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1.
Nature ; 615(7952): 490-498, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36890227

RESUMO

Metabolic rewiring underlies the effector functions of macrophages1-3, but the mechanisms involved remain incompletely defined. Here, using unbiased metabolomics and stable isotope-assisted tracing, we show that an inflammatory aspartate-argininosuccinate shunt is induced following lipopolysaccharide stimulation. The shunt, supported by increased argininosuccinate synthase (ASS1) expression, also leads to increased cytosolic fumarate levels and fumarate-mediated protein succination. Pharmacological inhibition and genetic ablation of the tricarboxylic acid cycle enzyme fumarate hydratase (FH) further increases intracellular fumarate levels. Mitochondrial respiration is also suppressed and mitochondrial membrane potential increased. RNA sequencing and proteomics analyses demonstrate that there are strong inflammatory effects resulting from FH inhibition. Notably, acute FH inhibition suppresses interleukin-10 expression, which leads to increased tumour necrosis factor secretion, an effect recapitulated by fumarate esters. Moreover, FH inhibition, but not fumarate esters, increases interferon-ß production through mechanisms that are driven by mitochondrial RNA (mtRNA) release and activation of the RNA sensors TLR7, RIG-I and MDA5. This effect is recapitulated endogenously when FH is suppressed following prolonged lipopolysaccharide stimulation. Furthermore, cells from patients with systemic lupus erythematosus also exhibit FH suppression, which indicates a potential pathogenic role for this process in human disease. We therefore identify a protective role for FH in maintaining appropriate macrophage cytokine and interferon responses.


Assuntos
Fumarato Hidratase , Interferon beta , Macrófagos , Mitocôndrias , RNA Mitocondrial , Humanos , Argininossuccinato Sintase/metabolismo , Ácido Argininossuccínico/metabolismo , Ácido Aspártico/metabolismo , Respiração Celular , Citosol/metabolismo , Fumarato Hidratase/antagonistas & inibidores , Fumarato Hidratase/genética , Fumarato Hidratase/metabolismo , Fumaratos/metabolismo , Interferon beta/biossíntese , Interferon beta/imunologia , Lipopolissacarídeos/farmacologia , Lipopolissacarídeos/metabolismo , Lúpus Eritematoso Sistêmico/enzimologia , Macrófagos/enzimologia , Macrófagos/imunologia , Macrófagos/metabolismo , Potencial da Membrana Mitocondrial , Metabolômica , Mitocôndrias/genética , Mitocôndrias/metabolismo , RNA Mitocondrial/metabolismo
2.
Am J Physiol Cell Physiol ; 327(3): C684-C697, 2024 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-39010842

RESUMO

Cancer cachexia, the unintentional loss of lean mass, contributes to functional dependency, poor treatment outcomes, and decreased survival. Although its pathogenicity is multifactorial, metabolic dysfunction remains a hallmark of cachexia. However, significant knowledge gaps exist in understanding the role of skeletal muscle lipid metabolism and dynamics in this condition. We examined skeletal muscle metabolic dysfunction, intramyocellular lipid droplet (LD) content, LD morphology and subcellular distribution, and LD-mitochondrial interactions using the Lewis lung carcinoma (LLC) murine model of cachexia. C57/BL6 male mice (n = 20) were implanted with LLC cells (106) in the right flank or underwent PBS sham injections. Skeletal muscle was excised for transmission electron microscopy (TEM; soleus), oil red O/lipid staining [tibialis anterior (TA)], and protein (gastrocnemius). LLC mice had a greater number (232%; P = 0.006) and size (130%; P = 0.023) of intramyocellular LDs further supported by increased oil-red O positive (87%; P = 0.0109) and "very high" oil-red O positive (178%; P = 0.0002) fibers compared with controls and this was inversely correlated with fiber size (R2 = 0.5294; P < 0.0001). Morphological analyses of LDs show increased elongation and complexity [aspect ratio: intermyofibrillar (IMF) = 9%, P = 0.046) with decreases in circularity [circularity: subsarcolemmal (SS) = 6%, P = 0.042] or roundness (roundness: whole = 10%, P = 0.033; IMF = 8%, P = 0.038) as well as decreased LD-mitochondria touch (-15%; P = 0.006), contact length (-38%; P = 0.036), and relative contact (86%; P = 0.004). Furthermore, dysregulation in lipid metabolism (adiponectin, CPT1b) and LD-associated proteins, perilipin-2 and perilipin-5, in cachectic muscle (P < 0.05) were observed. Collectively, we provide evidence that skeletal muscle myosteatosis, altered LD morphology, and decreased LD-mitochondrial interactions occur in a preclinical model of cancer cachexia.NEW & NOTEWORTHY We sought to advance our understanding of skeletal muscle lipid metabolism and dynamics in cancer cachexia. Cachexia increased the number and size of intramyocellular lipid droplets (LDs). Furthermore, decreases in LD-mitochondrial touch, contact length, and relative contact along with increased LD shape complexity with decreases in circularity and roundness. Dysregulation in lipid metabolism and LD-associated proteins was also documented. Collectively, we show that myosteatosis, altered LD morphology, and decreased LD-mitochondrial interactions occur in cancer cachexia.


