Vibhor Gupta

Human pyruvate kinase M2: A multifunctional protein

Glycolysis, a central metabolic pathway, harbors evolutionary conserved enzymes that modulate and potentially shift the cellular metabolism on requirement. Pyruvate kinase, which catalyzes the last but rate‐limiting step of glycolysis, is expressed in four isozymic forms, depending on the tissue requirement. M2 isoform (PKM2) is exclusively expressed in embryonic and adult dividing/tumor cells. This tetrameric allosterically regulated isoform is intrinsically designed to downregulate its activity by subunit dissociation (into dimer), which results in partial inhibition of glycolysis at the last step. This accumulates all upstream glycolytic intermediates as an anabolic feed for synthesis of lipids and nucleic acids, whereas reassociation of PKM2 into active tetramer replenishes the normal catabolism as a feedback after cell division. In addition, involvement of this enzyme in a variety of pathways, protein–protein interactions, and nuclear transport suggests its potential to perform multiple nonglycolytic functions with diverse implications, although multidimensional role of this protein is as yet not fully explored. This review aims to provide an overview of the involvement of PKM2 in various physiological pathways with possible functional implications.

RNF4-Dependent Hybrid SUMO-Ubiquitin Chains are Signals for RAP80 and thereby Mediate the Recruitment of BRCA1 to Sites of DNA Damage

DNA repair proteins find sites of damage marked by hybrid SUMO-ubiquitin chains. Hybrid Chains Mark the Spot Posttranslational modifications play a key role in marking sites of DNA damage so that the DNA repair machinery can find the damaged area and effect repair. Guzzo et al. report a role for hybrid chains consisting of SUMO attached to a Lys63-linked diubiquitin as contributing to the recruitment of the protein RAP80, which in turn recruits the DNA repair protein BRCA1, to sites of damaged DNA. Knockdown of the E3 ligase RNF4, which synthesizes hybrid SUMO-ubiquitin linkages, prevented efficient recruitment of RAP80 and BRCA1 to sites of DNA damage induced by irradiation. This study defined a high-affinity interaction between a closely positioned pair of ubiquitin-interacting motifs and a SUMO-interacting motif in RAP80 that contributes to the recognition of sites of DNA damage. The DNA repair function of the breast cancer susceptibility protein BRCA1 depends in part on its interaction with RAP80, which targets BRCA1 to DNA double-strand breaks (DSBs) through recognition of K63-linked polyubiquitin chains. The localization of BRCA1 to DSBs also requires sumoylation. We demonstrated that, in addition to having ubiquitin-interacting motifs, RAP80 also contains a SUMO-interacting motif (SIM) that is critical for recruitment to DSBs. In combination with the ubiquitin-binding activity of RAP80, this SIM enabled RAP80 to bind with nanomolar affinity to hybrid chains consisting of ubiquitin conjugated to SUMO. Furthermore, RNF4, a SUMO-targeted ubiquitin E3 ligase that synthesizes hybrid SUMO-ubiquitin chains, localized to DSBs and was critical for the recruitment of RAP80 and BRCA1 to sites of DNA damage. Our findings, therefore, connect ubiquitin- and SUMO-dependent DSB recognition, revealing that RNF4-synthesized hybrid SUMO-ubiquitin chains are recognized by RAP80 to promote BRCA1 recruitment and DNA repair.

Pyruvate kinase M2 and cancer: an updated assessment

Cancer cells are characterized by high glycolytic rates to support energy regeneration and anabolic metabolism, along with the expression of pyruvate kinase isoenzyme M2 (PKM2). The latter catalyzes the last step of glycolysis and reprograms the glycolytic flux to feed the special metabolic demands of proliferating cells. Besides, PKM2 has moonlight functions, such as gene transcription, favoring cancer. Accumulating evidence suggests a critical role played by the low‐activity‐dimeric PKM2 in tumor progression, supported by the identification of mutations which result in the down‐regulation of its activity and tumorigenesis in a nude mouse model. This review discusses PKM2 regulation and the benefits it confers to cancer cells. Further, conflicting views on PKM2s role in cancer, its therapeutic relevance and future directions in the field are also discussed.

