Active Dimer Research Articles

Abstract 2514: Most clinically approved anti-EGFR antibodies fail to neutralize EGFRvIII, explaining their lack of efficacy in high-grade glioma

Abstract The epidermal growth factor receptor (EGFR), a cell-surface receptor tyrosine kinase, is amplified or overexpressed in 60% of gliomas. Furthermore, in 30% of gliomas, an autoactive, disulphide-bonded truncation mutant, EGFRvIII, is co-expressed. Both the wild-type (wt) and EGFRvIII are central to driving gliomagenesis. Multiple anti-EGFR antibodies have been developed to neutralise these receptors; however, despite promising preclinical results, their efficacy against glioma in the clinic has been disappointing for reasons that are not understood. We have tested the efficacy and mechanism of action of the majority of known FDA-approved or preclinical anti-EGFR antibodies [panitumumab, cetuximab, necitumumab, nimotuzumab, matuzumab and chimeric 806 (ch806)] against five primary EGFRvIII-bearing glioma lines. We find that cetuximab, necitumumab, nimotuzumab and matuzumab all fail to fully neutralize EGFRvIII activity, leading to sustained downstream signalling and modest antiproliferative effects. ch806 neutralized EGFRvIII activity but did not engage and neutralize wtEGFR. Only panitumumab was able to completely neutralize both wtEGFR and EGFRvIII activation, resulting in a superior antiproliferative response. Not all EGFRvIII-bearing cell lines were responsive to panitumumab (3 out of 5), suggesting that the expression of EGFRvIII is not indicative of sensitivity to anti-EGFR therapy and that additional mechanisms of resistance are present when EGFRvIII is expressed. Only when cetuximab or necitumumab was combined with ch806, to neutralize both wtEGFR and EGFRvIII, was an antiproliferative response similar to panitumumab achieved. Mechanistically, panitumumab neutralized p-ERK but not p-AKT or p-STAT3 signaling. Panitumumab, cetuximab and ch806 induced EGFRvIII internalization and recycling by Rab11-positive endosomes back to the cell surface, while wtEGFR was degraded. Subsequent wtEGFR translation replaced the degraded wtEGFR, ensuring receptor pool replenishment. Finally, panitumumab does not exert its effects by breaking the active EGFRvIII disulphide-bonded dimer; rather, it may work by trapping EGFRvIII into an inactive high molecular weight receptor cluster, an effect that was not observed for cetuximab. Animal trials to compare the efficacy of these antibodies against one another in intracranial orthografts of patient-derived cell lines are ongoing. Collectively, our results challenge the existing premise that EGFRvIII is neutralized and degraded by EGFR antibodies and demonstrate that both wtEGFR and EGFRvIII must be neutralized for maximum antitumor effect. These results may help explain why existing antibodies such as cetuximab and nimotuzumab have failed in clinical trials in glioma and provide rationale that panitumumab may offer a superior and effective candidate for a further clinical trial in glioma. Citation Format: Sameer A. Greenall, Timothy E. Adams, Terrance G. Johns. Most clinically approved anti-EGFR antibodies fail to neutralize EGFRvIII, explaining their lack of efficacy in high-grade glioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2514.

Acetylation within the N- and C-Terminal Domains of Src Regulates Distinct Roles of STAT3-Mediated Tumorigenesis.

Posttranslational modifications of mammalian c-Src N-terminal and C-terminal domains regulate distinct functions. Myristoylation of G2 controls its cell membrane association and phosphorylation of Y419/Y527 controls its activation or inactivation, respectively. We provide evidence that Src-cell membrane association-dissociation and catalytic activation-inactivation are both regulated by acetylation. In EGF-treated cells, CREB binding protein (CBP) acetylates an N-terminal lysine cluster (K5, K7, and K9) of c-Src to promote dissociation from the cell membrane. CBP also acetylates the C-terminal K401, K423, and K427 of c-Src to activate intrinsic kinase activity for STAT3 recruitment and activation. N-terminal domain phosphorylation (Y14, Y45, and Y68) of STAT3 by c-Src activates transcriptionally active dimers of STAT3. Moreover, acetyl-Src translocates into nuclei, where it forms the Src-STAT3 enhanceosome for gene regulation and cancer cell proliferation. Thus, c-Src acetylation in the N-terminal and C-terminal domains play distinct roles in Src activity and regulation.Significance: CBP-mediated acetylation of lysine clusters in both the N-terminal and C-terminal regions of c-Src provides additional levels of control over STAT3 transcriptional activity. Cancer Res; 78(11); 2825-38. ©2018 AACR.