Assuntos
Caquexia , Carcinoma Pulmonar de Lewis , Gotículas Lipídicas , Camundongos Endogâmicos C57BL , Músculo Esquelético , Animais , Caquexia/metabolismo , Caquexia/patologia , Caquexia/etiologia , Masculino , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Carcinoma Pulmonar de Lewis/metabolismo , Carcinoma Pulmonar de Lewis/patologia , Carcinoma Pulmonar de Lewis/complicações , Gotículas Lipídicas/metabolismo , Gotículas Lipídicas/patologia , Camundongos , Metabolismo dos Lipídeos , Mitocôndrias Musculares/metabolismo , Mitocôndrias Musculares/patologia , Mitocôndrias Musculares/ultraestrutura , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Mitocôndrias/ultraestrutura
3.
Am J Physiol Endocrinol Metab ; 326(4): E407-E416, 2024 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-38324261

RESUMO

The tricarboxylic acid (TCA) cycle metabolite fumarate nonenzymatically reacts with the amino acid cysteine to form S-(2-succino)cysteine (2SC), referred to as protein succination. The immunometabolite itaconate accumulates during lipopolysaccharide (LPS) stimulation of macrophages and microglia. Itaconate nonenzymatically reacts with cysteine residues to generate 2,3-dicarboxypropylcysteine (2,3-DCP), referred to as protein dicarboxypropylation. Since fumarate and itaconate levels dynamically change in activated immune cells, the levels of both 2SC and 2,3-DCP reflect the abundance of these metabolites and their capacity to modify protein thiols. We generated ethyl esters of 2SC and 2,3-DCP from protein hydrolysates and used stable isotope dilution mass spectrometry to determine the abundance of these in LPS-stimulated Highly Aggressively Proliferating Immortalized (HAPI) microglia. To quantify the stoichiometry of the succination and dicarboxypropylation, reduced cysteines were alkylated with iodoacetic acid to form S-carboxymethylcysteine (CMC), which was then esterified. Itaconate-derived 2,3-DCP, but not fumarate-derived 2SC, increased in LPS-treated HAPI microglia. Stoichiometric measurements demonstrated that 2,3-DCP increased from 1.57% to 9.07% of total cysteines upon LPS stimulation. This methodology to simultaneously distinguish and quantify both 2SC and 2,3-DCP will have broad applications in the physiology of metabolic diseases. In addition, we find that available anti-2SC antibodies also detect the structurally similar 2,3-DCP, therefore "succinate moiety" may better describe the antigen recognized.NEW & NOTEWORTHY Itaconate and fumarate have roles as immunometabolites modulating the macrophage response to inflammation. Both immunometabolites chemically modify protein cysteine residues to modulate the immune response. Itaconate and fumarate levels change dynamically, whereas their stable protein modifications can be quantified by mass spectrometry. This method distinguishes itaconate and fumarate-derived protein modifications and will allow researchers to quantify their contributions in isolated cell types and tissues across a range of metabolic diseases.


Assuntos
Compostos Alílicos , Cisteína , Cisteína/análogos & derivados , Hidrocarbonetos Clorados , Doenças Metabólicas , Succinatos , Humanos , Cisteína/metabolismo , Lipopolissacarídeos/farmacologia , Proteínas , Fumaratos/metabolismo
4.
J Neurovirol ; 2024 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-38884890

RESUMO

HIV-associated neurological disorder (HAND) is a serious complication of HIV infection marked by neurotoxicity induced by viral proteins like Tat. Substance abuse exacerbates neurocognitive impairment in people living with HIV. There is an urgent need for therapeutic strategies to combat HAND comorbid with Cocaine Use Disorder (CUD). Our analysis of HIV and cocaine-induced transcriptomes in primary cortical cultures revealed significant overexpression of the macrophage-specific gene aconitate decarboxylase 1 (Acod1). The ACOD1 protein converts the tricarboxylic acid intermediate cis-aconitate into itaconate during the activation of inflammation. Itaconate then facilitates cytokine production and activates anti-inflammatory transcription factors, shielding macrophages from infection-induced cell death. However, the immunometabolic function of itaconate was unexplored in HIV and cocaine-exposed microglia. We assessed the potential of 4-octyl-itaconate (4OI), a cell-penetrable ester form of itaconate known for its anti-inflammatory properties. When primary cortical cultures exposed to Tat and cocaine were treated with 4OI, microglial cell number increased and the morphological altercations induced by Tat and cocaine were reversed. Microglial cells also appeared more ramified, resembling the quiescent microglia. 4OI treatment inhibited secretion of the proinflammatory cytokines IL-1α, IL-1ß, IL-6, and MIP1-α induced by Tat and cocaine. Transcriptome profiling determined that Nrf2 target genes were significantly activated in Tat and 4OI treated cultures relative to Tat alone. Further, genes associated with cytoskeleton dynamics in inflammatory microglia were downregulated by 4OI treatment. Together, the results strongly suggest 4-octyl-itaconate holds promise as a potential candidate for therapeutic development to treat HAND coupled with CUD comorbidities.

5.
Nat Chem Biol ; 15(4): 391-400, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30718813

RESUMO

Hereditary cancer disorders often provide an important window into novel mechanisms supporting tumor growth. Understanding these mechanisms thus represents a vital goal. Toward this goal, here we report a chemoproteomic map of fumarate, a covalent oncometabolite whose accumulation marks the genetic cancer syndrome hereditary leiomyomatosis and renal cell carcinoma (HLRCC). We applied a fumarate-competitive chemoproteomic probe in concert with LC-MS/MS to discover new cysteines sensitive to fumarate hydratase (FH) mutation in HLRCC cell models. Analysis of this dataset revealed an unexpected influence of local environment and pH on fumarate reactivity, and enabled the characterization of a novel FH-regulated cysteine residue that lies at a key protein-protein interface in the SWI-SNF tumor-suppressor complex. Our studies provide a powerful resource for understanding the covalent imprint of fumarate on the proteome and lay the foundation for future efforts to exploit this distinct aspect of oncometabolism for cancer diagnosis and therapy.


