We report NMR and kinetic studies of antibody AZ-28, which was generated against the diaryl-substituted cyclohexanol derivative 3 and catalyzes the oxy-Cope rearrangement of the corresponding ...hexadiene 1 to aldehyde 2 (Braisted, A. C., Schultz, P. G. J. Am. Chem. Soc. 1994, 116, 2211−2212). Conformational studies of free substrate and the antibody−substrate complex using transferred-NOE experiments demonstrate that the antibody binds the substrate in a cyclic conformation, consistent with the chair-like geometry of 3. In contrast, free substrate adopts an extended conformation in solution. In spite of the conformational restriction of the bound substrate revealed by NMR, the temperature dependence of the reaction indicates that the antibody functions primarily by lowering ΔH ⧧, offsetting a decrease in ΔS ⧧; the values of ΔH ⧧ and ΔS ⧧ are 15.4 ± 2.4 kcal/mol and −23 ± 8 cal/mol K, respectively. A secondary kinetic isotope effect (k catH/k catD) of 0.61 ± 0.1 was measured for substrate 23 in which both allylic termini are dideuterated, indicating that the chemical rearrangement step is wholly or partially rate limiting under saturating conditions. The magnitude of this secondary isotope effect is consistent with a significant degree of bond formation between C1 and C6 of the substrate in the transition state. These results, together with the recently reported three-dimensional crystal structure for the antibody−hapten 3 complex (Ulrich, H.; Mundroff, E.; Santarsiero, B. D.; Driggers, E. M.; Stevens, R. C.; Schultz, P. G. Nature 1997, 389, 271−275), provide a detailed mechanistic model for this antibody-catalyzed reaction.
Cancer cells need to meet the metabolic demands of rapid cell growth within a continually changing microenvironment. Genetic mechanisms for reprogramming cellular metabolism toward proliferative, ...pro-survival pathways are well-reported. However, post-translational mechanisms, which would enable more rapid, reversible adaptations of cellular metabolism in response to protein signaling or environmental sensing systems, are less well understood. Here we demonstrate that the post-translational modification
O
-linked β-
N
-acetylglucosamine (
O
-GlcNAc) is a key metabolic regulator of glucose metabolism.
O
-GlcNAc is dynamically induced at Ser529 of phosphofructokinase 1 (PFK1) in response to hypoxia. Glycosylation inhibits PFK1 activity and redirects the flux of glucose from glycolysis through the pentose phosphate pathway (PPP), thereby conferring a selective growth advantage to cancer cells. Blocking glycosylation of PFK1 at Ser529 reduced cancer cell proliferation
in vitro
and impaired tumor formation
in vivo
. These studies reveal an unexpected mechanism for the regulation of metabolic enzymes and pathways, and pinpoint a new therapeutic approach for combating cancer.
Metabolism has been shown to integrate with epigenetics and transcription to modulate cell fate and function. Beyond meeting the bioenergetic and biosynthetic demands of T-cell differentiation, ...whether metabolism might control T-cell fate by an epigenetic mechanism is unclear. Here, through the discovery and mechanistic characterization of a small molecule, (aminooxy)acetic acid, that reprograms the differentiation of T helper 17 (T
17) cells towards induced regulatory T (iT
) cells, we show that increased transamination, mainly catalysed by GOT1, leads to increased levels of 2-hydroxyglutarate in differentiating T
17 cells. The accumulation of 2-hydroxyglutarate resulted in hypermethylation of the Foxp3 gene locus and inhibited Foxp3 transcription, which is essential for fate determination towards T
17 cells. Inhibition of the conversion of glutamate to α-ketoglutaric acid prevented the production of 2-hydroxyglutarate, reduced methylation of the Foxp3 gene locus, and increased Foxp3 expression. This consequently blocked the differentiation of T
17 cells by antagonizing the function of transcription factor RORγt and promoted polarization into iT
cells. Selective inhibition of GOT1 with (aminooxy)acetic acid ameliorated experimental autoimmune encephalomyelitis in a therapeutic mouse model by regulating the balance between T
17 and iT
cells. Targeting a glutamate-dependent metabolic pathway thus represents a new strategy for developing therapeutic agents against T
17-mediated autoimmune diseases.
Abstract
Mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2), are present in most gliomas and secondary glioblastomas, but are rare in other neoplasms. IDH1/2 mutations are heterozygous, and ...affect a single arginine residue. Recently, IDH1 mutations were identified in 8% of acute myelogenous leukemia (AML) patients. Our previous study revealed that IDH1 mutations cause a gain of function, resulting in the production and accumulation of 2-hydroxyglutarate (2-HG). Genotyping of 145 AML biopsies identified 11 IDH1 R132 mutant samples. Liquid chromatography-mass spectrometry metabolite screening revealed increased 2-HG levels in IDH1 R132 mutant cells and sera, and uncovered two IDH2 R172K mutations. IDH1/2 mutations were associated with normal karyotypes. Recombinant IDH1 R132C and IDH2 R172K proteins catalyze the novel NADPH -dependent reduction of alpha-ketoglutarate (a-KG) to 2-HG. The IDH1 R132C mutation commonly found in AML reduces the affinity for isocitrate, and increases the affinity for NADPH and a-KG. This prevents the oxidative decarboxylation of isocitrate to a-KG, and facilitates the conversion of a-KG to 2-HG. IDH1/2 mutations confer an enzymatic gain of function that dramatically increases 2-HG in AML. This provides an explanation for the heterozygous acquisition of these mutations during tumorigenesis. 2-HG is a tractable metabolic biomarker of mutant IDH1/2 enzyme activity.