P-34 - Kinetics and biological consequences of quinone-induced protein adduction

Quinones are a major class of toxicological compounds, that can both generate radicals, and act as Michael acceptors with nucleophiles including Cys residues on proteins. This study aimed to quantitatively assess quinone-protein reactions and examine the relationship between quinone adduction and protein dysfunction. A range of quinones were incubated with glyceraldehyde-3-phosphate dehydrogenase (GAPDH), creatine kinase (CK), papain, bovine serum albumin (BSA), and human serum albumin (HSA), with both the kinetics of adduction and effects on protein structure and activity determined. Adduction rate constants at Cys residues, which were dependent on the quinone and protein structures, and thiol pKa, were in the range 103 -105 M-1s-1. p-Benzoquinone (BQ) induced dimerization of GAPDH and CK (but not BSA, HSA, or papain) in a dose- and time-dependent manner, as well as a loss of enzyme activity. Glutathione (GSH) reacts competitively with BQ, and can thereby reverse the activity loss and dimerization of GAPDH and CK. Mass spectrometry, after digestion to peptide, showed that BQ preferentially forms adducts with GAPDH at Cys149 and Cys244. These data suggest that quinones can induce toxicity by rapidly and selectively forming adducts with Cys residues in proteins.

Inhibitor-induced HER2-HER3 heterodimerisation promotes proliferation through a novel dimer interface.

While targeted therapy against HER2 is an effective first-line treatment in HER2+ breast cancer, acquired resistance remains a clinical challenge. The pseudokinase HER3, heterodimerisation partner of HER2, is widely implicated in the resistance to HER2-mediated therapy. Here, we show that lapatinib, an ATP-competitive inhibitor of HER2, is able to induce proliferation cooperatively with the HER3 ligand neuregulin. This counterintuitive synergy between inhibitor and growth factor depends on their ability to promote atypical HER2-HER3 heterodimerisation. By stabilising a particular HER2 conformer, lapatinib drives HER2-HER3 kinase domain heterocomplex formation. This dimer exists in a head-to-head orientation distinct from the canonical asymmetric active dimer. The associated clustering observed for these dimers predisposes to neuregulin responses, affording a proliferative outcome. Our findings provide mechanistic insights into the liabilities involved in targeting kinases with ATP-competitive inhibitors and highlight the complex role of protein conformation in acquired resistance.

Reactive Chemicals and Electrophilic Stress in Cancer: A Minireview.

Exogenous reactive chemicals can impair cellular homeostasis and are often associated with the development of cancer. Significant progress has been achieved by studying the macromolecular interactions of chemicals that possess various electron-withdrawing groups and the elucidation of the protective responses of cells to chemical interventions. However, the formation of electrophilic species inside the cell and the relationship between oxydative and electrophilic stress remain largely unclear. Derivatives of nitro-benzoxadiazole (also referred as nitro-benzofurazan) are potent producers of hydrogen peroxide and have been used as a model to study the generation of reactive species in cancer cells. This survey highlights the pivotal role of Cu/Zn superoxide dismutase 1 (SOD1) in the production of reactive oxygen and electrophilic species in cells exposed to cell-permeable chemicals. Lipophilic electrophiles rapidly bind to SOD1 and induce stable and functionally active dimers, which produce excess hydrogen peroxide leading to aberrant cell signalling. Moreover, reactive oxygen species and reactive electrophilic species, simultaneously generated by redox reactions, behave as independent entities that attack a variety of proteins. It is postulated that the binding of the electrophilic moiety to multiple proteins leading to impairing different cellular functions may explain unpredictable side effects in patients undergoing chemotherapy with reactive oxygen species (ROS)-inducing drugs. The identification of proteins susceptible to electrophiles at early steps of oxidative and electrophilic stress is a promising way to offer rational strategies for dealing with stress-related malignant tumors.

