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1.
Chem Sci ; 15(7): 2509-2517, 2024 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-38362406

RESUMO

Patients with alcoholism and type 2 diabetes manifest altered metabolism, including elevated aldehyde levels and unusually low asparagine levels. We show that asparagine synthetase B (ASNS), the only human asparagine-forming enzyme, is inhibited by disease-relevant reactive aldehydes, including formaldehyde and acetaldehyde. Cellular studies show non-cytotoxic amounts of reactive aldehydes induce a decrease in asparagine levels. Biochemical analyses reveal inhibition results from reaction of the aldehydes with the catalytically important N-terminal cysteine of ASNS. The combined cellular and biochemical results suggest a possible mechanism underlying the low asparagine levels in alcoholism and diabetes. The results will stimulate research on the biological consequences of the reactions of aldehydes with nucleophilic residues.

2.
Science ; 382(6670): eabp9201, 2023 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-37917677

RESUMO

One-carbon metabolism is an essential branch of cellular metabolism that intersects with epigenetic regulation. In this work, we show how formaldehyde (FA), a one-carbon unit derived from both endogenous sources and environmental exposure, regulates one-carbon metabolism by inhibiting the biosynthesis of S-adenosylmethionine (SAM), the major methyl donor in cells. FA reacts with privileged, hyperreactive cysteine sites in the proteome, including Cys120 in S-adenosylmethionine synthase isoform type-1 (MAT1A). FA exposure inhibited MAT1A activity and decreased SAM production with MAT-isoform specificity. A genetic mouse model of chronic FA overload showed a decrease n SAM and in methylation on selected histones and genes. Epigenetic and transcriptional regulation of Mat1a and related genes function as compensatory mechanisms for FA-dependent SAM depletion, revealing a biochemical feedback cycle between FA and SAM one-carbon units.


Assuntos
Carbono , Cisteína , Epigênese Genética , Formaldeído , Metionina Adenosiltransferase , S-Adenosilmetionina , Animais , Camundongos , Carbono/metabolismo , Epigênese Genética/efeitos dos fármacos , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/metabolismo , S-Adenosilmetionina/antagonistas & inibidores , S-Adenosilmetionina/metabolismo , Formaldeído/metabolismo , Formaldeído/toxicidade , Exposição Ambiental , Metionina Adenosiltransferase/antagonistas & inibidores , Metionina Adenosiltransferase/genética , Metionina Adenosiltransferase/metabolismo , Cisteína/metabolismo , Humanos , Células Hep G2
3.
Mol Cell ; 83(14): 2417-2433.e7, 2023 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-37348497

RESUMO

Aged hematopoietic stem cells (HSCs) display diminished self-renewal and a myeloid differentiation bias. However, the drivers and mechanisms that underpin this fundamental switch are not understood. HSCs produce genotoxic formaldehyde that requires protection by the detoxification enzymes ALDH2 and ADH5 and the Fanconi anemia (FA) DNA repair pathway. We find that the HSCs in young Aldh2-/-Fancd2-/- mice harbor a transcriptomic signature equivalent to aged wild-type HSCs, along with increased epigenetic age, telomere attrition, and myeloid-biased differentiation quantified by single HSC transplantation. In addition, the p53 response is vigorously activated in Aldh2-/-Fancd2-/- HSCs, while p53 deletion rescued this aged HSC phenotype. To further define the origins of the myeloid differentiation bias, we use a GFP genetic reporter to find a striking enrichment of Vwf+ myeloid and megakaryocyte-lineage-biased HSCs. These results indicate that metabolism-derived formaldehyde-DNA damage stimulates the p53 response in HSCs to drive accelerated aging.


Assuntos
Envelhecimento , Aldeídos , Dano ao DNA , Hematopoese , Proteína Supressora de Tumor p53 , Animais , Camundongos , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Aldeídos/metabolismo , Transcriptoma , Análise da Expressão Gênica de Célula Única , Células-Tronco Hematopoéticas/citologia , Células Mieloides/citologia , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia
4.
Blood ; 139(14): 2119-2129, 2022 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-35148375

RESUMO

Reactive aldehydes are potent genotoxins that threaten the integrity of hematopoietic stem cells and blood production. To protect against aldehydes, mammals have evolved a family of enzymes to detoxify aldehydes, and the Fanconi anemia DNA repair pathway to process aldehyde-induced DNA damage. Loss of either protection mechanisms in humans results in defective hematopoiesis and predisposition to leukemia. This review will focus on the impact of genotoxic aldehydes on hematopoiesis, the sources of endogenous aldehydes, and potential novel protective pathways.


