Robert N. Cole

Direct evidence that sulfhydryl groups of Keap1 are the sensors regulating induction of phase 2 enzymes that protect against carcinogens and oxidants

Coordinate induction of phase 2 proteins and elevation of glutathione protect cells against the toxic and carcinogenic effects of electrophiles and oxidants. All inducers react covalently with thiols at rates that are closely related to their potencies. Inducers disrupt the cytoplasmic complex between the actin-bound protein Keap1 and the transcription factor Nrf2, thereby releasing Nrf2 to migrate to the nucleus where it activates the antioxidant response element (ARE) of phase 2 genes and accelerates their transcription. We cloned, overexpressed, and purified murine Keap1 and demonstrated on native gels the formation of complexes of Keap1 with the Neh2 domain of Nrf2 and their concentration-dependent disruption by inducers such as sulforaphane and bis(2-hydroxybenzylidene)acetone. The kinetics, stoichiometry, and order of reactivities of the most reactive of the 25 cysteine thiol groups of Keap1 have been determined by tritium incorporation from [3H]dexamethasone mesylate (an inducer and irreversible modifier of thiols) and by UV spectroscopy with sulforaphane, 2,2′-dipyridyl disulfide and 4,4′-dipyridyl disulfide (titrants of thiol groups), and two closely related Michael reaction acceptors [bis(2- and 4-hydroxybenzylidene)acetones] that differ 100-fold in inducer potency and the UV spectra of which are bleached by thiol addition. With large excesses of these reagents nearly all thiols of Keap1 react, but sequential reaction with three successive single equivalents (per cysteine residue) of dipyridyl disulfides revealed excellent agreement with pseudo-first order kinetics, rapid successive declines in reaction velocity, and the stoichiometric formation of two equivalents of thiopyridone per reacted cysteine. This finding suggests that reaction of cysteine thiols is followed by rapid formation of protein disulfide linkages. The most reactive residues of Keap1 (C257, C273, C288, and C297) were identified by mapping the dexamethasone-modified cysteines by mass spectrometry of tryptic peptides. These residues are located in the intervening region between BTB and Kelch repeat domains of Keap1 and probably are the direct sensors of inducers of the phase 2 system.

Pyruvate Kinase M2 Is a PHD3-Stimulated Coactivator for Hypoxia-Inducible Factor 1

The pyruvate kinase isoforms PKM1 and PKM2 are alternatively spliced products of the PKM2 gene. PKM2, but not PKM1, alters glucose metabolism in cancer cells and contributes to tumorigenesis by mechanisms that are not explained by its known biochemical activity. We show that PKM2 gene transcription is activated by hypoxia-inducible factor 1 (HIF-1). PKM2 interacts directly with the HIF-1α subunit and promotes transactivation of HIF-1 target genes by enhancing HIF-1 binding and p300 recruitment to hypoxia response elements, whereas PKM1 fails to regulate HIF-1 activity. Interaction of PKM2 with prolyl hydroxylase 3 (PHD3) enhances PKM2 binding to HIF-1α and PKM2 coactivator function. Mass spectrometry and anti-hydroxyproline antibody assays demonstrate PKM2 hydroxylation on proline-403/408. PHD3 knockdown inhibits PKM2 coactivator function, reduces glucose uptake and lactate production, and increases O(2) consumption in cancer cells. Thus, PKM2 participates in a positive feedback loop that promotes HIF-1 transactivation and reprograms glucose metabolism in cancer cells.

Mapping Sites of O-GlcNAc Modification Using Affinity Tags for Serine and Threonine Post-translational Modifications

Identifying sites of post-translational modifications on proteins is a major challenge in proteomics. O-Linked β-N-acetylglucosamine (O-GlcNAc) is a dynamic nucleocytoplasmic modification more analogous to phosphorylation than to classical complex O-glycosylation. We describe a mass spectrometry-based method for the identification of sites modified by O-GlcNAc that relies on mild β-elimination followed by Michael addition with dithiothreitol (BEMAD). Using synthetic peptides, we also show that biotin pentylamine can replace dithiothreitol as the nucleophile. The modified peptides can be efficiently enriched by affinity chromatography, and the sites can be mapped using tandem mass spectrometry. This same methodology can be applied to mapping sites of serine and threonine phosphorylation, and we provide a strategy that uses modification-specific antibodies and enzymes to discriminate between the two post-translational modifications. The BEMAD methodology was validated by mapping three previously identified O-GlcNAc sites, as well as three novel sites, on Synapsin I purified from rat brain. BEMAD was then used on a purified nuclear pore complex preparation to map novel sites of O-GlcNAc modification on the Lamin B receptor and the nucleoporin Nup155. This method is amenable for performing quantitative mass spectrometry and can also be adapted to quantify cysteine residues. In addition, our studies emphasize the importance of distinguishing between O-phosphate versus O-GlcNAc when mapping sites of serine and threonine post-translational modification using β-elimination/Michael addition methods.