Neuraminidase 1 activates insulin receptor and reverses insulin resistance in obese mice

ObjectivesNeuraminidase 1 (NEU1) cleaves terminal sialic acids of glycoconjugates during lysosomal catabolism. It also modulates the structure and activity of cellular surface receptors affecting diverse pathways. Previously we demonstrated that NEU1 activates the insulin receptor (IR) and that NEU1-deficient CathAS190A-Neo mice (hypomorph of the NEU1 activator protein, cathepsin A/CathA) on a high-fat diet (HFD) develop hyperglycaemia and insulin resistance faster than wild-type animals. The major objective of the current work was to reveal the molecular mechanism by which NEU1 desialylation activates the IR and to test if increase of NEU1 activity in insulin target tissues reverses insulin resistance and glucose intolerance. MethodsTo test if desialylation causes a conformational change in the IR dimer we measured interaction between the receptor subunits by Bioluminescence Resonance Energy Transfer in the HEK293T cells either overexpressing NEU1 or treated with the NEU1 inhibitor. The influence of NEU1 overexpression on insulin resistance was studied in vitro in palmitate-treated HepG2 cells transduced with NEU1-expressing lentivirus and in vivo in C57Bl6 mice treated with HFD and either pharmacological inducer of NEU1, Ambroxol or NEU1-expressing adenovirus. NEU1-deficient CathAS190A-Neo mice were used as a control. ResultsBy desialylation of IR, NEU1 induced formation of its active dimer leading to insulin signaling. Overexpression of NEU1 in palmitate-treated HepG2 cells restored insulin signaling, suggesting that increased NEU1 levels may reverse insulin resistance. Five-day treatment of glycemic C57Bl6 mice receiving HFD with the activator of the lysosomal gene network, Ambroxol, increased NEU1 expression and activity in muscle tissue, normalized fasting glucose levels, and improved physiological and molecular responses to glucose and insulin. Ambroxol did not improve insulin sensitivity in obese insulin-resistant CathAS190A-Neo mice indicating that the Ambroxol effect is mediated through NEU1 induction. Sustained increase of liver NEU1 activity through adenovirus-based gene transfer failed to attenuate insulin resistance most probably due to negative feedback regulation of IR expression. ConclusionTogether our results demonstrate that increase of NEU1 activity in insulin target tissues reverses insulin resistance and glucose intolerance suggesting that a pharmacological modulation of NEU1 activity may be potentially explored for restoring insulin sensitivity and resolving hyperglycemia associated with T2DM.

Autoinhibitory regulation of S100A8/S100A9 alarmin activity locally restricts sterile inflammation.

Autoimmune diseases, such as psoriasis and arthritis, show a patchy distribution of inflammation despite systemic dysregulation of adaptive immunity. Thus, additional tissue-derived signals, such as danger-associated molecular patterns (DAMPs), are indispensable for manifestation of local inflammation. S100A8/S100A9 complexes are the most abundant DAMPs in many autoimmune diseases. However, regulatory mechanisms locally restricting DAMP activities are barely understood. We now unravel for the first time, to our knowledge, a mechanism of autoinhibition in mice and humans restricting S100-DAMP activity to local sites of inflammation. Combining protease degradation, pull-down assays, mass spectrometry, and targeted mutations, we identified specific peptide sequences within the second calcium-binding EF-hands triggering TLR4/MD2-dependent inflammation. These binding sites are free when S100A8/S100A9 heterodimers are released at sites of inflammation. Subsequently, S100A8/S100A9 activities are locally restricted by calcium-induced (S100A8/S100A9)2 tetramer formation hiding the TLR4/MD2-binding site within the tetramer interphase, thus preventing undesirable systemic effects. Loss of this autoinhibitory mechanism in vivo results in TNF-α-driven fatal inflammation, as shown by lack of tetramer formation in crossing S100A9-/- mice with 2 independent TNF-α-transgene mouse strains. Since S100A8/S100A9 is the most abundant DAMP in many inflammatory diseases, specifically blocking the TLR4-binding site of active S100 dimers may represent a promising approach for local suppression of inflammatory diseases, avoiding systemic side effects.