Citation Format: {Authors}. {Abstract title} abstract. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5452.
Cancer cells adapt their metabolic processes to drive macromolecular biosynthesis for rapid cell growth and proliferation (
1
,
2
). RNAi-based loss of function screening has proven powerful for the ...identification of novel and interesting cancer targets, and recent studies have used this technology
in vivo
to identify novel tumor suppressor genes (
3
). Here, we developed a method for identifying novel cancer targets via negative selection RNAi screening in solid tumours. Using this method, we screened a set of metabolic genes associated with aggressive breast cancer and stemness to identify those required for
in vivo
tumourigenesis. Among the genes identified, phosphoglycerate dehydrogenase (
PHGDH
) is in a genomic region of recurrent copy number gain in breast cancer and PHGDH protein levels are elevated in 70% of ER-negative breast cancers. PHGDH catalyzes the first step in the serine biosynthesis pathway, and breast cancer cells with high PHGDH expression have elevations in serine synthesis flux. Suppression of PHGDH in cell lines with elevated PHGDH expression, but not those without, causes a strong decrease in cell proliferation and a reduction in serine synthesis. We find that PHGDH suppression does not affect intracellular serine levels, but causes a drop in the levels of alpha-ketoglutarate, another output of the pathway and a TCA cycle intermediate. In cells with high PHGDH expression, the serine synthesis pathway contributes approximately 50% of the total anaplerotic flux of glutamine into the TCA cycle. These results reveal that certain breast cancers are dependent upon increased serine pathway flux caused by PHGDH over-expression and demonstrate the utility of
in vivo
negative selection RNAi screens for finding potential anticancer targets.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract
Mutations in the enzyme isocitrate dehydrogenase 1 (IDH1) are a common feature of most gliomas and secondary glioblastomas, as well as approx 10% acute myeloid leukemias. This event results ...in loss of the enzyme's ability to catalyze conversion of isocitrate to α -ketoglutarate. However, these mutations are all heterozygous and occur at a single amino acid residue of the IDH1 active site consistent with an enzymatic gain of function rather than a simple loss of function. To test this hypothesis we characterized mutant IDH1 (IDH1m) biochemically. We have shown that cancer-associated IDH1 mutations result in a new ability of the enzyme to catalyze the NADPH-dependent reduction of α-ketoglutarate to R(-)-2-hydroxyglutarate (2-HG). Patients with an inherited, neurometabolic disorders called 2-hydroxyglutaric aciduria exhibit an accumulation of 2-HG in their CNS, and an increased risk of developing malignant brain tumors. Similarly, in human malignant gliomas harboring IDH1 mutations, we find elevated levels of 2-HG. Altogether our data demonstrate that the IDH1 mutations result in production of 2-HG, and suggest that the excess 2HG which accumulates in vivo contributes to the formation and malignant progression of gliomas.
Citation Format: {Authors}. {Abstract title} abstract. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 33.
Antibody catalysis provides an opportunity to examine the evolution of binding energy and its relation to catalytic function in a system that has many parallels with natural enzymes. Here we report ...such a study involving an antibody AZ-28 that catalyses an oxy-Cope rearrangement, a pericyclic reaction that belongs to a well studied and widely used class of reactions in organic chemistry. Immunization with transition state analogue 1 results in a germline-encoded antibody that catalyses the rearrangement of hexadiene 2 to aldehyde 3 with a rate approaching that of a related pericyclic reaction catalysed by the enzyme chorismate mutase. Affinity maturation gives antibody AZ-28, which has six amino acid substitutions, one of which results in a decrease in catalytic rate. To understand the relationship between binding and catalytic rate in this system we characterized a series of active-site mutants and determined the three-dimensional crystal structure of the complex of AZ-28 with the transition state analogue. This analysis indicates that the activation energy depends on a complex balance of several stereoelectronic effects which are controlled by an extensive network of binding interactions in the active site. Thus in this instance the combinatorial diversity of the immune system provided both an efficient catalyst for a reaction where no enzyme is known, as well as an opportunity to explore the mechanisms and evolution of biological catalysis.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
In an effort to increase our insight into the catalysis of pericyclic reactions we have initiated a detailed study of an antibody that catalyzes an oxy-Cope rearrangement. We have determined the ...stereochemistry of the antibody-catalyzed reaction, and experiments are in progress to determine the conformation of the substrate bound in the antibody combining site. The genes encoding the variable regions of this antibody have been cloned and sequenced, and we have made use of a bacterial expression system to produce this antibody as a Fab fragment in recombinant form, making it amenable to genetic manipulations such as site-directed mutagenesis. The recombinant Fab fragment has been crystallized in the presence of its transition state analog, and we are now in the process of determining its active site structure.