Insulin enhances metabolic capacities of cancer cells by dual regulation of glycolytic enzyme pyruvate kinase M2

BackgroundInsulin is tightly associated with cancer progression; however, mechanistic insights into such observations are poorly understood. Recent studies show that metabolic transformation is critical to cancer cell proliferation. Here, we attempt to understand the role of insulin in promotion of cancer metabolism. To this end, the role of insulin in regulating glycolytic enzyme pyruvate kinase M2 (PKM2) was examined.ResultsWe observed that insulin up-regulated PKM2 expression, through PI3K/mTOR mediated HIF1α induction, but significantly reduced PKM2 activity independent of this pathway. Drop in PKM2 activity was attributed to subunit dissociation leading to formation of low activity PKM2 oligomers, as assessed by density gradient centrifugation. However, tyrosine 105 phosphorylation of PKM2, known for inhibiting PKM2 activity, remained unaffected on insulin treatment. Interestingly, insulin-induced ROS was found responsible for PKM2 activity reduction. The observed changes in PKM2 status led to augmented cancer metabolism. Insulin-induced PKM2 up-regulation resulted in enhanced aerobic glycolysis as confirmed by PKM2 knockdown studies. Further, PKM2 activity reduction led to characteristic pooling of glycolytic intermediates and increased accumulation of NADPH; suggesting diversion of glucose flux towards macromolecular synthesis, necessary for cancer cell growth.ConclusionThe study identifies new PKM2-mediated effects of insulin on cancer metabolism, thus, advancing the understanding of insulin’s role in cancer.

Dominant negative mutations affect oligomerization of human pyruvate kinase M2 isozyme and promote cellular growth and polyploidy.

This study was designed to understand the mechanism and functional implication of the two heterozygous mutations (H391Y and K422R) of human pyruvate kinase M2 isozyme (PKM2) observed earlier in a Bloom syndrome background. The co-expression of homotetrameric wild type and mutant PKM2 in the cellular milieu resulting in the interaction between the two at the monomer level was substantiated further by in vitro experiments. The cross-monomer interaction significantly altered the oligomeric state of PKM2 by favoring dimerization and heterotetramerization. In silico study provided an added support in showing that hetero-oligomerization was energetically favorable. The hetero-oligomeric populations of PKM2 showed altered activity and affinity, and their expression resulted in an increased growth rate of Escherichia coli as well as mammalian cells, along with an increased rate of polyploidy. These features are known to be essential to tumor progression. This study provides insight in understanding the modulated role of large oligomeric multifunctional proteins such as PKM2 by affecting cellular behavior, which is an essential observation to understand tumor sustenance and progression and to design therapeutic intervention in future.

Differential Behavior of Missense Mutations in the Intersubunit Contact Domain of the Human Pyruvate Kinase M2 Isozyme

In this study, we attempted to understand the mechanism of regulation of the activity and allosteric behavior of the pyruvate kinase M2 enzyme and two of its missense mutations, H391Y and K422R, found in cells from Bloom syndrome patients, prone to develop cancer. Results show that despite the presence of mutations in the intersubunit contact domain, the K422R and H391Y mutant proteins maintained their homotetrameric structure, similar to the wild-type protein, but showed a loss of activity of 75 and 20%, respectively. Interestingly, H391Y showed a 6-fold increase in affinity for its substrate phosphoenolpyruvate and behaved like a non-allosteric protein with compromised cooperative binding. However, the affinity for phosphoenolpyruvate was lost significantly in K422R. Unlike K422R, H391Y showed enhanced thermal stability, stability over a range of pH values, a lesser effect of the allosteric inhibitor Phe, and resistance toward structural alteration upon binding of the activator (fructose 1,6-bisphosphate) and inhibitor (Phe). Both mutants showed a slight shift in the pH optimum from 7.4 to 7.0. Although this study signifies the importance of conserved amino acid residues in long-range communications between the subunits of multimeric proteins, the altered behavior of mutants is suggestive of their probable role in tumor-promoting growth and metabolism in Bloom syndrome patients with defective pyruvate kinase M2.

Missense Mutations in Pyruvate Kinase M2 Promote Cancer Metabolism, Oxidative Endurance, Anchorage Independence, and Tumor Growth in a Dominant Negative Manner

Background: We earlier reported novel mutations in PKM2 that reduce its activity. Results: These mutations promoted cancer features and tumor growth in a dominant negative manner. Conclusion: Impaired PKM2 activity due to mutations benefits cancer. Significance: This study provides the first evidence linking natural mutations in PKM2 with cancer. The present study was designed to examine the functional relevance of two heterozygous mutations (H391Y and K422R), observed earlier by us in the Bloom syndrome condition. Cells stably expressing exogenous wild-type or mutant PKM2 (K422R or H391Y) or co-expressing both wild type and mutant (PKM2-K422R or PKM2-H391Y) were assessed for cancer metabolism and tumorigenic potential. Interestingly, cells co-expressing PKM2 and mutant (K422R or H391Y) showed significantly aggressive cancer metabolism as compared with cells expressing either wild-type or mutant PKM2 independently. A similar trend was observed for oxidative endurance, tumorigenic potential, cellular proliferation, and tumor growth. These observations signify the dominant negative nature of mutations. Remarkably, PKM2-H391Y co-expressed cells showed a maximal effect on all the studied parameters. Such a dominant negative impaired function of PKM2 in tumor development is not known; this study demonstrates for the first time the possible predisposition of Bloom syndrome patients with impaired PKM2 activity to cancer and the importance of studying genetic variations in PKM2 in the future to understand their relevance in cancer in general.