Mutagenesis of the Human α‐Galactosidase A Active Site, Dimer Interface, and Glycosylation Region

Fabry disease is an X‐linked lysosomal storage disorder caused by the deficiency of the enzyme, α‐galactosidase A, which results in the accumulation of the lipid substrate. This accumulation results in obstruction of blood flow in patients and early demise at approximately 40–60 years of age. Currently approved enzyme replacement therapy using recombinant human α‐galactosidase A improves patient symptoms but storage of the toxic enzyme substrate remains in the brain and other affected organs. The structure of α‐galactosidase A has been previously determined to be a homodimer with six N‐linked glycosylation sites, and the catalytic mechanism determined to be a ping pong bi bi with the second substrate being water. The purpose of this research is to generate a more efficient enzyme replacement therapy alternative. A more efficient therapy was investigated utilizing Pichia pastoris, as well as site directed mutagenesis of the human enzyme. Three locations on the structure of α‐galactosidase A protein were targeted for mutation: the active site; the dimer interface; and the hydrophobic loop between the active site and the third glycosylation site. Viable candidates for further study into a therapeutic were determined based on catalytic efficiency (kcat/Km), thermostability, and possible glycosylation independence. Three mutations were identified, M208E (hydrophobic loop), W277C (dimer interface), and Y207W (active site) to be of potential therapeutic significance.Support or Funding InformationNIH SBIR Grants and Royalty Income From PatentsThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Central Angiotensin II regulates Protein Inhibitor of Neuronal Nitric Oxide Synthase through post‐translational mechanisms in the Paraventricular Nucleus resulting in increased Sympathetic outflow

Increased activation of Renin‐Angiotensin System in the paraventricular nucleus (PVN) of the hypothalamus has been associated with the increased sympathetic outflow observed in hypertension and chronic heart failure (CHF). Previously we have shown increased protein expression of the PIN (a protein inhibitor of nNOS: neuronal nitric oxide synthase, known to dissociate nNOS dimers into monomers) with concomitantly reduced levels of catalytically active dimers of nNOS in the PVN of rats with CHF. However, PIN transcripts levels remain unchanged. To elucidate the molecular mechanism of increased PIN expression post‐transcriptionally, we used Sprague‐Dawley rats (250–300 g) subjected to intracerebroventricular infusion of Ang II (20 ng/min, 14 days, 0.5 μl/h) through osmotic mini‐pumps and NG108‐15 hybrid neuronal cell line treated with Ang II as an in vitro model. Ang II infusion significantly increased baseline mean arterial pressure (126 ± 9 vs. 84 ± 4 mmHg) and baseline renal sympathetic nerve activity (20.5 ± 2.3 vs. 6.4 ± 1.9 % of Max activity). Ang II infusion increased the expression of the PIN (1.36 ± 0.04* Ang II vs. 0.81 ± 0.02 Veh) a with concomitant decrease in PIN‐Ub conjugates (0.73 ± 0.04* Ang II vs. 1 ± 0.03 Veh) in the PVN. Further, PIN translation was inhibited in vitro using protein synthesis inhibitor cycloheximide (CHX) for 0–4h after 20h of pretreatment with Ang II. Ang II‐mediated increase in PIN expression(0.67±0.06* CHX AngII vs. 0.40±0.08 CHX 0h) was independent of CHX mediated decrease in PIN expression (0.41±0.06* CHX AngII vs. 0.19±0.04 CHX 4h) suggesting Ang II‐mediated post‐translational stabilization of PIN. Ubiquitination assay in cells transfected with pCMV‐(HA‐Ub) 8 vector revealed a reduction of HA‐Ub‐PIN conjugates after Ang II and proteasome inhibitor lactacystin (LC) treatment (4.5 ± 0.6* LC Ang II vs. 9.2 ± 2.2 LC). TUBE (Tandem Ubiquitin‐Binding Entities) assay showed decrease PIN‐Ub conjugates in Ang II‐treated cells (0.82 ± 0.12* LC Ang II vs. 1.23 ± 0.05 LC) while AT1R blocker Losartan(Los) treatment diminishes the Ang II‐mediated stabilization of PIN (1.21 ± 0.07 LC Los vs. 1.14 ± 0.04* LC AngII Los). These results suggest that PIN is targeted for rapid degradation by the ubiquitin‐proteasome and Ang II via AT1R delays the rate of degradation resulting in accumulation of PIN. It of interest to note that previously we observed that there was a decreased accumulation of PIN‐Ub conjugates in the PVN of CHF rats compared to Sham (0.63 ± 0.05* CHF vs. 1.00 ± 0.10 Sham). Taken together, our studies suggest that increased central levels of Ang II contribute to the upregulation of PIN expression in the PVN via decreasing the ubiquitination which may be involved in reducing the expression of dimeric nNOS of pre‐autonomic neurons in the PVN resulting in the increased sympathetic outflow.Support or Funding InformationSupported by AHA ‐ 14SDG19980007 and NIH grants HL124104 & HL62222.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Quantitative Characterization of the Membrane Dynamics of Newly Delivered TGF-β Receptors by Single-Molecule Imaging

