A1 are more selective and bind only a few of the anti-apoptotic proteins in the cell (Chen et al. 2005). With the exception of Bid and Bad, all known BH3-only proteins contain a hydrophobic C-terminal domain that localizes these proteins to intracellular membranes (Kelekar and Thompson 1998). As mammals contain at least thirteen members of the BH3-only family, it is theorized that each member may have a specific function in a stage of development for an organism (Lomonosova and Chinnadurai 2008). For example, Hrk is expressed in neurons targeted for apoptosis, suggesting its role in neuronal development (Imaizumi et al. 1999). 9

16 In addition to BH3-only proteins, the pro-apoptotic Bcl-2 family also includes the multi-bh domain proteins Bax, Bak, and Bok (Kelly and Strasser 2011). All of these proteins contain every one of the four BH domains as well as the transmembrane region, making them more similar in sequence to the anti-apoptotic proteins than their pro-apoptotic BH3-only relatives (Kelly and Strasser 2011; Suzuki 2000; Oltval et al. 1993). These proteins are essential to the cell death pathway, and many studies have shown that without activation of Bax and/or Bak, apoptosis will not occur via the intrinsic pathway (Lindsten et al. 2000; Wei et al. 2001; Zong et al. 2001). While BH3-only proteins appear to initiate cell death indirectly, via inhibition of anti-apoptotic proteins or activation of multi-bh domain pro-apoptotic proteins, Bak and Bax appear to be able to induce cell death more directly (Reed 2006). As previously stated, the structure of Bax is very similar to that of the anti-apoptotic proteins Bcl-2 and Bcl-xL, as each of these proteins appears to serve as an ion-pore forming protein in cell membranes (Muchmore et al. 1996; Schendel et al. 1997; Minn et al. 1997; Schlesinger et al. 1997). The different ion selectivity, conductance, and voltage dependence between Bax and its anti-apoptotic relatives are presumed to explain the opposing functions of these proteins despite their structural similarities (Schlesinger et al. 1997). Bak is thought to have a similar structure to Bax and is known to also contain a hydrophobic C-terminal domain that allows for its insertion into intracellular membranes (Reed 2006). In order to initiate apoptosis, it is thought that BH3-only proteins activate multi-bh domain proteins to induce a conformational change in these proteins at the outer mitochondrial membrane surface to promote their oligomerization and the 10

17 formation of a pore through which pro-apoptotic factors (such as cytochrome c) can be released from the mitochondrial intermembrane space (Waterhouse et al. 2002; Wei et al. 2000). Bax dimers were demonstrated to have ion-channel features, whereas Bax tetramers were shown to have a larger pore diameters demonstrated to transport cytochrome c (Korsmeyer et al. 2000). Two models have been proposed to explain the interactions between Bcl-2 family members that regulate apoptosis: the direct and indirect activation models (Kelly and Strasser 2011). According to the indirect activation model, binding to the anti-apoptotic Bcl-2 proteins sequesters Bax and Bak in a primed state in the cell. When BH3-only proteins are activated by transcriptional up-regulation and/or posttranslational modification, the BH3-only proteins displace Bax and Bak and preferentially bind the anti-apoptotic Bcl-2 proteins via insertion of their BH3 domain into the hydrophobic groove of the anti-apoptotic proteins formed by domains BH1, BH2, and BH3. This frees the primed Bax and Bak proteins to oligomerize at the mitochondrial outer membrane surface and induce permeabilization (Kelly and Strasser 2011; Willis et al. 2007). The direct activation model postulates that the BH3-only proteins can be categorized into activators (Bim, tbid, and Puma) and sensitizers (Bad, Bmf, Noxa). In this scenario, it is the activators that are sequestered by binding to the anti-apoptotic Bcl-2 proteins. When apoptosis is initiated, the sensitizers are activated once again by transcriptional up-regulation or posttranslational modification to preferentially bind to the anti-apoptotic Bcl-2 proteins via their BH3 domains and free up the activator proteins. The activators then bind to Bax and Bak, inducing their activation and oligomerization at the mitochondrial 11

18 surface (Kelly and Strasser 2011; Chipuk and Green 2008). Interestingly, studies performed on both cultured cells and in vivo suggest that aspects of both models are present in the pathways of intrinsic apoptosis that occur in cells (Kuwana et al. 2005; Mérino et al. 2009). 1.3 Apoptotic Misregulation and Cancer As mentioned before, misregulation of the apoptotic pathways is known to result in several different disease states. In particular, alterations that result in a suppression of apoptosis are known to aid in the development and progression of cancers (Elmore 2007). Cancer cells are able to mount resistance to apoptosis via an up-regulation of anti-apoptotic proteins such as Bcl-2 or a mutation or downregulation of pro-apoptotic proteins such as Bax (Elmore 2007). The tumor suppressor gene p53 initiates apoptosis in response to DNA damage via transcriptional regulation of these proteins (Elmore 2007). In addition to transcriptional regulation, p53 also directly activates Bcl-2 proteins at the mitochondrial surface both via inhibition of anti-apoptotic proteins and activation of Bax and Bak oligomerization in response to apoptotic stimuli (Mihara et al. 2003; Chipuk et al. 2004; Moll et al. 2005). Various studies have documented the connection between apoptotic misregulation and the development of cancer. In 1984, it was found that 85% of human follicular lymphomas contain a translocation of the Bcl-2 gene into the locus of the immunoglobulin heavy chain (Tsujimoto 1984). It was later documented that up-regulation of Bcl-2 in B-cell precursors promotes cell proliferation and results in 12

19 tumorigenic cell populations when working in conjunction with c-myc (Vaux et al. 1988). These findings were later verified in vivo through the use of transgenic mice (McDonnell and Korsmeyer 1991). Inactivating Bax mutations have been documented in more than fifty percent of mismatch repair-deficient colorectal tumors, suggesting that an inactivation of its pro-apoptotic function is selected for in colorectal carcinogenesis (Rampino et al. 1997). In B cells, it was shown that an inactivation of even a single allele of Bim accelerates Myc-induced development of tumors (Egle et al. 2004). RNAi induced suppression of Puma promoted oncogenic transformation of primary murine fibroblasts and accelerated myc-induced lymphomagenesis (Hemann et al. 2004). An extensive study performed in 2010 that surveyed over 3,000 cancer copy-number profiles from published abstracts found that the most significantly enriched literature term associated with protein copy number amplification peaks was apoptosis. Anti-apoptotic Bcl-2 proteins were always found in amplification peaks and pro-apoptotic Bcl-2 proteins were always found in deletion peaks in the cancer profiles. Such studies provide strong evidence for the idea that genetic alterations in Bcl-2 proteins that inhibit the apoptotic pathway are selected for in the development of cancer (Beroukhim et al. 2010). The majority of chemotherapeutic therapies against cancers induce apoptosis by activating the intrinsic pathway. It is therefore understandable that mutations to proteins affecting the regulation of this pathway are frequently associated with drug resistance in cancers (Fulda and Debatin 2006). Many anti-cancer therapies have therefore been designed to target the Bcl-2 family of proteins. Antisense techniques, BH3-domain peptides, and synthetic molecule drugs designed to interfere with the 13

20 function of anti-apoptotic Bcl-2 proteins have all been explored as methods to combat the frequent up-regulation of anti-apoptotic proteins in cancer cells (Fulda and Debatin 2006). An eighteen base antisense oligonucleotide targeted against Bcl-2 mrna was shown to reduce the expression of Bcl-2 and increase tumor cell apoptosis in patients with advanced malignant melanoma expressing Bcl-2 (Jansen et al. 2000). Bispecific antisense oligonucleotides directed against a sequence shown to be highly homologous between Bcl-2 and Bcl-xL have also been developed and demonstrated to induce apoptosis and increase chemosensitivity in tumor cells in vitro (Zangemeister-Wittke 2000; Yamanaka et al. 2005). BH3 domain peptides were also shown to sensitize or activate mitochondrial apoptosis in cancer cells in vitro (Letai et al. 2002). Small molecule inhibitors of Bcl-2 and related anti-apoptotic proteins have also been proposed. Computer screens of small molecules with potential to bind the hydrophobic BH3 domain binding pocket in the anti-apoptotic Bcl-2 protein resulted in the discovery of a small molecule effector that induces apoptosis of human acute myeloid leukemia in vitro via inactivation of the Bcl-2 protein (Wang et al. 2000). Clearly success has been achieved in the in vitro targeting of Bcl-2 proteins with anticancer therapeutics. These findings underscore the potential for vivo treatments that target chemoresistant cancers by modifying the expression or activity of Bcl-2 family proteins. 14

21 1.4 Cancer Cell Metabolism and the Connection to Apoptosis Cancer is by nature a metabolic disease. One of the hallmarks of cancer cells is their altered metabolic state that preferentially metabolizes glucose via glycolysis (as opposed to oxidative phosphorylation) even in oxygen-rich environments. This metabolic alteration results in increased glucose consumption and lactate production in these cells (Warburg 1956). This phenotype of cancer cells has now come to be known as the Warburg effect. An outline of the major metabolic pathways involved in the Warburg effect and general cancer cell metabolism are outlined in the figure below. 15

22 Figure 1.3: Metabolic Pathways Associated with Rapid Cell Proliferation and their Regulation by Oncogenes and Tumor Suppressor Genes Figure 1.3: Metabolic Pathways Associated with Rapid Cell Proliferation and their Regulation by Oncogenes and Tumor Suppressor Genes: This schematic displays the major metabolic pathways known to influence proliferation in cells. How these metabolic pathways are regulated and how they relate to biosynthetic pathways essential for proliferation are all shown. Many of the enzymes and processes outlined in this diagrammed will be referred to throughout the remainder of this thesis (Vander Heiden et al. 2009). Although an exact explanation as to why the Warburg effect is observed in cancers has not been agreed upon, several theories attempt to explain this seemingly odd observation. 16