Assuntos
Fumaratos/metabolismo , Leiomiomatose/metabolismo , Síndromes Neoplásicas Hereditárias/metabolismo , Neoplasias Cutâneas/metabolismo , Neoplasias Uterinas/metabolismo , Linhagem Celular Tumoral , Cromatografia Líquida/métodos , Cisteína , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Leiomiomatose/genética , Modelos Biológicos , Síndromes Neoplásicas Hereditárias/genética , Proteômica , Transdução de Sinais , Neoplasias Cutâneas/genética , Espectrometria de Massas em Tandem/métodos , Neoplasias Uterinas/genética
6.
Mol Cell Proteomics ; 18(3): 504-519, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30587509

RESUMO

The fumarate ester dimethyl fumarate (DMF) has been introduced recently as a treatment for relapsing remitting multiple sclerosis (RRMS), a chronic inflammatory condition that results in neuronal demyelination and axonal loss. DMF is known to act by depleting intracellular glutathione and modifying thiols on Keap1 protein, resulting in the stabilization of the transcription factor Nrf2, which in turn induces the expression of antioxidant response element genes. We have previously shown that DMF reacts with a wide range of protein thiols, suggesting that the complete mechanisms of action of DMF are unknown. Here, we investigated other intracellular thiol residues that may also be irreversibly modified by DMF in neurons and astrocytes. Using mass spectrometry, we identified 24 novel proteins that were modified by DMF in neurons and astrocytes, including cofilin-1, tubulin and collapsin response mediator protein 2 (CRMP2). Using an in vitro functional assay, we demonstrated that DMF-modified cofilin-1 loses its activity and generates less monomeric actin, potentially inhibiting its cytoskeletal remodeling activity, which could be beneficial in the modulation of myelination during RRMS. DMF modification of tubulin did not significantly impact axonal lysosomal trafficking. We found that the oxygen consumption rate of N1E-115 neurons and the levels of proteins related to mitochondrial energy production were only slightly affected by the highest doses of DMF, confirming that DMF treatment does not impair cellular respiratory function. In summary, our work provides new insights into the mechanisms supporting the neuroprotective and remyelination benefits associated with DMF treatment in addition to the antioxidant response by Nrf2.


Assuntos
Astrócitos/metabolismo , Cisteína/efeitos dos fármacos , Fumarato de Dimetilo/farmacologia , Fator 2 Relacionado a NF-E2/metabolismo , Neurônios/metabolismo , Células 3T3-L1 , Animais , Astrócitos/citologia , Astrócitos/efeitos dos fármacos , Células Cultivadas , Cofilina 1/química , Cofilina 1/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular , Espectrometria de Massas , Camundongos , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Neurônios/efeitos dos fármacos , Ratos , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo
7.
Int J Gynecol Pathol ; 37(5): 421-430, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28863073

RESUMO

Leiomyoma with bizarre nuclei (LM-BN), is a variant of uterine smooth muscle tumor with atypical histologic features. Although some LM-BN share several significant genetic alterations with leiomyosarcoma, including p16 and p53, the underlying tumorigenesis of LM-BN remains largely unknown. As we previously reported, LM-BN can be divided into 2 subtypes, type I and type II, based on different nuclear features. Type I LM-BN have similar histologic features as uterine smooth muscle tumors with fumarate hydratase (FH) alterations. In this study, we examined FH expression and FH mutations in 77 LM-BN (40 type I cases and 37 type II cases). FH expression was examined by immunohistochemistry using S-(2-succino)-cysteine antibodies (2SC, a protein modification associated with FH inactivation and subsequent fumarate accumulation) and FH antibodies (FH gene products). Seventy-two LM-BN tumors underwent Sanger sequencing to detect FH mutations. We found that 51% (39/77) of LM-BN showed FH alterations detected by immunohistochemistry with both 2SC and FH. Mutational analysis showed that 21% (15/72) of LM-BN harbored FH gene mutations. Further analysis revealed that 85% (34/40) of those with FH alterations were type I LM-BN while 19% (7/37) were type II LM-BN. Our findings suggest that over half of histologically diagnosed LM-BN may be related to FH alterations or FH mutations and the majority of these have the characteristic histologic features of type I LM-BN.


Assuntos
Fumarato Hidratase/genética , Leiomioma/enzimologia , Leiomioma/genética , Neoplasias Uterinas/enzimologia , Neoplasias Uterinas/genética , Adulto , Idoso , Biomarcadores Tumorais/análise , Núcleo Celular/patologia , Análise Mutacional de DNA , Feminino , Humanos , Leiomioma/patologia , Pessoa de Meia-Idade , Mutação , Neoplasias Uterinas/patologia , Adulto Jovem
8.
Mol Cell Proteomics ; 15(2): 445-61, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26450614