Induced Pluripotent Stem Cells from Patients with Huntington’s Disease : Show CAG Repeat-Expansion-Associated Phenotypes

Huntingtons disease (HD) is an inherited neurodegenerative disorder caused by an expanded stretch of CAG trinucleotide repeats that results in neuronal dysfunction and death. Here, The HD Consortium reports the generation and characterization of 14 induced pluripotent stem cell (iPSC) lines from HD patients and controls. Microarray profiling revealed CAG-repeat-expansion-associated gene expression patterns that distinguish patient lines from controls, and early onset versus late onset HD. Differentiated HD neural cells showed disease-associated changes in electrophysiology, metabolism, cell adhesion, and ultimately cell death for lines with both medium and longer CAG repeat expansions. The longer repeat lines were however the most vulnerable to cellular stressors and BDNF withdrawal, as assessed using a range of assays across consortium laboratories. The HD iPSC collection represents a unique and well-characterized resource to elucidate disease mechanisms in HD and provides a human stem cell platform for screening new candidate therapeutics.

The Microtubule-associated Protein Tau Is Extensively Modified with O-linked N-acetylglucosamine

Tau is a family of phosphoproteins that are important in modulating microtubule stability in neurons. In Alzheimers disease tau is abnormally hyperphosphorylated, no longer binds microtubules, and self-assembles to form paired helical filaments that likely contribute to neuron death. Here we demonstrate that normal bovine tau is multiply modified by Ser(Thr)-O-linked N-acetylglucosamine, a dynamic and abundant post-translational modification that is often reciprocal to Ser(Thr)-phosphorylation. O-GlcNAcylation of tau was demonstrated by blotting with succinylated wheat germ agglutinin and by probing with bovine milk β(1,4)galactosyltransferase. Structural analyses confirm the linkage and the saccharide structure. Tau splicing variants are multiply O-GlcNAcylated at similar sites, with an average stoichiometry of greater than 4 mol of O-linked N-acetylglucosamine/mol of tau. However, the number of sites occupied appears to be greater than 12, suggesting substoichiometric occupancy at any given site. A similar relationship between average stoichiometry and site-occupancy has also been described for the phosphorylation of tau. Site-specific or stoichiometric changes in O-GlcNAcylation may not only modulate tau function but may also play a role in the formation of paired helical filaments.

Cytosolic O-glycosylation is abundant in nerve terminals.

Phosphorylation plays a key role in regulating growth cone migration and protein trafficking in nerve terminals. Here we show that nerve terminal proteins contain another abundant post‐translational modification: β‐N‐acetylglucosamine linked to hydroxyls of serines or threonines (O‐GlcNAc1). O‐GlcNAc modifications are essential for embryogenesis and mounting evidence suggests that O‐GlcNAc is a regulatory modification that affects many phosphorylated proteins. We show that the activity and expression of O‐GlcNAc transferase (OGT) and N‐acetyl‐β‐d‐glucosaminidase (O‐GlcNAcase), the two enzymes regulating O‐GlcNAc modifications, are present in nerve terminal structures (synaptosomes) and are particularily abundant in the cytosol of synaptosomes. Numerous synaptosome proteins are highly modified with O‐GlcNAc. Although most of these proteins are present in low abundance, we identified by proteomic analysis three neuron‐specific O‐GlcNAc modified proteins: collapsin response mediator protein‐2 (CRMP‐2), ubiquitin carboxyl hydrolase‐L1 (UCH‐L1) and β‐synuclein. CRMP‐2, which is involved in growth cone collapse, is a major O‐GlcNAc modified protein in synaptosomes. All three proteins are implicated in regulatory cascades that mediate intracellular signaling or neurodegenerative diseases. We propose that O‐GlcNAc modifications in the nerve terminal help regulate the functions of these and other synaptosome proteins, and that O‐GlcNAc may play a role in neurodegenerative disease.

Identification and Quantification of S-Nitrosylation by Cysteine Reactive Tandem Mass Tag Switch Assay

Redox-switches are critical cysteine thiols that are modified in response to changes in the cells environment conferring a functional effect. S-nitrosylation (SNO) is emerging as an important modulator of these regulatory switches; however, much remains unknown about the nature of these specific cysteine residues and how oxidative signals are interpreted. Because of their labile nature, SNO-modifications are routinely detected using the biotin switch assay. Here, a new isotope coded cysteine thiol-reactive multiplex reagent, cysTMT6, is used in place of biotin, for the specific detection of SNO-modifications and determination of individual protein thiol-reactivity. S-nitrosylation was measured in human pulmonary arterial endothelia cells in vitro and in vivo using the cysTMT6 quantitative switch assay coupled with mass spectrometry. Cell lysates were treated with S-nitrosoglutathione and used to identify 220 SNO-modified cysteines on 179 proteins. Using this approach it was possible to discriminate potential artifacts including instances of reduced protein disulfide bonds (6) and S-glutathionylation (5) as well as diminished ambiguity in site assignment. Quantitative analysis over a range of NO-donor concentrations (2, 10, 20 μm; GSNO) revealed a continuum of reactivity to SNO-modification. Cysteine response was validated in living cells, demonstrating a greater number of less sensitive cysteine residues are modified with increasing oxidative stimuli. Of note, the majority of available cysteines were found to be unmodified in the current treatment suggesting significant additional capacity for oxidative modifications. These results indicate a possible mechanism for the cell to gauge the magnitude of oxidative stimuli through the progressive and specific accumulation of modified redox-switches.