23 To start, a metabolic rearrangement is necessary in order to sustain the incredible rate of proliferation observed in cancers. Rapidly dividing cells have a greater requirement to provide substrates for biosynthetic pathways necessary to maintain cell growth, and the altered metabolic profile of cancer cells is ideal for generating such substrates. In fact, the higher rate of glycolysis in cancer cells appears to be directed towards the synthesis of amino acids, nucleotides and lipids as opposed to oxidative phosphorylation. This altered metabolic flux appears to be necessary for and may even promote tumorigenesis (Hatzivassiliou 2005). It is known that tumor cells exclusively express an alternative form of pyruvate kinase that catalyzes the conversion of phosphoenolpyruvate to pyruvate more slowly than the normally expressed isoform. Although this may seem contrary to the Warburg effect, this alteration is in fact necessary for the growth of tumor cells and was demonstrated to increase the incorporation of carbon from glucose molecules into biosynthetic products, perhaps due to a build-up of glycolytic intermediates with potential to be utilized in biosynthetic processes (Christofk et al. 2008). Some have also suggested that the altered metabolism of cancer cells is selected for by the tumor microenvironment. As a tumor expands, the blood flow that supplied the original tumorigenic cells with oxygen eventually becomes insufficient to supply oxygen to all of the cells of the tumor. This leads to hypoxia and a stabilization of the hypoxia inducible factor, HIF. HIF subsequently alters the transcriptional make-up of the cell to provide solutions to the hypoxic stress experienced by the tumorigenic cells. This includes activation of enzymes that promote glycolysis or suppress oxidative phosphorylation thereby uncoupling 17

24 glycolysis from oxygen levels and reducing the oxygen requirements of the cells (Kaelin and Ratcliffe 2008; Hsu and Sabatini 2008). Yet another theory proposes that oncogenic activation results in the alterations in metabolism that are noted as the key aspects of the Warburg effect. Activation of the oncogene Ras was shown to increase rates of aerobic glycolysis (Dang and Semenza 1999). The transcription factor Myc has also been shown to increase the transcription of metabolic genes (Gordan et al. 2007). In addition to inhibiting the function of Bcl-2 pro-apoptotic BH3-only proteins, Akt kinase was demonstrated to increase glucose consumption and the rate of aerobic glycolysis in cancer cells (Manning and Cantley 2007; Elstrom et al. 2004). However, these observations are not unique to oncogenes, as tumor suppressors have also been implicated in the regulation of metabolism. Perhaps one of the clearest connections between cancer metabolism and the apoptotic response can be made through an analysis of the tumor suppressor protein p53. This protein is almost universally inactivated in cancers, presumably as a way to prevent cell cycle arrest and apoptosis. Various studies have shown that p53 also directly opposes the Warburg effect by decreasing glucose uptake, down-regulating glycolysis, and up-regulating oxidative phosphorylation. To start, p53 represses the transcription of the glucose transporters GLUT1 and GLUT4 (Schwartzenberg-Bar- Yoseph et al. 2004). P53 also induces expression of TIGAR (TP-53-induced glycolysis and apoptosis regulator) and directly down-regulates the glycolytic enzyme phosphoglycerate mutase, both of which decrease the rate of glycolysis in cells (Bensaad et al. 2006; Kondoh et al. 2005). In addition, p53 activates the pyruvate 18

25 dehydrogenase complex (thereby inducing production of acetyl-coa and upregulating the TCA cycle in favor of lactate production) through an inhibition of pyruvate dehydrogenase kinase (Contractor and Harris 2012). Lastly, p53 influences the respiratory chain in oxidative phosphorylation by transcriptionally activating two proteins essential for the assembly of complex IV of the mitochondrial electron transport chain (Okamura et al. 1999; Matoba et al. 2006). A recent study of p53 in mouse models demonstrated that the regulatory metabolic functions and tumor suppressing functions of p53 are inextricably linked. In this study, mice were generated with a p53 mutant that lacked its canonical cell cycle arrest, senescence and apoptosis inducing functions. These mice did not display early-onset tumor formation. Only p53 null mice experienced spontaneous development of cancers. This finding indicates that the non-canonical metabolic regulatory functions of p53 are essential to its tumor suppressor function, providing a clear link between both metabolic and apoptotic pathways in cancer formation (Li et al. 2012). Further studies have enhanced this link by demonstrating that metabolic pathways also regulate apoptosis. Initially, it was demonstrated that increased expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) the enzyme that catalyzes the sixth step in glycolysis prevented caspase-independent cell death. GAPDH inhibition of caspase-independent cell death was hypothesized to occur by autophagic removal of damaged mitochondria a process fueled by elevated ATP levels from an up-regulated glycolytic pathway in tumors (Colell et al. 2007). With the breakdown of glucose via glycolysis found to be implicated in the inhibition of 19

26 the caspase-independent pathway of cell death, metabolism of glucose via the pentose phosphate pathway (PPP) was then discovered to be implicated in the inhibition of the caspase-dependent pathway. In 2008, it was shown that the pro-apoptotic activity of cytochrome c is dependent on its redox state. It was then found that, in cancer cells where the PPP is up-regulated for biosynthesis and intracellular glutathione levels are high due to a high production of NADPH via the PPP, cytochrome c is reduced and rendered inactive by intracellular glutathione (Vaughn and Deshmukh 2008). Both of these studies display a clear connection between the regulation of metabolism and apoptosis. They demonstrate that adopting the metabolic phenotype of the Warburg effect imparts a selective advantage to cancer cells by inhibiting the induction of apoptosis. In support of this argument, it has been shown that altering the metabolic profile of cancer cells to up-regulate oxidative phosphorylation in favor of aerobic glycolysis facilitates apoptosis and inhibits tumor growth. This is proposed to occur via an increase in mitochondrial ROS levels and therefore oxidative stress in the mitochondria which inhibits the function of the electron transport chain, reducing the unusually high mitochondrial membrane potential observed in cancers (Bonnet et al. 2007). Sustained reduction in mitochondrial membrane potential has been established as an early irreversible step for the induction of mitochondrial apoptosis in cancer cells (Zamzami et al. 1995). In any sense, it once again illustrates the selective advantage of an up-regulated aerobic glycolytic pathway for cancers to evade apoptosis. 20

27 1.5 The Dual Functionality of the Bcl-2 Superfamily Due to the very clear connections that have been established between the apoptotic and metabolic pathways, it may be no surprise that members of the Bcl-2 superfamily have recently been implicated in the regulation of metabolism under conditions in which apoptosis is not induced. A dual functionality for the Bcl-2 proteins was first suggested when a 1996 study found that the BH3-only protein Bad was phosphorylated in response to the presence of survival factor IL-3 in cells. Only nonphosphorylated Bad was shown to heterodimerize with Bcl-xL. Phosphorylated Bad was sequestered in the cytosol bound to cytosolic phosphoserine binding protein, , and its pro-apoptotic function was thus inhibited (Zha et al. 1996). Later studies showed that in liver mitochondria, Bad resides in a protein complex with four other proteins: the catalytic units of protein kinase A and protein phosphatase 1, WAVE-1 as an A kinase anchoring protein, and glucokinase (hexokinase IV) (Danial et al. 2003). Bad was shown to be necessary for the formation of this complex. In Bad-deficient hepatocytes, mitochondrial glucokinase activity was diminished. Additionally, mice bearing a non-phosphorylatable Bad mutant displayed abnormal glucose homeostasis and defects in glucose tolerance (Danial et al. 2003). In this way, Bad and the protein complex it forms when phosphorylated has been shown to have a clear regulatory role in the metabolism of glucose. Although the dual-functionality of Bad may be the best established of the Bcl- 2 proteins, emerging evidence points to regulatory metabolic roles for other members of this superfamily. Interest in a novel function for Bcl-xL was first sparked when a study found that Bcl-xL had a Bax-independent mechanism for the inhibition of 21

28 apoptosis. It was previously thought that, in order to inhibit apoptosis, Bcl-xL needed to bind directly to Bax to inactivate its pro-apoptotic function. However, mutations in Bcl-xL to prevent Bax-binding still preserved 70-80% of the anti-apoptotic activity of Bcl-xL (Cheng et al. 1996). Later studies found that Bcl-xL induced outer mitochondrial membrane permeability to anionic metabolites without allowing for the release of cytochrome c a necessary process for coupled cellular respiration (Vander Heiden et al. 2001). Then, in 2011, it was shown that Bcl-xL overexpression leads to a reduction in acetyl-coa levels, which, in turn, results in decreased levels of N- alpha-acetylated proteins in the cell. N-alpha-acetylation is known to increase cell sensitivity to apoptotic stimuli. In this way, Bcl-xL was shown to mediate apoptosis through a metabolic pathway (Yi et al. 2011). This metabolic influence was further revealed when it was found that Bcl-xL increases the efficiency of mitochondrial metabolism by decreasing an ion leak within the F1F0-ATP synthase complex, thereby increasing the net transport of H+ by F1F0 and thus the overall production of ATP. Inhibition of Bcl-xL also leads to decreases in F1F0-ATP synthase activity, confirming the influence this anti-apoptotic protein has on metabolism (Alavian et al. 2011). Another anti-apoptotic Bcl-2 protein with strong evidence for a role in metabolic regulation is Mcl-1. While the full-length, anti-apoptotic isoform of Mcl-1 is known to reside on the outer mitochondrial membrane, a smaller isoform lacking 33 amino acids on the N-terminus appeared to localize in the mitochondrial matrix. This matrix-localized smaller isoform failed to interact with BH3-only proteins and exhibited attenuated anti-apoptotic activity. Interestingly, when ectopically expressed, 22