Assuntos
Aldeídos , Anemia de Fanconi , Aldeídos/metabolismo , Animais , Dano ao DNA , Anemia de Fanconi/genética , Hematopoese/genética , Células-Tronco Hematopoéticas/metabolismo , Humanos , Mamíferos/metabolismo
5.
Nature ; 600(7887): 158-163, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34819667

RESUMO

Endogenous DNA damage can perturb transcription, triggering a multifaceted cellular response that repairs the damage, degrades RNA polymerase II and shuts down global transcription1-4. This response is absent in the human disease Cockayne syndrome, which is caused by loss of the Cockayne syndrome A (CSA) or CSB proteins5-7. However, the source of endogenous DNA damage and how this leads to the prominent degenerative features of this disease remain unknown. Here we find that endogenous formaldehyde impedes transcription, with marked physiological consequences. Mice deficient in formaldehyde clearance (Adh5-/-) and CSB (Csbm/m; Csb is also known as Ercc6) develop cachexia and neurodegeneration, and succumb to kidney failure, features that resemble human Cockayne syndrome. Using single-cell RNA sequencing, we find that formaldehyde-driven transcriptional stress stimulates the expression of the anorexiogenic peptide GDF15 by a subset of kidney proximal tubule cells. Blocking this response with an anti-GDF15 antibody alleviates cachexia in Adh5-/-Csbm/m mice. Therefore, CSB provides protection to the kidney and brain against DNA damage caused by endogenous formaldehyde, while also suppressing an anorexic endocrine signal. The activation of this signal might contribute to the cachexia observed in Cockayne syndrome as well as chemotherapy-induced anorectic weight loss. A plausible evolutionary purpose for such a response is to ensure aversion to genotoxins in food.


Assuntos
Síndrome de Cockayne , Dano ao DNA , Formaldeído/efeitos adversos , Estresse Fisiológico/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Álcool Desidrogenase/deficiência , Álcool Desidrogenase/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Caquexia/complicações , Síndrome de Cockayne/induzido quimicamente , Síndrome de Cockayne/complicações , Síndrome de Cockayne/genética , Síndrome de Cockayne/patologia , Enzimas Reparadoras do DNA/deficiência , Modelos Animais de Doenças , Feminino , Formaldeído/metabolismo , Fator 15 de Diferenciação de Crescimento/antagonistas & inibidores , Fator 15 de Diferenciação de Crescimento/biossíntese , Fator 15 de Diferenciação de Crescimento/genética , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/patologia , Masculino , Camundongos , Proteínas de Ligação a Poli-ADP-Ribose/deficiência , Insuficiência Renal/complicações , Transcrição Gênica/genética
6.
Mol Cell ; 80(6): 996-1012.e9, 2020 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-33147438

RESUMO

Reactive aldehydes arise as by-products of metabolism and are normally cleared by multiple families of enzymes. We find that mice lacking two aldehyde detoxifying enzymes, mitochondrial ALDH2 and cytoplasmic ADH5, have greatly shortened lifespans and develop leukemia. Hematopoiesis is disrupted profoundly, with a reduction of hematopoietic stem cells and common lymphoid progenitors causing a severely depleted acquired immune system. We show that formaldehyde is a common substrate of ALDH2 and ADH5 and establish methods to quantify elevated blood formaldehyde and formaldehyde-DNA adducts in tissues. Bone-marrow-derived progenitors actively engage DNA repair but also imprint a formaldehyde-driven mutation signature similar to aging-associated human cancer mutation signatures. Furthermore, we identify analogous genetic defects in children causing a previously uncharacterized inherited bone marrow failure and pre-leukemic syndrome. Endogenous formaldehyde clearance alone is therefore critical for hematopoiesis and in limiting mutagenesis in somatic tissues.


Assuntos
Álcool Desidrogenase/genética , Aldeído-Desidrogenase Mitocondrial/genética , Formaldeído/sangue , Leucemia/genética , Adolescente , Aldeídos/sangue , Animais , Criança , Pré-Escolar , Adutos de DNA/genética , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Feminino , Formaldeído/toxicidade , Hematopoese/genética , Células-Tronco Hematopoéticas/metabolismo , Humanos , Lactente , Leucemia/sangue , Leucemia/patologia , Masculino , Camundongos , Mutação/genética , Especificidade por Substrato
7.
Future Med Chem ; 11(19): 2491-2504, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31633398

RESUMO

Aim: The p53 cancer mutation Y220C creates a conformationally unstable protein with a unique elongated surface crevice that can be targeted by molecular chaperones. We report the structure-guided optimization of the carbazole-based stabilizer PK083. Materials & methods: Biophysical, cellular and x-ray crystallographic techniques have been employed to elucidate the mode of action of the carbazole scaffolds. Results: Targeting an unoccupied subsite of the surface crevice with heterocycle-substituted PK083 analogs resulted in a 70-fold affinity increase to single-digit micromolar levels, increased thermal stability and decreased rate of aggregation of the mutant protein. PK9318, one of the most potent binders, restored p53 signaling in the liver cancer cell line HUH-7 with homozygous Y220C mutation. Conclusion: The p53-Y220C mutant is an excellent paradigm for the development of mutant p53 rescue drugs via protein stabilization. Similar rescue strategies may be applicable to other cavity-creating p53 cancer mutations.