RESUMO

Elevated fumarate concentrations as a result of Krebs cycle inhibition lead to increases in protein succination, an irreversible post-translational modification that occurs when fumarate reacts with cysteine residues to generate S-(2-succino)cysteine (2SC). Metabolic events that reduce NADH re-oxidation can block Krebs cycle activity; therefore we hypothesized that oxidative phosphorylation deficiencies, such as those observed in some mitochondrial diseases, would also lead to increased protein succination. Using the Ndufs4 knockout (Ndufs4 KO) mouse, a model of Leigh syndrome, we demonstrate for the first time that protein succination is increased in the brainstem (BS), particularly in the vestibular nucleus. Importantly, the brainstem is the most affected region exhibiting neurodegeneration and astrocyte and microglial proliferation, and these mice typically die of respiratory failure attributed to vestibular nucleus pathology. In contrast, no increases in protein succination were observed in the skeletal muscle, corresponding with the lack of muscle pathology observed in this model. 2D SDS-PAGE followed by immunoblotting for succinated proteins and MS/MS analysis of BS proteins allowed us to identify the voltage-dependent anion channels 1 and 2 as specific targets of succination in the Ndufs4 knockout. Using targeted mass spectrometry, Cys(77) and Cys(48) were identified as endogenous sites of succination in voltage-dependent anion channels 2. Given the important role of voltage-dependent anion channels isoforms in the exchange of ADP/ATP between the cytosol and the mitochondria, and the already decreased capacity for ATP synthesis in the Ndufs4 KO mice, we propose that the increased protein succination observed in the BS of these animals would further decrease the already compromised mitochondrial function. These data suggest that fumarate is a novel biochemical link that may contribute to the progression of the neuropathology in this mitochondrial disease model.


Assuntos
Complexo I de Transporte de Elétrons/genética , Doença de Leigh/genética , Proteômica , Succinatos/metabolismo , Animais , Tronco Encefálico/metabolismo , Tronco Encefálico/patologia , Ciclo do Ácido Cítrico , Cisteína/metabolismo , Modelos Animais de Doenças , Complexo I de Transporte de Elétrons/metabolismo , Fumaratos/metabolismo , Humanos , Doença de Leigh/metabolismo , Doença de Leigh/patologia , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Processamento de Proteína Pós-Traducional/genética , Espectrometria de Massas em Tandem
9.
Am J Physiol Cell Physiol ; 313(5): C487-C500, 2017 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-28768641

RESUMO

IL-6 and leukemia inhibitory factor (LIF), members of the IL-6 family of cytokines, play recognized paradoxical roles in skeletal muscle mass regulation, being associated with both growth and atrophy. Overload or muscle contractions can induce a transient increase in muscle IL-6 and LIF expression, which has a regulatory role in muscle hypertrophy. However, the cellular mechanisms involved in this regulation have not been completely identified. The induction of mammalian target of rapamycin complex 1 (mTORC1)-dependent myofiber protein synthesis is an established regulator of muscle hypertrophy, but the involvement of the IL-6 family of cytokines in this process is poorly understood. Therefore, we investigated the acute effects of IL-6 and LIF administration on mTORC1 signaling and protein synthesis in C2C12 myotubes. The role of glycoprotein 130 (gp130) receptor and downstream signaling pathways, including phosphoinositide 3-kinase (PI3K)-Akt-mTORC1 and signal transducer and activator of transcription 3 (STAT3)-suppressor of cytokine signaling 3 (SOCS3), was investigated by administration of specific siRNA or pharmaceutical inhibitors. Acute administration of IL-6 and LIF induced protein synthesis, which was accompanied by STAT3 activation, Akt-mTORC1 activation, and increased SOCS3 expression. This induction of protein synthesis was blocked by both gp130 siRNA knockdown and Akt inhibition. Interestingly, STAT3 inhibition or Akt downstream mTORC1 signaling inhibition did not fully block the IL-6 or LIF induction of protein synthesis. SOCS3 siRNA knockdown increased basal protein synthesis and extended the duration of the protein synthesis induction by IL-6 and LIF. These results demonstrate that either IL-6 or LIF can activate gp130-Akt signaling axis, which induces protein synthesis via mTORC1-independent mechanisms in cultured myotubes. However, IL-6- or LIF-induced SOCS3 negatively regulates the activation of myotube protein synthesis.


Assuntos
Interleucina-6/fisiologia , Fibras Musculares Esqueléticas/metabolismo , Mioblastos/metabolismo , Biossíntese de Proteínas/fisiologia , Animais , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Células Cultivadas , Citocinas/farmacologia , Citocinas/fisiologia , Interleucina-6/farmacologia , Fator Inibidor de Leucemia/farmacologia , Fator Inibidor de Leucemia/fisiologia , Camundongos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Mioblastos/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos
10.
Mol Cancer ; 16(1): 101, 2017 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-28592321

RESUMO

Uterine smooth muscle tumors range from benign leiomyomas to malignant leiomyosarcomas. Based on numerous molecular studies, leiomyomas and leiomyosarcomas mostly lack shared mutations and the majority of tumors are believed to develop through distinct mechanisms. To further characterize the molecular variability among uterine smooth muscle tumors, and simultaneously insinuate their potential malignant progression, we examined the frequency of known genetic leiomyoma driver alterations (MED12 mutations, HMGA2 overexpression, biallelic FH inactivation) in 65 conventional leiomyomas, 94 histopathological leiomyoma variants (18 leiomyomas with bizarre nuclei, 22 cellular, 29 highly cellular, and 25 mitotically active leiomyomas), and 51 leiomyosarcomas. Of the 210 tumors analyzed, 107 had mutations in one of the three driver genes. No tumor had more than one mutation confirming that all alterations are mutually exclusive. MED12 mutations were the most common alterations in conventional and mitotically active leiomyomas and leiomyosarcomas, while leiomyomas with bizarre nuclei were most often FH deficient and cellular tumors showed frequent HMGA2 overexpression. Highly cellular leiomyomas displayed the least amount of alterations leaving the majority of tumors with no known driver aberration. Our results indicate that based on the molecular background, histopathological leiomyoma subtypes do not only differ from conventional leiomyomas, but also from each other. The presence of leiomyoma driver alterations in nearly one third of leiomyosarcomas suggests that some tumors arise through leiomyoma precursor lesion or that these mutations provide growth advantage also to highly aggressive cancers. It is clinically relevant to understand the molecular background of various smooth muscle tumor subtypes, as it may lead to improved diagnosis and personalized treatments in the future.