29 the shorter isoform failed to enter the mitochondrial matrix and retained its antiapoptotic function and ability to interact with BH3-only proteins. This suggests that the anti-apoptotic function of Mcl-1 is inhibited upon entrance to the mitochondrial matrix (Huang and Yang-Yen 2010). After the discovery of this isoform, a separate study found that the matrix-localized Mcl-1 isoform played a role in the regulation of mitochondrial inner membrane structure and function. Upon import to the mitochondria, Mcl-1 is proteolytically cleaved, resulting in a smaller isoform that is tethered to the inner membrane of the mitochondria, exposed to the matrix. In the mitochondria, Mcl-1 maintains the structure of the inner membrane, regulates mitochondria fusion, and maintains levels of mtdna. Destabilization of complexes of the electron transport chain occurs in the absence of matrix associated Mcl-1, possibly due to loss of mitochondrial DNA that encodes the subunits for the complexes. Mcl-1 is also required for proper assembly of F1F0-ATP synthase into higher-order oligomers. Overall, it can be seen that the non-apoptotic functions of Mcl-1 regulate mitochondrial functioning in cells. This may facilitate generation of mitochondria-derived substrates required for biosynthesis, possibly explaining why, in addition to its anti-apoptotic function, Mcl-1 is one of the most highly amplified genes in various cancers (Perciavalle et al. 2012). Interestingly, in addition to its regulation of mitochondrial metabolism, it was found that Mcl-1 itself is a metabolically regulated protein. In the absence of glucose, Mcl-1 is phosphorylated by glucose synthase kinase-3 (GSK-3) targeting it for degradation by the proteasome. However, growth factor stimulated glucose uptake results in the inhibition of GSK-3 and therefore stabilization of Mcl-1 (Maurer et al. 2006; Zhao et al. 2007). 23

30 In a separate study of the BH3-only protein Puma, it was found that the activity and function of this protein is similarly metabolically regulated. This study found that withdrawal of the growth factor IL-3 caused a reduction of glucose uptake and subsequent increase in Puma expression. This up-regulation of Puma in the absence of IL-3 was attenuated upon expression of the glucose transporter GLUT1. Glucose deprivation in the presence of IL-3 also induced Puma expression and subsequent cell death. In this study, expression of Puma was found to be dependent on p53, which is known to be suppressed in response to high glycolytic activity and activated by withdrawal of IL-3 (Zhao et al. 2008). A similar finding has been reported with the BH3-only protein Noxa. A 2006 study found that activation of naïve human T cells up-regulated Noxa in a p53- independent fashion. In this study, a glucose-dependent interaction with the antiapoptotic Bcl-2 protein Mcl-1 was established. Knockdown of Noxa imparted protection, while knockdown of Mcl-1 imparted susceptibility, to apoptosis induced by glucose deprivation. While Noxa responded to glucose stress, it was not sensitive to other stresses presented to the cell (DNA damage, ER stress, oxidative stress, etc.) The study thereby established the Noxa/Mcl-1 pair as regulating the apoptosis response of dividing cells in response to nutrient stress (Alves et al. 2006; Andersen and Kornbluth 2013). Building off of this research, it was later discovered that Noxa is phosphorylated at Ser13 (outside of the BH3 domain of the protein) by the kinase Cdk5 when in the presence of glucose. This phosphorylation abrogates the proapoptotic function of Noxa and sequesters it to the cytosol, where it is seen to be in 24

31 association with high molecular weight complexes that also contain Mcl-1 (Lowman et al. 2010). Previously, it was known that when glucose is present in a cell, anti-apoptotic Mcl-1 binds Bak and inhibits its pro-apoptotic function. When glucose deprivation occurs, Noxa interrupts the Mcl-1/Bak interaction by displacing Bak and binding tightly to Mcl-1. In this way, Noxa promotes Bak activation in response to nutrient stress by displacing it from Mcl-1. In mice, Noxa binding to Mcl-1 also targets Mcl-1 for degradation via the proteasome. Overexpression of Noxa in transformed mouse embryonic fibroblasts triggered degradation of Mcl-1 while expression of a nonbinding Noxa mutant spared Mcl-1 in these cells (Willis et al. 2005). The finding that, in high glucose conditions, Noxa binds to Mcl-1 without promoting apoptosis or Mcl- 1 degradation is therefore a novel one (Lowman et al. 2010). In this study, it was also found that only Noxa co-immunoprecipitating with Mcl-1 in proliferating cells was phosphorylated. Glucose metabolism was also directly correlated with Noxa expression levels. In Noxa-overexpressing cell lines, it was found that glucose was metabolized much more quickly than in control cells. Lactate secretion was also increased in Noxa-overexpressing cells. When grown in glucose-free medium, Noxa-overexpressing cells were partly resistant to 2-deoxy glucose (2-DG) induced apoptosis while control cells were not. While 2-DG is unable to be metabolized via glycolysis beyond its conversion to 2-DG-6-phosphate, 2DG-6- P can be metabolized via the PPP in some instances (Zabos et al. 1978). Together these facts suggest that Noxa was diverting utilization of glucose for anabolic 25

32 purposes rather than through glycolysis to pyruvate and the TCA cycle for oxidative phosphorylation and ATP generation. 1.6 The Novel Noxa/Mcl-1 Protein Complexes The complexes identified in the Kelekar lab (Lowman et al.) will remain the center of focus for the remainder of this investigation. As mentioned above, these complexes were discovered while studying the phosphorylation of Noxa and its regulation in response to glucose levels. It was previously determined that Noxa levels decided the apoptotic response to glucose deprivation by interrupting the Mcl- 1/Bak interaction (Alves et al. 2006). This destabilization of the Mcl-1/Bak interaction frees the pro-apoptotic Bak to induce mitochondrial apoptosis (Willis et al. 2005). However, it was not known how the pro-apoptotic function of Noxa is suppressed in times without nutrient stress. When cultures of Jurkat cells (an immortalized T cell line from a patient with acute T cell leukemia) were grown in glucose-free (G-F) medium, Noxa could be immunoprecipitated with anti-noxa antibodies. However, when these cells were grown in glucose-rich (G-R) medium, immunoprecipitation of Noxa using the same antibodies was not possible. Thus, in cells grown in G-R medium, the Noxa epitope for antibody binding appeared to be rendered inaccessible. This suggested either an altered conformation of Noxa or its presence in a complex with other proteins. After twelve hours of culture in G-F medium, it was found that, if the cells were transferred to back to G-R medium, the Noxa antibody binding epitope would be once again sequestered and unavailable for antibody binding. These facts, in addition to fluorescence experiments identifying the 26

33 punctate nature of phosphorylated Noxa in the cytosol of growing cells, suggested the presence of Noxa in protein complexes that formed in response to the presence of glucose (Lowman et al. 2010). Given this information, cytosolic cell lysates from Jurkat cells grown in G-R medium were run by size-exclusion chromatography. Fractions from this column were resolved by SDS-PAGE and Western blotted for Noxa. It was in this experiment that Noxa was shown to cofractionate with two high molecular weight complexes, A and B, in the presence of glucose. When the same experiment was run with cells grown in G-F medium, Noxa was detected in fractions as both free Noxa and as a component of an even larger, presumably pro-apoptotic, complex C (see below, Lowman et al. 2010). 27

34 Figure 1.4: Western Blots of Column Fractionated Lysates Reveal Noxa Complexes Figure 1.4: Western Blots of Column Fractionated Lysates Reveal Noxa Complexes: TCA-precipitated proteins from Superose 6 column fractions of cytosolic lysates of unperturbed and glucose deprived Jurkat cells were immunoblotted for Noxa. Blots were densitometrically scanned and the values plotted as shown (Lowman et al. 2010). As mentioned before, it was also found that in presence of glucose, Noxa is phosphorylated on Ser13. This phosphorylation serves to inhibit the proteins proapoptotic function and sequester it into the large complexes observed. The antiapoptotic Mcl-1, which Noxa is known to target for degradation in response to 28

35 glucose deprivation, was also found with phospho-noxa in these complexes, as only Noxa that co-immunoprecipitated with Mcl-1 was found to be phosphorylated (Lowman et al. 2010). After the discovery of complexes A and B, they were enriched in extracts from Jurkat cells grown in G-R medium by Mcl-1 co-immunoprecipitation and subsequently resolved via non-denaturing gel electrophoresis. The gels were stained with a fluorescent protein stain SYPRO Ruby Red and ultraviolet light was used to identify the complexes within the gel. The complexes were then excised, isolated, and subjected to mass spectroscopy for identification of the components of the complexes. This approach revealed the components listed below (Lowman and Keleker, unpublished data). A cartoon of complex B with its components is shown as well. Figure 1.5: A Graphic Model and Chart Representation of the Described Complexes Complex B: Complex A Noxa Mcl-1 GAPDH PGK1 Ade2 Complex B Noxa Mcl-1 GAPDH EF-1ϒ PHLPP2 hc8 (PSMA3) 29