The dynamics and stoichiometry of receptors newly delivered on the plasma membrane play a vital role in cell signal transduction, yet knowledge of this process is limited because of the lack of suitable analytical methods. Here we developed a new strategy that combines single-molecule imaging (SMI) and fluorescence recovery after photobleaching (FRAP), named FRAP-SMI, to monitor and quantify individual newly delivered and inserted transmembrane receptors on plasma membranes of living cells. Transforming-growth-factor-β type II receptor (TβRII), a typical serine/threoninekinase receptor, was studied with this method. We first eliminated the fluorescence signals from the pre-existing EGFP-labeled TβRII molecules on the plasma membrane, and then we recorded the individual newly appeared TβRII-GFP by total-internal-reflection fluorescence imaging. The fluorescence-intensity distributions, photobleaching steps, and diffusion rates of the single TβRII-GFP molecules were analyzed. We reported, for the first time, that TβRII was transported to the plasma membrane mainly in the monomeric form in both resting and TGF-β1stimulated cells. This strongly supported our former discovery that TβRII could exist as a monomer on the cell membrane. We also found that ligand stimulation resulted in enhanced delivery rates and prolonged membrane-association times for the TβRII molecules. On the basis of these observations, we proposed a mechanism of TGF-β1-induced TβRII dimerization for receptor activation. Our method provides a useful tool for the real-time quantification of the spatial arrangement, mobility, and oligomerization of cell-surface proteins in living cells, thus providing a better understanding of cell signaling.

SIRT5 inhibits peroxisomal ACOX1 to prevent oxidative damage and is downregulated in liver cancer.

Peroxisomes account for ~35% of total H2O2 generation in mammalian tissues. Peroxisomal ACOX1 (acyl-CoA oxidase 1) is the first and rate-limiting enzyme in fatty acid β-oxidation and a major producer of H2O2 ACOX1 dysfunction is linked to peroxisomal disorders and hepatocarcinogenesis. Here, we show that the deacetylase sirtuin 5 (SIRT5) is present in peroxisomes and that ACOX1 is a physiological substrate of SIRT5. Mechanistically, SIRT5-mediated desuccinylation inhibits ACOX1 activity by suppressing its active dimer formation in both cultured cells and mouse livers. Deletion of SIRT5 increases H2O2 production and oxidative DNA damage, which can be alleviated by ACOX1 knockdown. We show that SIRT5 downregulation is associated with increased succinylation and activity of ACOX1 and oxidative DNA damage response in hepatocellular carcinoma (HCC). Our study reveals a novel role of SIRT5 in inhibiting peroxisome-induced oxidative stress, in liver protection, and in suppressing HCC development.

Caspase-1 self-cleavage is an intrinsic mechanism to terminate inflammasome activity.

Host-protective caspase-1 activity must be tightly regulated to prevent pathology, but mechanisms controlling the duration of cellular caspase-1 activity are unknown. Caspase-1 is activated on inflammasomes, signaling platforms that facilitate caspase-1 dimerization and autoprocessing. Previous studies with recombinant protein identified a caspase-1 tetramer composed of two p20 and two p10 subunits (p20/p10) as an active species. In this study, we report that in the cell, the dominant species of active caspase-1 dimers elicited by inflammasomes are in fact full-length p46 and a transient species, p33/p10. Further p33/p10 autoprocessing occurs with kinetics specified by inflammasome size and cell type, and this releases p20/p10 from the inflammasome, whereupon the tetramer becomes unstable in cells and protease activity is terminated. The inflammasome-caspase-1 complex thus functions as a holoenzyme that directs the location of caspase-1 activity but also incorporates an intrinsic self-limiting mechanism that ensures timely caspase-1 deactivation. This intrinsic mechanism of inflammasome signal shutdown offers a molecular basis for the transient nature, and coordinated timing, of inflammasome-dependent inflammatory responses.