Assuntos
Biomarcadores Tumorais , Fumarato Hidratase/genética , Proteína HMGA2/genética , Complexo Mediador/genética , Tumor de Músculo Liso/genética , Tumor de Músculo Liso/patologia , Neoplasias Uterinas/genética , Neoplasias Uterinas/patologia , Análise Mutacional de DNA , Feminino , Fumarato Hidratase/metabolismo , Expressão Gênica , Proteína HMGA2/metabolismo , Humanos , Complexo Mediador/metabolismo , Mutação , Gradação de Tumores , Estudos Retrospectivos , Tumor de Músculo Liso/metabolismo , Neoplasias Uterinas/metabolismo
11.
Biochim Biophys Acta ; 1853(1): 213-21, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25448036

RESUMO

While the 3T3-L1 adipocyte model is routinely used for the study of obesity and diabetes, the mitochondrial respiratory profile in normal versus high glucose has not been examined in detail. We matured adipocytes in normal (5mM) or high (30 mM) glucose and insulin and examined the mitochondrial bioenergetics. We also assessed the requirement for the Unfolded Protein Response (UPR) and ER stress under these conditions. Basal respiration was ~1.7-fold greater in adipocytes that had matured in 30 mM glucose; however, their ability to increase oxygen consumption in response to stress was impaired. Adipogenesis proceeded in both normal and high glucose with concomitant activation of the UPR, but only high glucose was associated with increased levels of ER stress and mitochondrial stress as observed by parallel increases in CHOP and protein succination. Treatment of adipocytes with sodium phenylbutyrate relieved mitochondrial stress through a reduction in mitochondrial respiration. Our data suggests that mitochondrial stress, protein succination and ER stress are uniquely linked in adipocytes matured in high glucose.


Assuntos
Estresse do Retículo Endoplasmático/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Glucose/farmacologia , Mitocôndrias/efeitos dos fármacos , Fenilbutiratos/farmacologia , Células 3T3-L1 , Adipogenia/efeitos dos fármacos , Animais , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Mitocôndrias/metabolismo , Consumo de Oxigênio , Dobramento de Proteína , Fator de Transcrição CHOP/análise , Resposta a Proteínas não Dobradas
12.
Br J Cancer ; 114(12): 1405-11, 2016 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-27187686

RESUMO

BACKGROUND: Uterine leiomyomas from hereditary leiomyomatosis and renal cell cancer (HLRCC) patients are driven by fumarate hydratase (FH) inactivation or occasionally by mediator complex subunit 12 (MED12) mutations. The aim of this study was to analyse whether MED12 mutations and FH inactivation are mutually exclusive and to determine the contribution of MED12 mutations on HLRCC patients' myomagenesis. METHODS: MED12 exons 1 and 2 mutation screening and 2SC immunohistochemistry indicative for FH deficiency was performed on a comprehensive series of HLRCC patients' (122 specimens) and sporadic (66 specimens) tumours. Gene expression analysis was performed using Affymetrix GeneChip Human Exon Arrays (Affymetrix, Santa Clara, CA, USA). RESULTS: Nine tumours from HLRCC patients harboured a somatic MED12 mutation and were negative for 2SC immunohistochemistry. All remaining successfully analysed lesions (107/116) were deficient for FH. Of sporadic tumours, 35/64 were MED12 mutation positive and none displayed a FH defect. In global gene expression analysis FH-deficient tumours clustered together, whereas HLRCC patients' MED12 mutation-positive tumours clustered together with sporadic MED12 mutation-positive tumours. CONCLUSIONS: Somatic MED12 mutations and biallelic FH inactivation are mutually exclusive in both HLRCC syndrome-associated and sporadic uterine leiomyomas. The great majority of HLRCC patients' uterine leiomyomas are caused by FH inactivation, but incidental tumours driven by somatic MED12 mutations also occur. These MED12 mutation-positive tumours display similar expressional profiles with their sporadic counterparts and are clearly separate from FH-deficient tumours.


Assuntos
Biomarcadores Tumorais/genética , Fumarato Hidratase/metabolismo , Leiomioma/enzimologia , Leiomioma/genética , Complexo Mediador/genética , Neoplasias Uterinas/enzimologia , Neoplasias Uterinas/genética , Ativação Enzimática , Feminino , Mutação em Linhagem Germinativa , Humanos , Imuno-Histoquímica , Complexo Mediador/metabolismo , Mutação , Transcriptoma
13.
Biochem Biophys Res Commun ; 470(4): 783-91, 2016 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-26801556