Assuntos
Carbazóis/farmacologia , Chaperonas Moleculares/metabolismo , Ativação Transcricional/genética , Proteína Supressora de Tumor p53/genética , Carbazóis/síntese química , Carbazóis/química , Humanos , Chaperonas Moleculares/síntese química , Chaperonas Moleculares/química , Estrutura Molecular , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/metabolismo
8.
Eur J Med Chem ; 152: 101-114, 2018 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-29702446

RESUMO

Many cancers have the tumor suppressor p53 inactivated by mutation, making reactivation of mutant p53 with small molecules a promising strategy for the development of novel anticancer therapeutics. The oncogenic p53 mutation Y220C, which accounts for approximately 100,000 cancer cases per year, creates an extended surface crevice in the DNA-binding domain, which destabilizes p53 and causes denaturation and aggregation. Here, we describe the structure-guided design of a novel class of small-molecule Y220C stabilizers and the challenging synthetic routes developed in the process. The synthesized chemical probe MB710, an aminobenzothiazole derivative, binds tightly to the Y220C pocket and stabilizes p53-Y220C in vitro. MB725, an ethylamide analogue of MB710, induced selective viability reduction in several p53-Y220C cancer cell lines while being well tolerated in control cell lines. Reduction of viability correlated with increased and selective transcription of p53 target genes such as BTG2, p21, PUMA, FAS, TNF, and TNFRSF10B, which promote apoptosis and cell cycle arrest, suggesting compound-mediated transcriptional activation of the Y220C mutant. Our data provide a framework for the development of a class of potent, non-toxic compounds for reactivating the Y220C mutant in anticancer therapy.


Assuntos
Antineoplásicos/farmacologia , Benzotiazóis/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Antineoplásicos/síntese química , Antineoplásicos/química , Benzotiazóis/síntese química , Benzotiazóis/química , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Estrutura Molecular , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Relação Estrutura-Atividade , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genética
9.
Nature ; 548(7669): 549-554, 2017 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-28813411

RESUMO

The folate-driven one-carbon (1C) cycle is a fundamental metabolic hub in cells that enables the synthesis of nucleotides and amino acids and epigenetic modifications. This cycle might also release formaldehyde, a potent protein and DNA crosslinking agent that organisms produce in substantial quantities. Here we show that supplementation with tetrahydrofolate, the essential cofactor of this cycle, and other oxidation-prone folate derivatives kills human, mouse and chicken cells that cannot detoxify formaldehyde or that lack DNA crosslink repair. Notably, formaldehyde is generated from oxidative decomposition of the folate backbone. Furthermore, we find that formaldehyde detoxification in human cells generates formate, and thereby promotes nucleotide synthesis. This supply of 1C units is sufficient to sustain the growth of cells that are unable to use serine, which is the predominant source of 1C units. These findings identify an unexpected source of formaldehyde and, more generally, indicate that the detoxification of this ubiquitous endogenous genotoxin creates a benign 1C unit that can sustain essential metabolism.


Assuntos
Carbono/metabolismo , Ácido Fólico/química , Ácido Fólico/metabolismo , Formaldeído/química , Formaldeído/metabolismo , Redes e Vias Metabólicas , Mutagênicos/química , Mutagênicos/metabolismo , Álcool Desidrogenase/metabolismo , Animais , Carbono/deficiência , Linhagem Celular , Galinhas , Coenzimas/metabolismo , Reagentes de Ligações Cruzadas/metabolismo , Dano ao DNA , Reparo do DNA , Humanos , Inativação Metabólica , Camundongos , Nucleotídeos/biossíntese , Oxirredução , Serina/química , Serina/metabolismo , Tetra-Hidrofolatos/metabolismo
10.
Science ; 357(6347): 130-131, 2017 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-28706026
12.
Cell Rep ; 7(4): 1130-42, 2014 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-24813893

RESUMO

T helper 2 (Th2) cells regulate helminth infections, allergic disorders, tumor immunity, and pregnancy by secreting various cytokines. It is likely that there are undiscovered Th2 signaling molecules. Although steroids are known to be immunoregulators, de novo steroid production from immune cells has not been previously characterized. Here, we demonstrate production of the steroid pregnenolone by Th2 cells in vitro and in vivo in a helminth infection model. Single-cell RNA sequencing and quantitative PCR analysis suggest that pregnenolone synthesis in Th2 cells is related to immunosuppression. In support of this, we show that pregnenolone inhibits Th cell proliferation and B cell immunoglobulin class switching. We also show that steroidogenic Th2 cells inhibit Th cell proliferation in a Cyp11a1 enzyme-dependent manner. We propose pregnenolone as a "lymphosteroid," a steroid produced by lymphocytes. We speculate that this de novo steroid production may be an intrinsic phenomenon of Th2-mediated immune responses to actively restore immune homeostasis.