Pressure Induced SOS Response in Escherichia coli Involves Mrr Restriction Endonuclease Dissociation

Subjecting the mesophilic Escherichia coli bacterium to a pressure of ∼100 MPa elicits an SOS response, despite the fact that pressure itself cannot compromise DNA covalent integrity. Screens for pressure shock resistance revealed the constitutive presence in several strains of E. coli of a cryptic, endogenous type IV restriction endonuclease, Mrr (Methylated adenine Recognition and Restriction), that cleaves methylated DNA. Since the enzyme is present and non-toxic in the absence of pressure treatment, activation must be pressure dependent. Although types IV REases do not have a cognate methyltransferase (MTase), HhaII has been shown to activate Mrr and induce an SOS response through methylation of high affinity sites, recognized by Mrr. Our previous in vivo fluorescence results using scanning Number and Brightness suggest that pressure shock “pushes” the equilibrium from the inactive tetrameric form of Mrr toward the active dimer which can bind cryptic sites on E. coli chromosome and cleave DNA. In contrast, HhaII induction appears to “pull” the equilibrium toward the dimeric Mrr active forms bind to DNA. To test this model, we used the same approach to evaluate Mrr stoichiometry in both catalytically defective mutants as well as a constitutive mutant which is lethal for E. coli. Some of the mutants were found to respond only to pressure and not to HhaII MTase induction, or vice versa. Homology modeling of the tetrameric Mrr catalytic domain supports the proposed mechanism of action drawn from our in vivo results of the mutants. sN&B confirmed the role of the tetramer-dimer equilibrium in modulating Mrr activity. We are now attempting to study association/dissociation process of purified Mrr under pressure by fluorescence correlation spectroscopy (FCS) thanks to a high-pressure set-up we recently implemented on our 2-photon microscope.

Regulation of Kinase Activity in the Caenorhabditis elegans EGF Receptor, LET-23

In the active HER receptor dimers, kinases play distinct roles; one is the catalytically active kinase and the other is its allosteric activator. This specialization enables signaling by the catalytically inactive HER3, which functions exclusively as an allosteric activator upon heterodimerization with other HER receptors. It is unclear whether the allosteric activation mechanism evolved before HER receptors functionally specialized. We determined the crystal structure of the kinase domain of the only EGF receptor in Caenorhabditis elegans, LET-23. Our structure of a non-human EGFR kinase reveals autoinhibitory features conserved in the human counterpart. Strikingly, mutations within the putative allosteric dimer interface abrogate activity of the isolated LET-23 kinase and of the full-length receptor despite these regions being only partially conserved with human EGFR. Our results indicate that ancestral EGFRs have built-in features that poise them for allosteric activation that could facilitate emergence of the catalytically dead, yet functional, orthologs.

Cholecystokinin is involved in triglyceride fatty acid uptake by rat adipose tissue.

The incorporation of plasma triglyceride (TG) fatty acids to white adipose tissue (WAT) depends on lipoprotein lipase (LPL), which is regulated by angiopoietin-like protein-4 (ANGPTL-4), an unfolding molecular chaperone that converts active LPL dimers into inactive monomers. The production of ANGPTL-4 is promoted by fasting and repressed by feeding. We hypothesized that the postprandial hormone cholecystokinin (CCK) facilitates the storage of dietary TG fatty acids in WAT by regulating the activity of the LPL/ANGPTL-4 axis and that it does so by acting directly on CCK receptors in adipocytes. We report that administration of CCK-8 (a bioactive fragment of CCK) to rats: (i) reduces plasma ANGTPL-4 levels; (ii) represses Angptl-4 expression in WAT and (iii) simultaneously enhances LPL activity in this tissue without inducing Lpl expression. In vivo CCK-8 effects are specifically antagonized by the CCK-2 receptor (CCK-2R) antagonist, L-365,260. Moreover, CCK-8 downregulates Angptl-4 expression in wild-type pre-adipocytes, an effect that is not observed in engineered pre-adipocytes lacking CCK-2R. These effects have functional consequences as CCK-8 was found to promote the uptake of dietary fatty acids by WAT, as demonstrated by means of proton nuclear magnetic resonance (1H-NMR). The efficacy of acute CCK-8 administration was not reduced after chronic CCK-8 treatment. Moreover, the effects of CCK-8 on WAT were not associated to the increase of circulating insulin. Our results show that cholecystokinin promotes lipid storage in WAT by acting on adipocyte CCK-2R, suggesting a pivotal role for CCK in TG homeostasis.