RESUMO

Recent studies have demonstrated that adult humans have substantial amounts of functioning brown adipose tissue (BAT). Since BAT has been implicated as an anti-obese and anti-diabetic tissue, it is important to understand the signaling molecules that regulate BAT function. There has been a link between insulin signaling and BAT metabolism as deletion or pharmaceutical inhibition of insulin signaling impairs BAT differentiation and function. Tribbles 3 (TRB3) is a pseudo kinase that has been shown to regulate metabolism and insulin signaling in multiple tissues but the role of TRB3 in BAT has not been studied. In this study, we found that TRB3 expression was present in BAT and overexpression of TRB3 in brown preadipocytes impaired differentiation and decreased expression of BAT markers. Furthermore, TRB3 overexpression resulted in significantly lower oxygen consumption rates for basal and proton leakage, indicating decreased BAT activity. Based on previous studies showing that deletion or pharmaceutical inhibition of insulin signaling impairs BAT differentiation and function, we assessed insulin signaling in brown preadipocytes and BAT in vivo. Overexpression of TRB3 in cells impaired insulin-stimulated IRS1 and Akt phosphorylation, whereas TRB3KO mice displayed improved IRS1 and Akt phosphorylation. Finally, deletion of IRS1 abolished the function of TRB3 to regulate BAT differentiation and metabolism. These data demonstrate that TRB3 inhibits insulin signaling in BAT, resulting in impaired differentiation and function.


Assuntos
Adipócitos Marrons/citologia , Adipócitos Marrons/metabolismo , Adipogenia/fisiologia , Proteínas de Ciclo Celular/metabolismo , Insulina/fisiologia , Animais , Diferenciação Celular/fisiologia , Células Cultivadas , Regulação para Baixo , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais/fisiologia
14.
Am J Dermatopathol ; 38(12): 887-891, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27097334

RESUMO

AIMS: Hereditary leiomyomatosis and renal cell cancer (HLRCC) syndrome is an autosomal dominant disorder caused by heterozygotic germline mutations in fumarate hydratase (FH) with incomplete penetrance, and clinically challenging to diagnose. Immunohistochemical stainings may favor an earlier diagnosis. METHODS AND RESULTS: The authors have tested 31 smooth muscle neoplasms. Ten of the 13 lesions from patients with HLRCC syndrome showed negative FH staining. Most sporadic piloleiomyomas presented strongly positive FH staining although 5 cases were negative. Sensitivity of FH staining in our series is 83.3% but specificity is 75%. Anti-S-(2-succino)-cysteine (2SC) showed the opposite intensity staining pattern and showed great correlation with anti-FH (rho spearman = -0.797). Anti-2SC staining increased the diagnostic accuracy in 19% of the cases. LIMITATIONS: The main limitation of this study is the lack additional clinical data to further classify the cases as the case inclusion was histopathological. CONCLUSIONS: Negative FH staining could indicate a high risk of HLRCC but it could also suggest the presence of a syndrome in up to 25% of sporadic cases. Thus, when there is a doubtful case, anti-2SC may be added to exclude the syndrome if a negative staining is found.


Assuntos
Biomarcadores Tumorais/análise , Cisteína/análogos & derivados , Fumarato Hidratase/análise , Imuno-Histoquímica , Leiomiomatose/enzimologia , Processamento de Proteína Pós-Traducional , Neoplasias Cutâneas/enzimologia , Neoplasias Uterinas/enzimologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Cisteína/análise , Análise Mutacional de DNA , Regulação para Baixo , Detecção Precoce de Câncer , Feminino , Fumarato Hidratase/genética , Humanos , Leiomiomatose/genética , Leiomiomatose/patologia , Masculino , Pessoa de Meia-Idade , Mutação , Síndromes Neoplásicas Hereditárias , Valor Preditivo dos Testes , Reprodutibilidade dos Testes , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Neoplasias Uterinas/genética , Neoplasias Uterinas/patologia
15.
Mass Spectrom Rev ; 33(2): 98-109, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24115015

RESUMO

The post-translational modifications (PTMs) of cysteine residues include oxidation, S-glutathionylation, S-nitrosylation, and succination, all of which modify protein function or turnover in response to a changing intracellular redox environment. Succination is a chemical modification of cysteine in proteins by the Krebs cycle intermediate, fumarate, yielding S-(2-succino)cysteine (2SC). Intracellular fumarate concentration and succination of proteins are increased by hyperpolarization of the inner mitochondrial membrane, in concert with mitochondrial, endoplasmic reticulum (ER) and oxidative stress in 3T3 adipocytes grown in high glucose medium and in adipose tissue in obesity and diabetes in mice. Increased succination of proteins is also detected in the kidney of a fumarase deficient conditional knock-out mouse which develops renal cysts. A wide range of proteins are subject to succination, including enzymes, adipokines, cytoskeletal proteins, and ER chaperones with functional cysteine residues. There is also some overlap between succinated and glutathionylated proteins, suggesting that the same low pKa thiols are targeted by both. Succination of adipocyte proteins in diabetes increases as a result of nutrient excess derived mitochondrial stress and this is inhibited by uncouplers, which discharge the mitochondrial membrane potential (ΔΨm) and relieve the electron transport chain. 2SC therefore serves as a biomarker of mitochondrial stress or dysfunction in chronic diseases, such as obesity, diabetes, and cancer, and recent studies suggest that succination is a mechanistic link between mitochondrial dysfunction, oxidative and ER stress, and cellular progression toward apoptosis. In this article, we review the history of the succinated proteome and the challenges associated with measuring this non-enzymatic PTM of proteins by proteomics approaches.