36 In the diagram above, the small central brown oval is representative of Noxa. The green triangle signifies its phosphorylation at Ser13 and the red line segment represents its BH3 domain (for which a clear BH3 binding pocket is seen in the oval structure of Mcl-1). As can be seen in the table above, each of the two complexes is composed of a core of the two Bcl-2 proteins Noxa and Mcl-1 and the glycolytic protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH). GAPDH is the enzyme that catalyzes the sixth step in the glycolytic pathway, from glyceraldehyde-3-phosphate (G3P) to 1,3-bisphosphoglycerate (1,3-BPG) in a two-step process. The first step reduces one equivalent of NAD + to NADH in the exergonic oxidation of the aldehyde at C1 of G3P to produce a carboxylic acid. The second step then utilizes an inorganic phosphate and the energy of the first step of the reaction to catalyze the endergonic phosphorylation of the produced carboxylic acid. The overall reaction is endergonic and is made possible via energy coupling with other glycolytic enzymes. The glycolytically active GAPDH is localized to the cytosol as a homotetramer with four catalytic thiol groups from four GAPDH subunits, each kda in size (Tristan et al. 2007; Tomokuni et al. 2010). In addition to its role as a metabolic enzyme, GAPDH has several other established functions in the cell that may include DNA repair, trna export, membrane fusion and transport, and cytoskeletal dynamics all of which depend on post-translational modification and subcellular localization of the enzyme. GAPDH has also been found to be up-regulated in some cancers and its expression has been reported to be influenced by the cell proliferative rate (Tristan et al. 2007). Recently, a novel role for GAPDH has been suggested in the induction of apoptosis (Hara et al. 2005; Tarze et 30

37 al. 2007). Initially it was found that nitric oxide generation following apoptotic stimulation leads to the nitrosylation of cytosolic GAPDH. GAPDH nitrosylation promotes its binding to Siah1 an E3 ubiquitin ligase with a nuclear translocation signal. The binding of GAPDH to Siah1 stabilizes Siah1. Upon binding, these proteins translocate to the nucleus, facilitating Siah1 mediated degradation of nuclear proteins and apoptosis (Hara et al. 2005). Later, a function for GAPDH in the facilitation of apoptotic mitochondrial membrane permeabilization was also established. A study found that, during apoptosis, GAPDH accumulates in the mitochondria. GAPDH was observed to interact with voltage-dependent anion channels in the mitochondria, mediating a loss of transmembrane potential and resulting in swelling of the matrix as well as a release of the pro-apoptotic proteins cytochrome c and AIF from the mitochondrial intermembrane space. These are all key events in the intrinsic pathway of apoptosis indicating that GAPDH plays a role in the induction of apoptosis in the mitochondria (Tarze et al. 2007). Of the proteins identified in the Noxa/Mcl-1 complexes described above, the only two proteins with a well-established interaction (apart from Noxa and Mcl-1) are GAPDH and PGK1. Phosphoglycerate kinase (PGK) catalyzes the seventh step in the glycolytic pathway: the exergonic conversion of 1,3-BPG to 3-phosphoglycerate (3- PG). In the process, PGK removes the phosphate group on C1 of 1,3-BPG and transfers it to a molecule of ADP in order to produce ATP. This reaction is highly exergonic and occurs directly after the endergonic GAPDH catalyzed conversion of G3P to 1,3-BPG, allowing energy coupling between the reactions to make both favorable. This is possible due to a complex formed by GAPDH and PGK that allows 31

38 direct metabolite transfer between the two proteins (Srivastava and Bernhard 1986; Tomokuni et al. 2010). While GAPDH is normally in a homotetramer form, PGK acts as a monomer of about 44 kda (Tomokuni et al. 2010). Both of these proteins are known to be transcriptionally up-regulated as part of the metabolic reprogramming by the hypoxia inducible factor HIF-1 in several different cancers (Semenza 2003). PGK1 is one of two isoforms of phosphoglycerate kinase: PGK1 and PGK2. PGK1 is the ubiquitously expressed isoform while PGK2 is only expressed during spermatogenesis (Danshina et al. 2010). In complex A, the only peripheral protein aside from PGK1 is Ade2. Very little is known about Ade2 or its role in cancers. What is known is that ADE2 gene encodes a protein involved in the purine biosynthesis pathway and that its expression is regulated by the presence of adenine in cells, although most studies on this gene have occurred in yeast (Gedvilaite and Sasnauskas 1994). The larger complex B contains three peripheral proteins in addition to its Noxa, Mcl-1, and GAPDH core. These include EF-1ϒ, PHLPP2 and hc8. EF-1ϒ (elongation factor 1 gamma) is a subunit of the EF-1 beta gamma delta complex. This complex functions as a nucleotide exchange factor for EF-1 alpha. EF-1 alpha facilitates the transfer of aminoacyl-trnas to the ribosome during the elongation phase of translation. In the process of transferring an aminoacyl-trna to the ribosome, EF-1 alpha hydrolyzes its bound GTP. The EF-1 beta gamma delta complex then exchanges the produced GDP molecule for a new GTP molecule. EF-1 alpha has been shown to have a diffuse cytosolic localization while EF-1 beta gamma delta was found to co-localize with the endoplasmic reticulum (Sanders et al. 1996). 32

39 One study found that EF-1ϒ-hybridizing RNA was overexpressed in 25 of 29 analyzed colorectal carcinomas (Chi et al. 1992). A subsequent study found that over 50% of adenomas from patients without familial adenomatous polyposis have at least a two-fold overexpression of EF-1ϒ-hybridizing RNA (Ender et al. 1993). Together, these findings indicate that overexpression of EF-1ϒ RNA is associated with the development of colorectal cancer (Ender et al. 1993). In addition to its role as an elongation factor, EF-1ϒ has also been found to contain a glutathione transferase domain. A computer sequence motif search and tertiary structural model indicates that EF-1ϒ contains an N-terminal domain related glutathione S-transferases (GSTs). In this region of the protein, two GST-related motifs are likely to form a fold very similar to that of known GSTs. One of these motifs is implicated glutathione binding while the other is predicted to maintain the proper conformation of this region. The domain is predicted to be enzymatically active and involved in the regulation of multi-subunit complexes containing EF-1ϒ (Koonin et al. 1994). S-thiolation of GAPDH has been shown to occur in response to increased levels of reactive oxygen species (ROS) as a mechanism to prevent permanent oxidative damage to the catalytic thiol group in GAPDH. However, in addition to protecting the protein from oxidative stress, S-thiolation of GAPDH at its catalytic cysteine inhibits its function. This S-thiolation functional inhibition can be spontaneously reversed upon the removal of the oxidative stimulus. In this way, S- thiolation of cysteinyl thiols of cellular proteins can both function as a protective and regulatory post-translational modification in response to increased ROS levels (Schuppe-Koistinen et al. 1994). 33

40 Cancer cells have long been associated with increased levels of ROS. This association goes two ways: ROS is thought to possibly induce cancer and cancer cells are known to generate more ROS than normal cells. The stimulation of cell cycle progression is also known to increase ROS signaling. However, cancer cells are also noted for their increased antioxidant defenses, namely higher intracellular glutathione levels. These increased levels of glutathione are thought of as inhibiting the potential induction of apoptosis despite such high ROS levels. The balance of ROS and glutathione is a well-regulated ratio that must be monitored in cancer cells in order to prevent the induction of apoptosis (Schumacker 2006). When cells are deprived of glucose, oxidative stress occurs. This induces protein S-thiolation and a subsequent reduction in glutathione levels that can influence the induction of an apoptotic response (Marambio et al. 2010). This idea will be explored in the context of the described complex B. As GAPDH is a known target of glutathionylation, a putative role for EF-1ϒ as a glutathione transferase to regulate the glycolytic activity of GAPDH in response to increased ROS as a result of glucose deprivation will be investigated. In addition to a proposed glutathione transferase, complex B also contains a phosphatase. PHLPP2 (Pleckstrin Homology Leucine Rich Repeat Protein Phosphatase 2) is a serine phosphatase member of the PPM (protein phosphatase magnesium or manganese dependent) family. It was first identified as the phosphatase responsible for the dephosphorylation of the hydrophobic motif of Akt/Protein Kinase B (Gao et al. 2005). Phosphorylation of Akt is known to activate the kinase, which has various roles in promoting cell survival, enabling cell cycle progression, altering 34

41 glucose metabolism, and controlling angiogenesis (Altomare and Testa 2005). Akt phosphorylation of the Bcl-2 protein Bad is known to inactivate this pro-apoptotic protein and to therefore contribute to an inhibition of the intrinsic apoptotic pathway (Datta et al. 1997). As a result, aberrant and frequently hyperactive Akt signaling has been observed in several different cancers (Altomare and Testa 2005). PHLPP levels are also notably reduced in several colon cancer and glioblastoma cell lines with elevated Akt phosphorylation. Reintroduction of PHLPP into a glioblastoma cell line has also been shown to significantly attenuate tumor growth (Gao et al. 2005). PHLPP2 is one of three PHLPP isoforms and is responsible for the dephosphorylation of the Akt1 and Akt3 isoforms of Akt (Brognard et al. 2007). In addition to Akt1 and Akt3, PHLPP2 is known to dephosphorylate Protein Kinase C. This targets the kinase for degradation, preventing it from carrying out its pro-survival and proliferative role (Gao et al. 2008). Mst1 has also been identified as a target of PHLPP phosphatase function. Dephosphorylation of Mst1 activates this protein and its downstream effectors to induce apoptosis (Qiao et al. 2010). These various functions of PHLPP are the reason this protein has become known as a tumor suppressor protein in humans. In the context of complex B, it is proposed that PHLPP2 may mediate the dephosphorylation of Noxa, thereby activating its pro-apoptotic function. Initial results appear to support this hypothesis (Theede and Kelekar, unpublished studies). Little is known about the last protein contained in complex B, referred to as hc8 or PSMA3 (proteasome subunit alpha type 3). HC8 as it will be referred to here on out is an alpha subunit of the 20S proteasome (Kinyamu et al. 2008). It has been shown to bind a mitosis regulatory protein, Aurora-B, and signal for its 35