Functional Impact of the N-terminal Arm of Proline Dehydrogenase from Thermus thermophilus.

Proline dehydrogenase (ProDH) is a ubiquitous flavoenzyme that catalyzes the oxidation of proline to Δ1-pyrroline-5-carboxylate. Thermus thermophilus ProDH (TtProDH) contains in addition to its flavin-binding domain an N-terminal arm, consisting of helices αA, αB, and αC. Here, we report the biochemical properties of the helical arm truncated TtProDH variants ΔA, ΔAB, and ΔABC, produced with maltose-binding protein as solubility tag. All three truncated variants show similar spectral properties as TtProDH, indicative of a conserved flavin-binding pocket. ΔA and ΔAB are highly active tetramers that rapidly react with the suicide inhibitor N-propargylglycine. Removal of the entire N-terminal arm (ΔABC) results in barely active dimers that are incapable of forming a flavin adduct with N-propargylglycine. Characterization of V32D, Y35F, and V36D variants of ΔAB established that a hydrophobic patch between helix αC and helix α8 is critical for TtProDH catalysis and tetramer stabilization.

The effect of phosphate group binding cup coordination on the stability of the amine transaminase from Chromobacterium violaceum

The amine transaminase from Chromobacterium violaceum (Cv-ATA) is a pyridoxal-5'-phosphate (PLP) dependent enzyme. The biological activity of this enzyme requires the formation of a holo homo dimer. The operational stability of Cv-ATA is, however, low due to dimer dissociation. At the enzyme dimeric interface, two phosphate group binding cups (PGBC) are located. Each cup coordinates the phosphate group of PLP by hydrogen bonds originating from both subunits. Hypothetically, molecular coordination of phosphate groups (PLP or free inorganic phosphate) into the PGBC can affect both dimer stabilization and enzyme activity. To test this assumption, the influence of phosphate (as a functional group in PLP or as free inorganic anions) on the stability and activity of Cv-ATA was explored by various biophysical techniques. The results show that Cv-ATA has a relatively low affinity towards PLP, which results in an excess of apo dimeric enzyme after enzyme purification. Incubation of the apo dimer in buffer solution supplemented with PLP restored the active holo dimer. The addition of PLP or inorganic phosphate into the enzyme storage solutions protected Cv-ATA from both chemical and long term storage unfolding. The use of phosphate buffer leads to faster inactivation of the holo enzyme, compared to the use of HEPES buffer. These results open up for new perspectives on how to improve the stability of PLP-dependent enzymes.

Abstract B024: PKM2 activation suppresses cellular ROS scavenging capacity and potentiates doxorubicin antitumor activity