Assuntos
Cisteína/análogos & derivados , Cisteína/metabolismo , Fumaratos/metabolismo , Proteoma/química , Proteoma/metabolismo , Animais , Ciclo do Ácido Cítrico , Cisteína/análise , Diabetes Mellitus/metabolismo , Humanos , Mitocôndrias/metabolismo , Neoplasias/metabolismo , Estresse Oxidativo
16.
Biochem J ; 462(2): 231-45, 2014 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-24909641

RESUMO

Protein succination is a stable post-translational modification that occurs when fumarate reacts with cysteine residues to generate 2SC [S-(2-succino)cysteine]. We demonstrate that both α- and ß-tubulin are increasingly modified by succination in 3T3-L1 adipocytes and in the adipose tissue of db/db mice. Incubation of purified tubulin from porcine brain with fumarate (50 mM) or the pharmacological compound DMF (dimethylfumarate, 500 µM) inhibited polymerization up to 35% and 59% respectively. Using MS we identified Cys347α, Cys376α, Cys12ß and Cys303ß as sites of succination in porcine brain tubulin and the relative abundance of succination at these cysteine residues increased in association with fumarate concentration. The increase in succination after incubation with fumarate altered tubulin recognition by an anti-α-tubulin antibody. Succinated tubulin in adipocytes cultured in high glucose compared with normal glucose also had reduced reactivity with the anti-α-tubulin antibody; suggesting that succination may interfere with tubulin-protein interactions. DMF reacted rapidly with 11 of the 20 cysteine residues in the αß-tubulin dimer, decreased the number of free thiols and inhibited the proliferation of 3T3-L1 fibroblasts. Our data suggest that inhibition of tubulin polymerization is an important undocumented mechanism of action of DMF. Taken together, our results demonstrate that succination is a novel post-translational modification of tubulin and suggest that extensive modification by fumarate, either physiologically or pharmacologically, may alter microtubule dynamics.


Assuntos
Ácido Succínico/metabolismo , Tubulina (Proteína)/metabolismo , Células 3T3-L1 , Adipócitos/metabolismo , Tecido Adiposo/metabolismo , Animais , Encéfalo/metabolismo , Bovinos , Proliferação de Células , Meios de Cultura , Diabetes Mellitus Tipo 2/metabolismo , Fumarato de Dimetilo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fumaratos/farmacologia , Glucose/metabolismo , Camundongos , Polimerização
17.
Mod Pathol ; 27(7): 1020-7, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24309325

RESUMO

Rare, sporadic uterine leiomyomas arise in the setting of severe metabolic aberration due to a somatic fumarate hydratase mutation. Germline mutations account for the hereditary leiomyomatosis and renal cell carcinoma syndrome, which predisposes for cutaneous and uterine leiomyomas and aggressive renal cell carcinomas. Altered fumarate hydratase leads to fumarate accumulation in affected cells with formation of S-(2-succino)-cysteine, which can be detected with the polyclonal antibody. High levels of these modified cysteine residues are found characteristically in fumarate hydratase-deficient cells but not in normal tissues or tumors unassociated with hereditary leiomyomatosis and renal cell carcinoma syndrome. We hypothesized that S-(2-succino)-cysteine-positive leiomyomas, indicating fumarate hydratase aberration, have morphologic features that differ from those without S-(2-succino)-cysteine positivity. Hematoxylin and eosin-stained slides of uterine smooth-muscle tumors were prospectively analyzed for features suggesting hereditary leiomyomatosis and renal cell carcinoma syndrome, such as prominent eosinophilic macronucleoli with perinucleolar halos, yielding nine cases. Germline genetic testing for fumarate hydratase mutations was performed in three cases. A detailed morphological analysis was undertaken, and S-(2-succino)-cysteine immunohistochemical analysis was performed with controls from a tissue microarray (leiomyomas (19), leiomyosarcomas (29), and endometrial stromal tumors (15)). Of the nine study cases, four had multiple uterine smooth muscle tumors. All cases had increased cellularity, staghorn vasculature, and fibrillary cytoplasm with pink globules. All cases had inclusion-like nucleoli with perinuclear halos (7 diffuse, 1 focal). All showed diffuse granular cytoplasmic labeling with the S-(2-succino)-cysteine antibody. Two of three tested patients had germline fumarate hydratase mutations. Only one leiomyoma from the tissue microarray controls was immunohistochemically positive, and it showed features similar to other immunohistochemically positive cases. Smooth-muscle tumors with fumarate hydratase aberration demonstrate morphological reproducibility across cases and S-(2-succino)-cysteine immuno-positivity. Although the features described are not specific for the germline fumarate hydratase mutation or the hereditary leiomyomatosis and renal cell carcinoma syndrome, their presence should suggest fumarate hydratase aberration. Identifying these cases is an important step in the diagnostic workup of patients with possible hereditary leiomyomatosis and renal cell carcinoma.


Assuntos
Cisteína/análogos & derivados , Fumarato Hidratase/genética , Tumor de Músculo Liso/genética , Neoplasias Uterinas/genética , Adulto , Cisteína/metabolismo , Feminino , Fumarato Hidratase/metabolismo , Predisposição Genética para Doença , Mutação em Linhagem Germinativa , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Tumor de Músculo Liso/metabolismo , Tumor de Músculo Liso/patologia , Neoplasias Uterinas/metabolismo , Neoplasias Uterinas/patologia
18.
Clin Chem Lab Med ; 52(1): 69-75, 2014 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-23612664

RESUMO

In a recent article, we presented the hypothesis that decompartmentalized metal ions are a major contributor to the development of diabetic complications and supported the use of chelation therapy for the treatment of diabetic complications [Nagai R, Murray DB, Metz TO, Baynes JW. Chelation: a fundamental mechanism of action of AGE inhibitors, AGE breakers, and other inhibitors of diabetes complications. Diabetes 2012;61:549-59]. Evidence in support of this hypothesis included the observation that many drugs used in the treatment of diabetes are chelators, that advanced glycation end product (AGE) inhibitors and AGE breakers lack carbonyl-trapping or AGE-breaker activity but are potent chelators, and that simple copper chelators inhibit vascular pathology in diabetes and aging. In the present article, we extend this hypothesis, proposing the interplay between copper and iron in the development of pathology in diabetes and other chronic age-related diseases, including atherosclerosis and neurodegenerative diseases. We also discuss the need and provide a framework for the development of a clinical laboratory test to assess plasma autoxidative catalytic activity and transition metal homeostasis in vivo.