In Silico Screening, Genotyping, Molecular Dynamics Simulation and Activity Studies of SNPs in Pyruvate Kinase M2

Role of, 29-non-synonymous, 15-intronic, 3-close to UTR, single nucleotide polymorphisms (SNPs) and 2 mutations of Human Pyruvate Kinase (PK) M2 were investigated by in-silico and in-vitro functional studies. Prediction of deleterious substitutions based on sequence homology and structure based servers, SIFT, PANTHER, SNPs&GO, PhD-SNP, SNAP and PolyPhen, depicted that 19% emerged common between all the mentioned programs. SNPeffect and HOPE showed three substitutions (C31F, Q310P and S437Y) in-silico as deleterious and functionally important. In-vitro activity assays showed C31F and S437Y variants of PKM2 with reduced activity, while Q310P variant was catalytically inactive. The allosteric activation due to binding of fructose 1-6 bisphosphate (FBP) was compromised in case of S437Y nsSNP variant protein. This was corroborated through molecular dynamics (MD) simulation study, which was also carried out in other two variant proteins. The 5 intronic SNPs of PKM2, associated with sporadic breast cancer in a case-control study, when subjected to different computational analyses, indicated that 3 SNPs (rs2856929, rs8192381 and rs8192431) could generate an alternative transcript by influencing splicing factor binding to PKM2. We propose that these, potentially functional and important variations, both within exons and introns, could have a bearing on cancer metabolism, since PKM2 has been implicated in cancer in the recent past.

Interplay between Epigenetics & Cancer Metabolism

Nutrient utilization is dramatically altered when cells receive signals to proliferate. Characteristic metabolic changes enable cells to meet the large biosynthetic demands associated with cell growth and division. Changes in rate-limiting glycolytic enzymes redirect metabolism to support growth and proliferation. Metabolic reprogramming in cancer is controlled largely by oncogenic activation of signal transduction pathways and transcription factors. Although less well understood, epigenetic mechanisms may seem to contribute to the regulation of metabolic gene expression in cancer. Reciprocally, accumulating evidence suggests that metabolic alterations may affect the epigenome. Understanding the relation between metabolism and epigenetics in cancer cells may open new avenues for anti-cancer strategies. In multi-cellular systems, molecular signals promoting cell growth and proliferation mediate the switch between catabolism and anabolism. Both normal proliferating and cancer cells must achieve high levels of macromolecular biosynthesis to provide the raw materials needed to produce new daughter cells. From a therapeutic view point, it is of great interest to determine metabolic differences that exist between normal proliferating cells and cancer cells. Cancer cells also exhibit significant alterations in the epigenome. Recent data indicate that cellular metabolism and epigenetic phenomenon are engaged in crosstalk. Considering current efforts to target both cancer metabolism and epigenetics, an understanding of the relationship between these two key features is of paramount importance. Here we discuss the role of cellular metabolism in regulation of the epigenome. Moreover, we discuss how epigenetic changes may contribute to establish cancer-specific metabolic features.

Pyruvate Kinase M2: A Metabolic Tuner

Metabolic enzymes are emerging as key mediators in modifying cell metabolism in response to extracellular signals, such as growth factors which trigger the upregulation of a specific isoform of pyruvate kinase (PK) M2. PKM2 alters its own catalytic activity by many different ways, and one of these involves the switch in the degree of oligomerization, shifting resting cell metabolism to a pro-proliferative one. A shift of enzymatic activity from high to low is generally considered as a trigger for cell division; however, such a change is guided under a delicate equilibrium, which if lost leads to uncontrolled cell metabolism, a reason for which PKM2 is associated with cancer and Warburg effect. Thus, a curiosity is aroused to understand the factors regulating PKM2 activity and expression and how these are affected by different metabolites and proteins. In this chapter, we discuss different levels of regulating PK expression and activity and possible therapeutic approaches, where PKM2 is proposed as a potential target for cancer.