Abstract Pyruvate kinase (PK) is a key mediator of the metabolic reprogramming and the Warburg effect observed in tumorigenesis. PK catalyzes the rate-limiting conversion of phosphoenolpyruvate (PEP) to pyruvate in glycolysis. There are fourteen known transcript variants of the PKM gene. In normal adult tissues, the M1 (PKM1) isoform predominates, promoting oxidative phosphorylation and generation of ATP. The M2 (PKM2) isoform, however, and indeed many of the PKM variants other than M1, predominate in tumor tissues. Unlike PKM1, PKM2 exists largely as a less active dimer. The decreased activity of dimer PKM2, relative to PKM1, in this key conversion step leads to an accumulation and shunting of upstream glycolytic intermediates into synthetic pathways needed for the building blocks necessary to maintain continued tumor cell growth. Thus, PKM2 is the focus of intense research into its utility as a target for the treatment of cancer. It has been hypothesized that PKM2 activation should starve cells of necessary building blocks, slowing tumor cell proliferation. We have developed a PKM2 activator, TP-1454, that activates PKM2 in biochemical assays with an AC50 of 11 nM. In cell viability assays, TP-1454 inhibited A549 lung cell growth with an IC50 of 3.4 μM. In conditions where additional nutrients, such as serine, are withdrawn but not growth limiting, TP-1454 inhibited cell growth with an IC50 of 20.3 nM. Consistent with the notion that PKM2 activation leads to a reduction in upstream building blocks, a mass spectrometry metabolomics study showed that TP-1454-induced PKM2 activation modulated several key cellular metabolic components, including key amino acids and glycolytic and citric acid intermediates. However, the single highest-modulated metabolite was glutathione, which was reduced more than 10-fold in treated cells. This finding was also confirmed using a luciferase-based approach with the GSH-Glo glutathione assay. These data led us to hypothesize that PKM2 activation may cause multiple changes in cellular metabolism, but that the greatest vulnerability induced by PKM2 activation was to compromise the cell’s ability to combat reactive oxygen species (ROS) due to the loss of glutathione levels. Therefore, we reasoned that TP-1454 would combine well with drugs known to induce ROS-mediated cell death, such as the anthracyclines. Doxorubicin inhibited A549 cell viability with an IC50 of 173 nM. We observed a four-fold reduction in IC50 of A549 cells treated with 4 μM TP-1454. The antioxidant N-acetyl cysteine partially rescued the synergistic effect of TP-1454 on doxorubicin activity, suggesting that the combination effect was ROS driven. Studies investigating TP-1454-induced glutathione suppression in combination with doxorubicin, in vivo, are currently under way. Taken together, these data support the hypothesis that PKM2 activators may combine well with anthracycline drugs and warrant clinical investigation of PKM2 as a potential therapeutic target for the treatment of multiple cancer types. Citation Format: Peter Peterson, Clifford J. Whatcott, David J. Bearss, Steven L. Warner, Adam Siddiqui-Jain. PKM2 activation suppresses cellular ROS scavenging capacity and potentiates doxorubicin antitumor activity [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B024.

ZWINT positively regulates TBK1-mediated RLR antiviral signaling

TANK-Binding Kinase 1 (TBK1) is a serine/threonine kinase ubiquitously expressed in many tissues and plays an important role in the RLR antiviral signaling pathway. Upon viral infection, various virus components, including viral DNA, RNA are sensed by different pattern recognition receptors (PRRs). Among these PRRs, TLR3, RIG-I/MDA5, and cGAS recruit different adaptors including TRIF, VISA/ MAVS, and MITA/STING to activate TBK1. Activated TBK1 then promote the phosphorylation and homodimerization of IFN-regulatory factors 3 and 7 (IRF3/7), in turn promoting type-I Interferon (IFNI) production. In this study, we reported that ZW10 Interacting Kinetochore Protein (ZWINT) positively regulates RLR antiviral signaling by targeting RIG-I/VISA/TBK1. Ectopic expression of ZWINT potentiated Sendai virus-induced, as well as TBK1- or VISA/MAVS-mediated IFN-β promoter activation by enhancing dimerization of IRF3. In contrast, knocking down of ZWINT expression with siRNA resulted in reduced IFN-β promoter activation and IRF3 dimerization induced by Sendai virus, indicating that ZWINT positively regulates TBK1-mediated RLR antiviral signaling.

Purification of fully active and crystallizable photosystem II from thermophilic cyanobacteria.

Photosystem II (PSII) is a membrane protein complex which functions to catalyze light-induced water oxidation in oxygenic photosynthesis. Through the water-splitting reaction of PSII, light energy is converted into biologically useful chemical energy, and molecular oxygen is formed which transformed the atmosphere into an aerobic one and sustained aerobic life on the Earth. The PSII core complex from cyanobacteria consists of 17 transmembrane subunits and 3 extrinsic subunits with a total molecular mass of approximately 350kDa per monomer, and PSII exists predominately in a dimeric form in vivo. This chapter describes the purification procedures leading to highly pure, homogenous, and highly active PSII core dimers from a thermophilic cyanobacterium, Thermosynechococcus vulcanus (T. vulcanus), that are used for successful crystallization and diffraction at atomic resolution. The purity and homogeneity of the PSII dimers thus obtained are characterized by absorption spectra, low-temperature fluorescence spectra, SDS-PAGE, clear native PAGE, blue native PAGE, gel filtration chromatography, and oxygen-evolving activity measurements. Finally, high-quality crystals obtained from the purified PSII dimers are shown.