Assuntos
Quelantes/uso terapêutico , Cobre/metabolismo , Complicações do Diabetes/tratamento farmacológico , Ferro/metabolismo , Terapia por Quelação , Cobre/química , Complicações do Diabetes/metabolismo , Complicações do Diabetes/patologia , Produtos Finais de Glicação Avançada/antagonistas & inibidores , Produtos Finais de Glicação Avançada/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Íons/química , Ferro/química , Cinética , Oxirredução , Estresse Oxidativo
19.
J Cachexia Sarcopenia Muscle ; 15(1): 124-137, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38062911

RESUMO

BACKGROUND: More than 650 million people are obese (BMI > 30) worldwide, which increases their risk for several metabolic diseases and cancer. While cachexia and obesity are at opposite ends of the weight spectrum, leading many to suggest a protective effect of obesity against cachexia, mechanistic support for obesity's benefit is lacking. Given that obesity and cachexia are both accompanied by metabolic dysregulation, we sought to investigate the impact of obesity on skeletal muscle mass loss and mitochondrial dysfunction in murine cancer cachexia. METHODS: Male C57BL/6 mice were given a purified high fat or standard diet for 16 weeks before being implanted with 106 Lewis lung carcinoma (LLC) cells. Mice were monitored for 25 days, and hindlimb muscles were collected for cachexia indices and mitochondrial assessment via western blotting, high-resolution respirometry and transmission electron microscopy (TEM). RESULTS: Obese LLC mice experienced significant tumour-free body weight loss similar to lean (-12.8% vs. -11.8%, P = 0.0001) but had reduced survival (33.3% vs. 6.67%, χ2  = 10.04, P = 0.0182). Obese LLC mice had reduced muscle weights (-24%, P < 0.0354) and mCSA (-16%, P = 0.0004) with similar activation of muscle p65 (P = 0.0337), and p38 (P = 0.0008). ADP-dependent coupled respiration was reduced in both Obese and Obese LLC muscle (-30%, P = 0.0072) consistent with reductions in volitional cage activity (-39%, P < 0.0001) and grip strength (-41%, P < 0.0001). TEM revealed stepwise reductions in intermyofibrillar and subsarcolemmal mitochondrial size with Obese (IMF: -37%, P = 0.0009; SS: -21%, P = 0.0101) and LLC (IMF: -40%, P = 0.0019; SS: -27%, P = 0.0383) mice. Obese LLC mice had increased pAMPK (T172; P = 0.0103) and reduced FIS1 (P = 0.0029) and DRP1 (P < 0.0001) mitochondrial fission proteins, which were each unchanged in Lean LLC. Further, mitochondrial TEM analysis revealed that Obese LLC mice had an accumulation of damaged and dysfunctional mitochondria (IMF: 357%, P = 0.0395; SS: 138%, P = 0.0174) in concert with an accumulation of p62 (P = 0.0328) suggesting impaired autophagy and clearance of damaged mitochondria. Moreover, we observed increases in electron lucent vacuoles only in Obese LLC muscle (IMF: 421%, P = 0.0260; SS: 392%, P = 0.0192), further supporting an accumulation of damaged materials that cannot be properly cleared in the obese cachectic muscle. CONCLUSIONS: Taken together, these results demonstrate that obesity is not protective against cachexia and suggest exacerbated impairments to mitochondrial function and quality control with a particular disruption in the removal of damaged mitochondria. Our findings highlight the need for consideration of the severity of obesity and pre-existing metabolic conditions when determining the impact of weight status on cancer-induced cachexia and functional mitochondrial deficits.


Assuntos
Caquexia , Carcinoma Pulmonar de Lewis , Humanos , Masculino , Animais , Camundongos , Caquexia/patologia , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Atrofia Muscular/patologia , Carcinoma Pulmonar de Lewis/complicações , Carcinoma Pulmonar de Lewis/patologia , Obesidade/complicações , Obesidade/patologia , Músculo Esquelético/patologia
20.
Amino Acids ; 45(5): 1243-7, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23892396

RESUMO

Protein succination, the non-enzymatic modification of cysteine residues by fumarate, is distinguishable from succinylation, an enzymatic reaction forming an amide bond between lysine residues and succinyl-CoA. Treatment of adipocytes with 30 mM glucose significantly increases protein succination with only a small change in succinylation. Protein succination may be significantly increased intracellularly after treatment with fumaric acid esters, however, the ester must be removed by saponification to permit 2SC-antibody detection of the fumarate adduct.


Assuntos
Adipócitos/metabolismo , Ciclo do Ácido Cítrico , Fumaratos/farmacologia , Proteínas/metabolismo , Células 3T3 , Adipócitos/efeitos dos fármacos , Animais , Ciclo do Ácido Cítrico/efeitos dos fármacos , Cisteína/metabolismo , Glucose/metabolismo , Camundongos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos
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