42 degradation in a proteasome-dependent manner (Shu et al. 2003). Since Noxa binding to Mcl-1 is known to target Mcl-1 for degradation by the proteasome, it is suggested that the function of hc8 in complex B might be to bind Noxa-bound Mcl-1 and facilitate its proteasome-dependent degradation. Mcl-1 is a highly regulated protein predominantly via post-translational modifications in its N-terminal region with a short half-life (Michels et al. 2005; Thomas et al. 2010). It is targeted for degradation via ubiquitination at five different lysines. This ubiquitination is facilitated by Mule (Mcl-1 ubiquitin ligase E3). Mule contains a region similar to the BH3 domain, allowing it to directly interact with Mcl- 1. When Mule expression is abrogated via RNA interference, Mcl-1 is stabilized and apoptosis is attenuated (Zhong et al. 2005). This ubiquitination can be removed by the deubiquitinase USP9X in order to stabilize the protein. Increased expression of USP9X correlates with increased expression of Mcl-1 in different types of lymphomas and a poorer prognosis for multiple myeloma patients (Schwickart et al. 2010). In addition to modification by ubiquitination, Mcl-1 can be phosphorylated at multiple sites. Four serine residues and two threonine residues are known to be potential sites of phosphorylation within the protein, by the protein kinases CDK1 and CDK2, JNK, ERK-1, and GSK-3. Phosphorylation of Mcl-1 can influence its stability, binding to other Bcl-2 family members, and its anti-apoptotic function (Thomas et al. 2010). Alternative isoforms of Mcl-1 with separate subcellular localizations and functions are also known to exist. Three different isoforms have been identified to date: 40 kda and 38 kda isoforms localize to the outer mitochondrial membrane and 36

43 exhibit the characteristic anti-apoptotic function of this protein; a shorter 36 kda isoform with no ability to bind other Bcl-2 proteins and no anti-apoptotic function is known to localize to the mitochondrial matrix where it maintains inner mitochondrial membrane structure and function (Perciavalle et al. 2012). 1.7 Specific Aims and Proposed Studies It is unknown which isoform of Mcl-1 exists in the identified complexes. The ubiquitination and phosphorylation state of the Mcl-1 in these complexes is also unknown. For that matter, the degree and type of post-translational modification to any of the identified components of the complexes (with the exception of Noxa, phosphorylated at Ser13) is unknown. This information could provide much insight in to the function of the individual complex components as well as the complexes as a whole. The potentially altered glutathionylated state of GAPDH would alter its glycolytic activity and therefore function between the two complexes. The phosphorylation and ubiquitination state of Mcl-1 in complex B as opposed to complex A could provide insight as to whether or not hc8 may facilitate the proteasome-dependent degradation of Mcl-1. The relationship between the two complexes is also not understood. Given their identical core structure, it is hypothesized that there may be a dynamic interplay between complex A and complex B through an exchange of peripheral proteins. What influences the potential exchange between the complexes is unknown. It is suggested that one form may be favored over another given different cellular conditions particularly the availability of glucose. Although it is suggested that these complexes 37

44 influence the metabolic profile of cancer cells (as well as other rapidly dividing cells) in order to allow for their increased proliferative rate, it is not known how they would do this. This study aims to provide an understanding of the regulation and modification of these complexes in response to nutrient stress prior to the induction of apoptosis. The relative levels of expression of the complexes were investigated given different time lengths of glucose deprivation. The effects of glucose availability on the post-translational modifications that occur to the components of the complexes were also analyzed. Studies were performed on Jurkat cells (an immortalized T cell leukemia line) as well as primary and activated T cells. Comparisons were thus made between cancer cells and rapidly dividing cells of the T cell type in order to provide new insight in to the potential roles that these complexes may play in the metabolic reprogramming of cancer cells as well as other rapidly dividing cell types. 38

45 Chapter Two: Materials and Methods 2.1 Cell lines, cell culture, and cell lysis Cells were grown in RPMI-1640 (25 mm glucose) supplemented with 10% fetal bovine serum, 2 mm L-glutamine, 100-units/mL penicillin, and 100 µg/ml streptomycin. Cells were cultured in a humidified atmosphere of 5% CO 2 at 37 o C and passaged when confluent. Jurkat cells were lysed with Dignam Buffer A (10 mm HEPES, 1.5 mm MgCl 2, 10 mm KCl, 0.05% IGEPAL, 0.5 mm DTT, ph 7.9) supplemented with a cocktail of protease and phosphatase inhibitor cocktails. Jenna Benson, a post-doctoral member of the Kelekar lab, performed all work with primary T cells to the point of production of cell lysate. Cell lysates were then received to subsequently perform column fractionation and Western Blotting analysis as a part of this study. Primary T cells were purified from normal peripheral blood monocytes by a negative selection protocol using the Pan T cell isolation kit from Miltenyi Biotech as per the manufacturer s instructions. To mimic activation that occurs via antigen exposure in vivo, CD3+ human primary T cells were stimulated for 24 hours at a density of 10 6 cells/ml with 1 µg/ml plate-bound anti-cd3 (OKT3, BioLegend) and 1 µg/ml soluble anti-cd28 (CD28.2, BioLegend). Primary T cells were lysed with RIPA buffer (50 mm Tris-HCl [ph 7.5], 150 mm NaCl, 0.5% v/v sodium deoxycholate, 1% v/v Nonidet P-40, 0.1% SDS) supplemented with protease and phosphatase inhibitors. 39

46 2.2 Glucose Deprivation In order to deprive cells of glucose, cell cultures grown in glucose-rich medium (RPMI-1640, supplemented as described above) were pelleted in a centrifuge at 4 o C and the glucose-rich medium was decanted. Cells were washed in glucose-free basal medium, pelleted once again at 4 o C, and resuspended in glucose-free RPMI also supplemented with 10% fetal bovine serum, 2 mm L-glutamine, 100-units/mL penicillin, and 100 µg/ml streptomycin pre-heated in a water bath to 37 o C. Cells were then returned to incubation with 5% CO 2 at 37 o C until the indicated length of deprivation was over and cell lysate was collected. 2.3 Immunoprecipitation and Antibodies Upon collection, cell lysate was incubated with 1-5 µg/ml of antibody for 6-18 hours at 4 o C with rotary agitation. Complexes were captured via incubation with protein G agarose beads (Invitrogen) for 1-2 hours at 4 o C with rotary agitation. Beads were collected at the bottom of the tubes via brief centrifugation at 4 o C and the supernatant was removed. Beads were washed with lysis buffer, centrifuged, and the supernatant removed. This process was repeated three times. When subsequently running gel electrophoresis, 2x Laemmli Sample Buffer (Bio-Rad) was added, the mixture was heated at 95 o C for 5 min to separate proteins from the beads, the samples were centrifuged, and the supernatant collected for use. Antibodies against Noxa, Mcl-1, PGK1, hc8 (PSM-α3), GAPDH, and EF-1ϒ were purchased from Santa Cruz Biotechnology (SCBT). Additional antibodies 40

47 against Mcl-1 were purchased from Cell Signaling Technology. PHLPP2 antibodies were purchased from Bethyl Laboratories. 2.4 Western Blotting After running lysates or immunoprecipitated proteins by polyacrylamide gel electrophoresis, these gels were transferred to nitrocellulose membrane in 1X transfer buffer (25 mm Tris-HCl [ph 7.6], 192 mm glycine, 20% methanol made from a 25X Novex solution). Membranes were blocked in 5% dry milk in TBS-T for one hour at room temperature (RT) and washed in TBS-T for 2 minutes. Membranes were then incubated with primary antibody in TBS-T at a dilution of 1:500 to 1:10,000 depending on the antibody and experiment at hand for one hour at RT. Membranes were washed three times for five minutes in TBS-T, incubated in secondary antibody in 5% milk in TBST at a dilution of 1:15,000 for one hour at RT, and subsequently washed again three times for five minutes. Chemiluminescence reactions were carried out using the ECL Plus kit (Amersham) or Super Signal West Femto kit (Pierce Biotech). Blots were wrapped in clear plastic wrap, taped in exposure cassettes, and exposed to film for anywhere between one second and five minutes (depending on the experiment at hand) before developing the film. Blots were stripped for reuse by washing three times for minutes in TBS-T (ph ). 2.5 Gel Exclusion Chromatography Cell lysate (0.5-1 mg protein) in Dignam Buffer A (described above) was loaded on to a Superose 6 column (GE/Amersham) previously equilibrated with the 41

48 lysis buffer A and eluted in the same buffer at a constant flow rate of 0.1 ml/min. 28 fractions of 1 ml were collected. Proteins were precipitated from the fractions using trichloroacetic acid (TCA) and acetone. One volume of 100% TCA was added to four volumes of protein sample. The mixture was vortexed and incubated for ten minutes at 4 o C. Tubes were then centrifuged at 14,000 rpm for five minutes. Supernatant was removed and 200 µl of acetone pre-chilled at -20 o C was added to the protein pellet. Tubes were centrifuged again at 14,000 rpm for five minutes and the supernatant removed. This process of adding acetone, centrifuging, and removing the supernatant was repeated for a total of two acetone washes. The protein pellet was dried by placing the tube in a 95 o C heat block for five to ten minutes. For SDS PAGE, 20 µl of 2x Laemmli Sample Buffer (Bio-Rad) was added to the precipitated protein solution and the solution was mixed, heated in a 95 o C heat block for ten minutes, and loaded in to the gel. 2.6 Two-Dimensional Gel Electrophoresis Prior to 2D gel electrophoresis, cell lysates were prepared using a 2D Cleanup Kit (GE Healthcare Life Sciences or Bio-Rad) following the manufacturers instructions and the final protein pellet was dissolved in 200 µl Sample Rehydration Buffer (Bio-Rad). If Mcl-1 immunoprecipitation was performed prior to 2D electrophoresis, beads were washed and resuspended in 250 µl of a 0.2 M Glycine, 1M Tris-HCl (ph 8.0) solution prior to heating. Beads were then heated at 95 o C for 5 minutes, centrifuged, and the supernatant collected and used for rehydration. The 200 µl of cleaned lysate or immunoprecipitated complexes was used for rehydration of 42