Assuntos
Pregnenolona/biossíntese , RNA/metabolismo , Células Th1/imunologia , Células Th2/imunologia , Animais , Homeostase/imunologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Pregnenolona/genética , Pregnenolona/imunologia , RNA/genética , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Células Th1/metabolismo , Células Th2/metabolismo , Transcriptoma
13.
Eur J Immunol ; 44(7): 1925-35, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24771041

RESUMO

Excision of uracil introduced into the immunoglobulin loci by AID is central to antibody diversification. While predominantly carried out by the UNG uracil-DNA glycosylase as reflected by deficiency in immunoglobulin class switching in Ung(-/-) mice, the deficiency is incomplete, as evidenced by the emergence of switched IgG in the serum of Ung(-/-) mice. Lack of switching in mice deficient in both UNG and MSH2 suggested that mismatch repair initiated a backup pathway. We now show that most of the residual class switching in Ung(-/-) mice depends upon the endogenous SMUG1 uracil-DNA glycosylase, with in vitro switching to IgG1 as well as serum IgG3, IgG2b, and IgA greatly diminished in Ung(-/-) Smug1(-/-) mice, and that Smug1 partially compensates for Ung deficiency over time. Nonetheless, using a highly MSH2-dependent mechanism, Ung(-/-) Smug1(-/-) mice can still produce detectable levels of switched isotypes, especially IgG1. While not affecting the pattern of base substitutions, SMUG1 deficiency in an Ung(-/-) background further reduces somatic hypermutation at A:T base pairs. Our data reveal an essential requirement for uracil excision in class switching and in facilitating noncanonical mismatch repair for the A:T phase of hypermutation presumably by creating nicks near the U:G lesion recognized by MSH2.


Assuntos
Switching de Imunoglobulina , Mutação , Uracila-DNA Glicosidase/fisiologia , Uracila/metabolismo , Animais , Imunoglobulina A/sangue , Imunoglobulina G/sangue , Camundongos , Proteína 2 Homóloga a MutS/fisiologia
14.
Nucleic Acids Res ; 40(13): 6016-25, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22447450

RESUMO

Deamination of cytosine (C), 5-methylcytosine (mC) and 5-hydroxymethylcytosine (hmC) occurs spontaneously in mammalian DNA with several hundred deaminations occurring in each cell every day. The resulting potentially mutagenic mispairs of uracil (U), thymine (T) or 5-hydroxymethyluracil (hmU) with guanine (G) are substrates for repair by various DNA glycosylases. Here, we show that targeted inactivation of the mouse Smug1 DNA glycosylase gene is sufficient to ablate nearly all hmU-DNA excision activity as judged by assay of tissue extracts from knockout mice as well as by the resistance of their embryo fibroblasts to 5-hydroxymethyldeoxyuridine toxicity. Inactivation of Smug1 when combined with inactivation of the Ung uracil-DNA glycosylase gene leads to a loss of nearly all detectable uracil excision activity. Thus, SMUG1 is the dominant glycosylase responsible for hmU-excision in mice as well as the major UNG-backup for U-excision. Both Smug1-knockout and Smug1/Ung-double knockout mice breed normally and remain apparently healthy beyond 1 year of age. However, combined deficiency in SMUG1 and UNG exacerbates the cancer predisposition of Msh2(-/-) mice suggesting that when both base excision and mismatch repair pathways are defective, the mutagenic effects of spontaneous cytosine deamination are sufficient to increase cancer incidence but do not preclude mouse development.


Assuntos
Reparo do DNA , Pentoxil (Uracila)/análogos & derivados , Uracila-DNA Glicosidase/genética , Uracila-DNA Glicosidase/metabolismo , Animais , Linhagem Celular , Fibroblastos/efeitos dos fármacos , Fluoruracila/metabolismo , Marcação de Genes , Predisposição Genética para Doença , Longevidade , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 2 Homóloga a MutS/genética , Neoplasias Experimentais/genética , Pentoxil (Uracila)/metabolismo , Timidina/análogos & derivados , Timidina/toxicidade , beta-Galactosidase/genética
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