Electrophilic tuning of the chemoprotective natural product sulforaphane

Sulforaphane [1-isothiocyanato-4-(methylsulfinyl)butane], a naturally occurring isothiocyanate derived from cruciferous vegetables, is a highly potent inducer of phase 2 cytoprotective enzymes and can protect against electrophiles including carcinogens, oxidative stress, and inflammation. The mechanism of action of sulforaphane is believed to involve modifications of critical cysteine residues of Keap1, which lead to stabilization of Nrf2 to activate the antioxidant response element of phase 2 enzymes. However, the dithiocarbamate functional group formed by a reversible reaction between isothiocyanate of sulforaphane and sulfhydryl nucleophiles of Keap1 is kinetically labile, and such modification in intact cells has not yet been demonstrated. Here we designed sulforaphane analogs with replacement of the reactive isothiocyanate by the more gentle electrophilic sulfoxythiocarbamate group that also selectively targets cysteine residues in proteins but forms stable thiocarbamate adducts. Twenty-four sulfoxythiocarbamate analogs were synthesized that retain the structural features important for high potency in sulforaphane analogs: the sulfoxide or keto group and its appropriate distance to electrophilic functional group. Evaluation in various cell lines including hepatoma cells, retinal pigment epithelial cells, and keratinocytes as well as in mouse skin shows that these analogs maintain high potency and efficacy for phase 2 enzyme induction as well as the inhibitory effect on lipopolysaccharide-induced nitric oxide formation like sulforaphane. We further show in living cells that a sulfoxythiocarbamate analog can label Keap1 on several key cysteine residues as well as other cellular proteins offering new insights into the mechanism of chemoprotection.

Calcineurin Promotes Hypoxia-inducible Factor 1α Expression by Dephosphorylating RACK1 and Blocking RACK1 Dimerization

Oxygen homeostasis represents an essential organizing principle of metazoan evolution and biology. Hypoxia-inducible factor 1 (HIF-1) is a master regulator of transcriptional responses to changes in O2 concentration. HIF-1 is a heterodimer of HIF-1α and HIF-1β subunits. O2-dependent degradation of the HIF-1α subunit is mediated by prolyl hydroxylase, von Hippel-Lindau protein (VHL)/Elongin-C E3 ubiquitin ligase, and the proteasome. O2-independent degradation of HIF-1α is regulated by the competition of RACK1 and HSP90 for binding to HIF-1α. RACK1 binding results in the recruitment of the Elongin-C E3 ubiquitin ligase, leading to VHL-independent ubiquitination and degradation of HIF-1α. In this report, we show that calcineurin inhibits the ubiquitination and proteasomal degradation of HIF-1α. Calcineurin is a serine/threonine phosphatase that is activated by calcium and calmodulin. The phosphatase activity of calcineurin is required for its regulation of HIF-1α. RACK1 binds to the catalytic domain of calcineurin and is required for HIF-1α degradation induced by the calcineurin inhibitor cyclosporine A. Elongin-C and HIF-1α each bind to RACK1 and dimerization of RACK1 is required to recruit Elongin-C to HIF-1α. Phosphorylation of RACK1 promotes its dimerization and dephosphorylation by calcineurin inhibits dimerization. Serine 146 within the dimerization domain is phosphorylated and mutation of serine 146 impairs RACK1 dimerization and HIF-1α degradation. These results indicate that intracellular calcium levels can regulate HIF-1α expression by modulating calcineurin activity and RACK1 dimerization.

A quantitative proteomic approach for identification of potential biomarkers in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide. In this study, our objective was to identify differentially regulated proteins in HCC through a quantitative proteomic approach using iTRAQ. More than 600 proteins were quantitated of which 59 proteins were overexpressed and 92 proteins were underexpressed in HCC as compared to adjacent normal tissue. Several differentially expressed proteins were not implicated previously in HCC. A subset of these proteins (six each from upregulated and downregulated groups) was further validated using immunoblotting and immunohistochemical labeling. Some of the overexpressed proteins with no previous description in the context of HCC include fibroleukin, interferon induced 56 kDa protein, milk fat globule-EGF factor 8, and myeloid-associated differentiation marker. Interestingly, all the enzymes of urea metabolic pathway were dramatically downregulated. Immunohistochemical labeling confirmed differential expression of fibroleukin, myeloid associated differentiation marker and ornithine carbamoyl transferase in majority of HCC samples analyzed. Our results demonstrate quantitative proteomics as a robust discovery tool for the identification of differentially regulated proteins in cancers.