49 11cm, ph 3-10, immobilized ph gradient (IPG) strips (Bio-Rad) for isoelectric focusing. Strips were rehydrated face down at room temperature for one hour in rehydration trays, overlaid with 2 ml mineral oil and further rehydrated for 8-10 hours. After rehydration, strips were placed in a focusing tray for focusing in a PROTEAN IEF Cell (Bio-Rad). Parameters were set to linearly increase the voltage to 500V over the period of 1 hour, followed by a linear increase to 1000V over another hour, and finally rapidly increase the voltage to a final value of 8000V over a period of 30,000 volt hours. After focusing, strips were equilibrated at room temperature using 1.5 ml Equilibration Buffers I and II (Bio-Rad) for 10 minutes each. Strips were dipped into a beaker of SDS running buffer to rinse off leftover oil and equilibration buffers before being placed in to a Criterion 4-20% Gradient IPG+1 Gel (Bio-Rad). Gels were run at 50V for 10 minutes followed by 120V for minutes. After being run, gels were either stained or transferred for Western Blotting. 2.7 Gel Staining After being run, gels were either stained with Sypro Ruby Red (Figure 3.1) or Coomassie Brilliant Blue stain (Figures 3.5 and 3.7) (both stains from Bio-Rad). For Sypro Ruby Red staining, gels were fixed in a 10% methanol, 7% acetic acid solution for 30 minutes. Gels were then stained for at least 3 hours, washed in the 10% methanol, 7% acetic acid solution for 30 minutes, and subsequently washed in HyClone Molecular Biology Grade water several times before imaging. For 43

50 Coomassie Brilliant Blue staining, gels were washed several times in Molecular Biology Grade water, stained for at least 3 hours, washed several times again in Molecular Biology Grade water, and subsequently imaged. 44

51 Chapter Three: Results 3.1 Dynamic interplay suggested to occur between complexes Given that the expression of the complexes was observed to be dependent on the presence of glucose and the fact that the functions of three of the complex components Noxa, GAPDH, and PGK1 have a known reliance on glucose, the responsiveness of the complexes to glucose deprivation was explored. Prior studies indicated that the complexes are present in human leukemic T cells grown in glucoserich medium and significantly diminished in these cells when deprived of glucose for twenty-four hours (Figure 1.4; Lowman et al. 2010). Initial characterization of the complexes thus focused on their expression under shorter lengths of deprivation. In order to run these experiments, large cultures of Jurkat cells were initially grown and expanded in glucose-rich medium. At the start of each experiment, these large cultures were split into smaller cultures in order to start deprivations of different lengths of time. These smaller cultures of cells were resuspended in glucose-free medium and further incubated for 1, 3, 6, 12, or 24 hours depending on the experiment at hand. After the indicated length of glucose deprivation, cytosolic lysates were formed from the cells. Protease and phosphatase inhibitors were added to the cell lysis buffer in order to inhibit any unwanted proteolysis or dephosphorylation of the complex components that could affect the composition or structure of the complexes. After this, the lysates were refrigerated at 4 o C until lysates from all deprivation time points had been collected. At this point, lysates were subjected to a variety of in vitro assays to characterize the complexes at hand. 45

52 Primary experiments centered on the differential expression of each of the complexes with various lengths of glucose deprivation. In order to isolate the complexes, immunoprecipitation of Mcl-1 was performed. As stated, the two complexes described both contain a core of Noxa, Mcl-1, and GAPDH. When previous studies had performed immunoprecipitation of Noxa in order to isolate the complexes in growing cells, nothing was pulled down with Noxa (Lowman et al. 2010). This is predicted to be true because Noxa is a small protein (~10 kda) and is thought of as being contained in the central core of the complex, with its antibody binding epitope covered by the other proteins in the complex. Noxa immunoprecipitation was therefore out of the question as a method to isolate the complexes. GAPDH, as a housekeeping protein, is contained in a plethora of protein complexes that would all be pulled down in immunoprecipitation of this protein. The imprecision of GAPDH immunoprecipitation, as well as the lack of success obtained by Noxa immunoprecipitation, led to the use of Mcl-1 immunoprecipitation to isolate the complexes. After the complexes were isolated from cell lysates via Mcl-1 immunoprecipitation, native (non-sds) polyacrylamide gel electrophoresis (PAGE) was performed in order to separate the two complexes based on their sizes (complex A being smaller than complex B). This type of non-denaturing gel electrophoresis had been run before in order to isolate the complexes from one another for mass spectrometry to initially identify their respective components. However, running these gels in conjunction with a series of glucose deprivations had not been explored. Cells were thus deprived of glucose for 0, 1, 3, 6, and 12 hours to explore the 46

53 expression levels of the two complexes at each of these time points prior to the known diminished expression of the complexes after twenty-four hours of deprivation. Figure 3.1: Native Gel Electrophoresis of Mcl-1 Immunoprecipitates of Cytosolic Lysates from Unperturbed and Glucose Deprived Jurkat Cells Deprivation (hrs): ! Complex B! Degrades with time! Apoptotic! Complex C! Complex A! Figure 3.1: Native Gel Electrophoresis of Mcl-1 Immunoprecipitates of Cytosolic Lysates from Unperturbed and Glucose Deprived Jurkat Cells: Mcl-1 immunoprecipitated lysates were resolved by non-denaturing gel electrophoresis and stained with Sypro Ruby Red Stain to visualize the response of the complexes to glucose deprivation. This experiment was run in triplicate with similar results from each trial. The higher molecular weight complex B appears to decrease in size with increasing lengths of glucose deprivation. Meanwhile, no significant changes appear to occur to 47

54 complex A. Complex C identified as an apoptotic complex that appears after twenty-four hours of glucose deprivation in prior studies (see Figure 1.4) is now shown to first appear after twelve hours of deprivation. The change in size that occurs to complex B is suggestive of its disassembly in response to glucose deprivation. As seen in Figure 1.4, it appears that in the presence of glucose the expression levels of the two complexes are about even. However, after twenty-four hours of glucose deprivation, the expression of complex A appears to predominate over the expression of complex B. The apparent disassembly of complex B with increasing amounts of glucose deprivation seen in Figure 1.4 agrees with this finding. Having observed that glucose deprivation induced a change in the size of complex B, the next step was to identify how these changes occur. This was done by once again depriving cells of glucose for 0, 1, 3, 6, and 12 hours, lysing the cells, immunoprecipitating Mcl-1, and running the immunoprecipitates by denaturing SDS PAGE. By utilizing SDS to break apart the complexes in question, it could then be determined if all of the known proteins remain associated with the complexes given glucose deprivation, or if a dissociation of peripheral proteins occurs (as would account for the observed decrease in the size of complex B). Western blotting was utilized with antibodies against the proteins of each of the complexes. It was predicted that, with lengths of glucose deprivation of up to twelve hours, all proteins associated with complex A (Ade2 and PGK1) would remain in association with Mcl- 1. At least some of the proteins associated with complex B (PHLPP2, hc8/psm-α 3, and EF-1ϒ), however, were predicted to dissociate from Mcl-1. 48

55 Figure 3.2: Western Blotting of Mcl-1 Immunoprecipitates of Cytosolic Lysates from Unperturbed and Glucose Deprived Jurkat Cells Resolved by SDS PAGE Deprivation (hrs): ! EF-1ϒ! PGK1! PSM-α3! Noxa! Figure 3.2: Western Blotting of Mcl-1 Immunoprecipitates of Cytosolic Lysates from Unperturbed and Glucose Deprived Jurkat Cells Resolved by SDS PAGE: Mcl-1 immunoprecipitated lysates were resolved by SDS PAGE and transferred to a membrane. The membrane was immunoblotted for every protein known to be present in the two complexes. A sample of these blots is shown here. Results indicate that all peripheral proteins between the two complexes other than EF-1ϒ remain in constant association with Mcl-1 through 12 hours of glucose deprivation. Interestingly, EF-1ϒ was observed to lose its association with Mcl-1 just one hour into glucose deprivation. The departure of EF-1ϒ from complex B partially explains the observed decrease in the size of complex B with glucose deprivation. The association of Noxa with Mcl-1 also appears to vary given different lengths of glucose deprivation. This is an unexpected result given that Noxa was initially shown to be present in both complexes A and B in the presence of glucose. The Noxa-Mcl-1 association appears to be strongest at one hour of deprivation with diminishing strength through to twelve hours. This result may suggest that, although these 49

56 complexes do sequester Noxa in glucose rich conditions, Noxa may be freed from the complexes as early as three hours after glucose deprivation is induced. This result also suggests that the complexes may remain stable without association with Noxa. Given that all of the peripheral proteins in the two complexes stay in constant association with Mcl-1 through twelve hours of glucose deprivation (with the exception of EF-1ϒ), the apparent loss of association of Noxa with the complexes may suggest that the departure of Noxa from the complexes does not immediately induce instability and disassembly of the complexes. Another interesting result obtained from this experiment is the apparent slight decrease in the size of Noxa that occurs after six and twelve hours of glucose deprivation. This result strongly suggests that Noxa is dephosphorylated at these time points in response to deprivation, as has been previously established to occur in order to induce apoptosis after twenty-four hours of deprivation. These experiments were run in duplicate with the same results obtained both times. 3.2 Shifts in complex expression shown with glucose To gain a more detailed view of how glucose deprivation affects the components of the two complexes, gel filtration chromatography was utilized in favor of Mcl-1 immunoprecipitation to better resolve the complexes prior to Western blotting. This method allows for the assessment of the presence and abundance of Mcl-1 between the two complexes not afforded by direct immunoprecipitation of Mcl-1. Large cultures of Jurkat cells were thus glucose deprived for 0, 3, or 24 hours, and cell extracts were fractionated via size exclusion chromatography on a Superose 6 50

57 gel filtration column. Protein was isolated from constant volumes of each fraction by TCA precipitation and resolved by SDS PAGE. The gels were transferred to a membrane, and the presence or absence of the components of the complexes was determined by Western blotting. The Western blots of each protein were then compiled by time point. The compiled Western blot images allowed the identification of fractions that contained specific components of the complexes at specific times after glucose deprivation. These images could also be used to analyze whether and how the expression of each protein was modified in relation to each of the two complexes given different lengths of glucose deprivation. 51

58 Figure 3.3: Western Blotted Fractions of Cytosolic Lysates of Unperturbed and Glucose Deprived Jurkat Cells Run by Column Chromatography 52

59 Figure 3.3: Western Blotted Fractions of Cytosolic Lysates of Unperturbed and Glucose Deprived Jurkat Cells Run by Column Chromatography: Cytosolic lysates were fractionated via gel filtration chromatography to separate protein complexes by size. Samples from each fraction were resolved by SDS PAGE. Gels were transferred to a membrane and immunoblotted for each of the proteins present in the complexes. The compiled blots from each length of glucose deprivation indicate the presence of complex A in fractions and complex B in fractions This is based on the presence of hc8 (PSM-α3), EF-1ϒ, Mcl-1 and Noxa in fractions and PGK1 and Mcl-1 in fractions Western blots for Ade2 and PHLPP2 were performed but did not give convincing results and are thus not included above. This experiment was run in duplicate with similar results. The Ade2 and PHLPP2 blots did not turn out in either run of the experiment. In the compiled blots, a clear distinction cannot be made as to exactly which fractions contain complex A as opposed to complex B. The results suggest the presence of several intermediary complexes that exist between the identified complexes A and B. This is seen given that the largest density of Noxa and of the lower molecular weight isoform of Mcl-1 is in fraction 15, between the defined boundaries of the two identified complexes. The wide range of fractions that each protein can be observed in suggests a wide range of complexes of varying sizes that each protein associates with. The overlap of peripheral proteins between complexes A and B also suggests a possible dynamic interplay among these complexes. This could 53

60 indicate the potential of complex B to switch to complex A (and vice versa) via an exchange of peripheral proteins and a transition through the conceivable intermediary complexes. An interesting finding through this study reveals that different isoforms of Mcl-1 are present between the two complexes. As stated before, Mcl-1 is known to exist in three isoforms of various sizes. These isoforms are 36, 38, and 40 kda in size. The 38 and 40 kda isoforms are observed in the complexes in question and are the two isoforms known to exhibit the anti-apoptotic function of Mcl-1 (Perciavalle et al. 2012). In the Western blots it can be seen that the 40 kda isoform only associates with complex A while the 38 kda isoform appears to associate with both complexes. A shift in the association of Mcl-1 can be observed from complex B to complex A with glucose deprivation. In glucose-rich medium (zero hours of glucose deprivation), Mcl-1 is associated with both complexes. However, after three hours of glucose deprivation, Mcl-1 is predominantly observed in the fractions that contain complex A. The majority of the 38 kda isoform of Mcl-1 is no longer observable after three hours of deprivation and there is very little Mcl-1 observed in the fractions associated with complex B. This trend continues to twenty-four hours of deprivation, where there is virtually no observable Mcl-1 present in the fractions corresponding to complex B but the 40 kda isoform of Mcl-1 is observable in the fractions of complex A. This data is in agreement with that of Figures 3.1 and 3.2 and suggests that, with shorter lengths of glucose deprivation, a shift occurs in the expression levels of the complexes so that complex B disassembles and complex A becomes the predominant form in the cells. This suggests a potential role for complex B in the metabolism of 54

61 the cells that allows for their rapid growth and division in the presence of glucose that is not necessary or functional in times of glucose deprivation. Complex A may thus play a more protective role in the cells in response to nutrient stress. 3.3 Primary T cells show similar complex expression patterns Although these complexes were initially discovered and characterized in leukemic T cells, they are known to exist in other rapidly dividing cells of the body. To allow for a comparison of cancerous and non-cancerous cell types, blood from a healthy adult was collected and the T cells isolated for study. As a population, these healthy T cells are unstimulated and thus not rapidly dividing. However, upon costimulation with antibodies against surface proteins CD3 and CD28, these T cells begin to divide rapidly as they would normally in response to stimulation in the body during an immune response. Therefore, an analysis of unstimulated and stimulated T cells allows for an interesting comparison: healthy non-rapidly dividing T cells, healthy rapidly dividing T cells, glucose-fed T leukemia cells, and glucose-deprived T leukemia cells. T cells isolated from a normal human blood sample were split in to two populations: one which would remain unstimulated and one which would undergo stimulation prior to cell lysate collection. Lysates from both samples were run on the same superpose 6 gel filtration column as the Jurkat cell lysates, TCA precipitated, resolved by SDS PAGE, and Western blotted for proteins in the complexes. Based on proliferation rates, unstimulated T cells were predicted to exhibit a similar metabolic profile as glucose-deprived Jurkat cells while stimulated T cells were predicted to 55

62 exhibit a similar metabolic profile as glucose-fed Jurkat cells. The predicted similarities in metabolic profiles were expected to be reflected in similarities in the expression levels and make-up of the identified complexes. 56

63 Figure 3.4: Western Blotted Fractions of Cytosolic Lysates of Unstimulated and Stimulated Primary T Cells Run by Column Chromatography Figure 3.4: Western Blotted Fractions of Cytosolic Lysates of Unstimulated and Stimulated Primary T Cells Run by Column Chromatography: Primary T cells were either left unstimulated or were stimulated with anti CD3 and anti CD28 antibodies. Cytosolic lysates were fractionated via gel filtration chromatography to separate protein complexes by size. Samples from each fraction were resolved by SDS PAGE. Gels were transferred to a membrane and immunoblotted for each of the proteins present in the complexes. 57

64 This experiment was run once. The PHLPP2, Ade2, and EF-1ϒ blots did not turn out. PGK1 and hc8 were thus the main indicators used to define boundaries between fractions containing complex A as opposed to complex B. GAPDH expression is also observed to be more profound in the fractions believed to contain these two predominating complexes. The results of this experiment agree entirely with the proposed hypothesis. In the unstimulated, non-rapidly dividing T cells, only the 40 kda isoform of Mcl-1 is observed in the fractions and is only contained in those fractions corresponding to complex A (17-19). Noxa does not appear to be expressed in the fractions of either complex, or in the loaded lysate. This lack of Mcl- 1 and Noxa expression in the fractions that contain complex B matches with that of glucose deprived Jurkat cells. This agrees with the proposed model that complex B may regulate glucose metabolism in order to drive proliferation of cells. In either nonrapidly dividing cells or in glucose-deprived cells, this complex would thus not be needed, explaining the lack of complete complex assembly in these cell types. In the stimulated T cell fractions, a major shift occurs in the expression of Mcl-1 and Noxa with respect to the complexes. The 38 kda isoform of Mcl-1 appears upon stimulation. The majority of this Mcl-1 is observed in fractions 15-18, largely between the defined boundaries of the complexes. The Noxa however is expressed almost entirely in the fractions corresponding to complex B fractions There is no observable Noxa expression in the complex A fractions. These results suggest a major up-regulation in the expression of complex B upon stimulation of the T cells. This agrees with the proposed hypothesis and matches with the observed results for glucose-fed Jurkat cells. It is only in these rapidly dividing cell types that complete 58

65 complex B formation is observed. This is as expected given the proposed model of metabolic regulation by complex B. Although these shifts are very clearly seen to occur in response to stimulation, a direct connection to the complexes cannot be absolutely established. These are not Mcl-1 immunoprecipitated fractions and, therefore, it cannot be concluded that these proteins are actually in the complexes. It is known that the complexes appear in these fractions and that the components immunoblotted for are contained in the complexes. These results are thus strongly suggestive of occurring in the complexes, but it is important to note that this is not completely conclusive. 3.4 Modifications to cellular components of the complexes The column fractionation experiments described above offered insight into the changes in expression levels of the components of the complexes in response to glucose deprivation. With altered expression levels explored, the next step in characterization of these complexes was to investigate potential post-translational modifications of the components of the complexes following glucose deprivation. Many of the proteins in the complexes are known to undergo several different types of post-translational modifications (e.g. phosphorylation, glutathionylation, nitrosylation, etc.). These modifications have the potential to influence the structures and functions of these proteins. The post-translational state of these proteins was thus explored in order to obtain insight into the possible functional states of each of these proteins when contained within the complexes. 59

66 In order to determine the post-translational state of each of the proteins in the complexes, the complexes were resolved by 2-Dimensional (2D) gel electrophoresis. While Western blotting techniques separate proteins by size and are largely used to determine expression levels of proteins in a cell, 2D gel electrophoresis also separates proteins by their isoelectric point (pi). This is done by initially rehydrating an immobilized ph gradient (IPG) gel strip with the lysate sample and running an electric current through the gel strip (via isoelectric focusing, IEF) so that the proteins in the solution separate based on charge. These strips are then subjected to SDS PAGE so that, in the end, proteins are separated by charge (indicative of posttranslational modifications) on the horizontal axis and size on the vertical axis of the resultant SDS PAGE gel. In theory, this will result in the same proteins with different post-translational modifications (e.g. Noxa and phospho-noxa) lining up next to each other along the same horizontal axis, but being represented by distinct spots along this axis given their different isoelectric points based on the acidity or basicity imparted by each modification. These gels are then stained and imaged to detect the changes in the isoelectric points of each of the proteins given various lengths of glucose deprivation. Initial studies were performed on cytosolic lysates in order to both test the validity of this approach to observing post-translational modifications in Jurkat cells and to determine whether any observable changes occurred to the proteome of the cell with glucose deprivation, regardless of specific changes to the complexes at hand. 60

67 Figure 3.5: 2D Gel Electrophoresis of Cytosolic Lysates of Unperturbed and Glucose Deprived Jurkat Cells Figure 3.5: 2D Gel Electrophoresis of Cytosolic Lysates of Unperturbed and Glucose Deprived Jurkat Cells: Lysate was collected from Jurkat cells deprived of glucose for 0, 12, and 24 hours. Isoelectric focusing was followed by SDS PAGE, separating proteins first by isoelectric point and then molecular weight. Gels were stained with Coomassie Blue stain, creating a proteomic map at each glucose deprivation time point. Major differences between deprivation lengths are pointed out with arrows; actin is circled in red; what is believed to be GAPDH is circled in orange. 61

68 This experiment was run in triplicate with similar results each time. The experiment was also run with a glucose deprivation length of three hours (results not shown). Clearly, given the changes highlighted by arrows in the figure above, there are significant changes that occur to the proteome of the cell given glucose deprivation. Post-translational modifications are seen to occur given the several proteins that appear to shift horizontally with different lengths of deprivation. Some post-translational states are seen to disappear and others to appear with deprivation as indicated by the disappearance and appearance of various spots along the same horizontal axis in the figure. Actin is labeled based on its molecular weight and isoelectric point as it is a highly expressed protein and is expected to focus in that location. GAPDH is labeled using the same logic. However, it is clearly not possible to definitively identify spots as specific proteins given the wide array of possible proteins each spot could represent. One way to exactly analyze these gels and the precise proteins represented by each spot is via Western blotting. Given that many of the proteins that are contained in the complexes in question are expressed at a level too low to be highlighted by Coomassie Blue staining, the 2D gels can be transferred to a membrane and probed with antibodies specific to the complex components to precisely identify the locations of each protein on the gel. This was the next step in the investigation. 2D gels were run with cytosolic lysates from Jurkat cells deprived of glucose for 0, 3, and 24 hours. These gels were then transferred and immunoblotted to identify specific proteins from 62

69 the complexes and possible modifications that could alter the charge on these proteins in response to glucose deprivation. It is important to note that because cytosolic lysates are being used as opposed to isolated complexes, the changes that are observed are representative of the proteins described in the proteome as a whole. These changes are not necessarily occurring to the proteins associated with the complexes, but merely the proteins that may or may not be contained in the complexes. For example, as a housekeeping protein, GAPDH is known to associate with several other proteins in the cell. Therefore, any changes to GAPDH observed in this study may not be directly related to the GAPDH proteins bound to the complexes being investigated here, but rather reflect overall changes to the total amount of GAPDH present in the cell. 63

70 Figure 3.6: Western Blotted 2D Gel Electrophoresis of Cytosolic Lysates of Unperturbed and Glucose Deprived Jurkat Cells 64

71 Figure 3.6: Western Blotted 2D Gel Electrophoresis of Cytosolic Lysates of Unperturbed and Glucose Deprived Jurkat Cells: 2D electrophoresis gels were transferred to a membrane. The membrane was immunoblotted for every protein known to be present in the two complexes. A sample of the performed blots is shown here. This experiment was run in triplicate. The Mcl-1, Noxa, Ade2, and PHLPP2 blots did not turn out, even with very high concentrations of antibody during the Western blotting process, presumably because the concentrations of these proteins are so low in the context of the proteome of the cell. However, results for the blots shown above were consistent across repeated trials. GAPDH, present in both of the complexes in question, underwent two noticeable changes in response to glucose deprivation. The first is quite obvious when comparing results between the time points: GAPDH appears to be degraded with ongoing glucose deprivation. This is not a surprising result, given that GAPDH is a glycolytic protein and would not be necessary in the absence of glucose. Jurkat cells are also known to be largely committed to apoptosis after twenty-four hours of glucose deprivation. This would induce many of the proteolysis mechanisms of apoptosis that would result in overall lower protein concentrations in the cell. The second result appears to suggest that the types of GAPDH present in the cell reduce from six to four different types as glucose deprivation occurs. This suggests that some of the post-translational states of GAPDH expressed in the presence of glucose are not necessary or functional in its absence. Although the canonical role of GAPDH is 65

72 as a glycolytic enzyme, it is known to have many other functions in cells. Perhaps the glycolytically active forms of GAPDH are the forms that are being degraded or modified as glucose deprivation occurs. Determination of which of these forms of GAPDH is present in the complexes, and if it is glycolytically active, will be an important future step in the functional characterization of these complexes. EF-1ϒ has a well understood role and a second, putative, role in cells: it is known to act as an elongation factor to promote the progression of protein synthesis and it is a putative glutathione transferase. This protein undergoes changes in both its expression as well as its isoelectric point in response to glucose deprivation. However, whereas GAPDH was observed to have a consistently decreasing level of expression with increasing glucose deprivation, EF-1ϒ appears to lose expression at three hours of deprivation and then regain expression at twenty-four hours of deprivation. This observation may be related to its dual function in cells. At three hours of glucose deprivation, EF-1ϒ would not be needed as an elongation factor since protein synthesis would be significantly down regulated in comparison to when the cells were rapidly dividing in glucose-rich conditions. However, glucose deprivation is known to cause oxidative stress in cells. Glutathione transfer is a known mechanism that attaches a glutathione molecule to proteins in response to oxidative stress so as to protect exposed thiol groups on proteins from oxidative damage. It is possible that this function of EF-1ϒ is needed in high levels at twentyfour hours of deprivation, explaining its re-expression after this length of time in these cells. 66

73 Proteomic hc8 is also seen to undergo modifications in response to glucose deprivation. The post-translational states of hc8 are seen to undergo a quite dramatic shift toward a more basic isoelectric point at twenty-four hours of glucose deprivation. As a sub-unit of the proteasome, a large amount of hc8 should become functionally active upon the induction of apoptosis in cell types deprived of glucose for twenty-four hours. The observed results may suggest a potential post-translational modification on hc8 in growing cells that imparts an acidic isoelectric point to the protein (e.g. phosphorylation) that inhibits its proteomic function in the presence of glucose. This modification would thus be removed in response to significant nutrient stress (e.g. twenty-four hours of glucose deprivation) and then allow for the proteins proteomic association. PGK1 does not appear to undergo dramatic changes in response to deprivation. Some alterations are observed to occur to the expression levels of some of the identified forms of PGK1 (both up-regulation and down-regulation). It would be predicted that the glycolytically active form(s) of PGK1 would be the spots downregulated in response to glucose deprivation. Although this information cannot be directly related to the complexes at hand, it does indicate that significant modifications do occur to the types of proteins found in the complexes in response to glucose deprivation. The next step in the study would thus be to investigate which, if any, of the observed changes occur to the proteins actually contained in the complexes. It would then be essential to identify exactly what kind of post-translational state each of the proteins of the complexes is in under various conditions. This should help to elucidate the individual functions of the 67

74 proteins in question so that a more specific function for these complexes in their relation to glucose metabolism and proliferation can be described. 3.5 Modifications to direct complex components In order to determine if the observed changes to protein components in 2D analysis of cytosolic lysates are also detected in the proteins within the described complexes, the complexes were first isolated from cell lysates before being resolved by 2D gel electrophoresis. The process of isolation of the complexes began with gel filtration chromatography of cell lysates. Portions of each collected fraction were resolved by SDS PAGE and Western blotted as previously shown (see Figures 3.3 and 3.4). Upon identification of the complex-containing fractions, the remaining volumes of these fractions were pooled. An immunoprecipitation was performed on these pooled fractions using antibodies against Mcl-1 to isolate the Mcl-1 complexes from the remaining lysate in the fractions. The isolated complexes were then focused by IEF, resolved by SDS PAGE, and the gels stained. 68

75 Figure 3.7: 2D Gel Electrophoresis of Mcl-1 IPs of Pooled Gel Filtrated Lysate Fractions Shown to Contain Mcl-1 from Lysate of Unperturbed and Glucose 100! 75!! Deprived Jurkat Cells Increasing Isoelectric Point! 50!!! 37!! 25! Glucose Deprivation: 0 hrs! Molecular Weight (kda)! 20!! 15!! 10! 100! 75! 50!!! 37!! 25! 20!! 15!! 10! 100! 75! 50!! 37!! 25! 20! 15!! 10!!!! Glucose Deprivation: 3 hrs! Glucose Deprivation: 12 hrs! 69