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Objectives: Chondrocyte metabolic dysfunction plays an important role in osteoarthritis (OA) development during aging and obesity. Protein post-translational modifications (PTMs) have recently emerged as an important regulator of cellular metabolism. We aim to study one type of PTM, lysine malonylation (MaK) and its regulator Sirt5 in OA development. Methods: Human and mouse cartilage tissues were used to measure SIRT5 and MaK levels. Both systemic and cartilage-specific conditional knockout mouse models were subject to high-fat diet (HFD) treatment to induce obesity and OA. Proteomics analysis was performed in Sirt5 -/- and WT chondrocytes. SIRT5 mutation was identified in the Utah Population Database (UPDB). Results: We found that SIRT5 decreases while MAK increases in the cartilage during aging. A combination of Sirt5 deficiency and obesity exacerbates joint degeneration in a sex dependent manner in mice. We further delineate the malonylome in chondrocytes, pinpointing MaK's predominant impact on various metabolic pathways such as carbon metabolism and glycolysis. Lastly, we identified a rare coding mutation in SIRT5 that dominantly segregates in a family with OA. The mutation results in substitution of an evolutionally invariant phenylalanine (Phe-F) to leucine (Leu-L) (F101L) in the catalytic domain. The mutant protein results in higher MaK level and decreased expression of cartilage ECM genes and upregulation of inflammation associated genes. Conclusions: We found that Sirt5 mediated MaK is an important regulator of chondrocyte cellular metabolism and dysregulation of Sirt5-MaK could be an important mechanism underlying aging and obesity associated OA development.
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Senescence emerged as a significant mechanism of aging and age-related diseases, offering an attractive target for clinical interventions. Senescent cells release a senescence-associated secretory phenotype (SASP), including exosomes that may act as signal transducers between distal tissues, propagating secondary or bystander senescence and signaling throughout the body. However, the composition of exosome SASP remains underexplored, presenting an opportunity for novel unbiased discovery. Here, we present a detailed proteomic and lipidomic analysis of exosome SASP using mass spectrometry from human plasma from young and older individuals and from tissue culture of senescent primary human lung fibroblasts. We identified ~1,300 exosome proteins released by senescent fibroblasts induced by three different senescence inducers causing most exosome proteins to be differentially regulated with senescence. In parallel, a human plasma cohort from young and old individuals revealed over 1,350 exosome proteins and 171 plasma exosome proteins were regulated when comparing old vs young individuals. Of the age-regulated plasma exosome proteins, we observed 52 exosome SASP factors that were also regulated in exosomes from the senescent fibroblasts, including serine protease inhibitors (SERPINs), Prothrombin, Coagulation factor V, Plasminogen, and Reelin. In addition, 247 lipids were identified with high confidence in all exosome samples. Following the senescence inducers, a majority of the identified phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin species increased significantly indicating cellular membrane changes. The most notable categories of significantly changed proteins were related to extracellular matrix remodeling and inflammation, both potentially detrimental pathways that can damage surrounding tissues and even induce secondary or bystander senescence. Our findings reveal mechanistic insights and potential senescence biomarkers, enabling a better approach to surveilling the senescence burden in the aging population and offering promising therapeutic targets for interventions.
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In a series of experiments involving beliefs and misinformation beliefs, we find that individuals who are prompted with a counterfactual mindset are significantly more likely to change their existing beliefs when presented with evidence that contradicts their beliefs. While research finds that beliefs that are considered part of one's identity are highly resistant to change in the face of evidence that challenges these beliefs, four experiments provide evidence that counterfactual generation causes individuals to adjust beliefs and correct misinformation beliefs in response to contradicting evidence. Indeed, we find that a counterfactual mindset was effective in promoting incorporation of accurate facts and causing individuals to revise misinformation beliefs about COVID vaccination safety for a large sample of individuals who have rejected COVID vaccinations. Finally, the results of the psychophysiological experiment reveal that counterfactual generation alters decision makers' search strategies, increases their cognitive arousal in response to evidence that challenges their beliefs, and increases their desire to seek out disconfirming evidence. Overall, the four experiments indicate that counterfactual generation can effectively activate mindsets that increase individuals' willingness to evaluate evidence that contradicts their beliefs and adjust their beliefs in response to evidence.
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COVID-19 , Comunicação , Humanos , Feminino , Masculino , COVID-19/psicologia , COVID-19/prevenção & controle , Adulto , Adulto Jovem , Vacinas contra COVID-19/administração & dosagem , SARS-CoV-2 , Tomada de Decisões , Vacinação/psicologia , Cultura , Conhecimentos, Atitudes e Prática em SaúdeRESUMO
Lysine malonylation is a protein posttranslational modification. We present a protocol to generate stable gene-knockdown K562 cell lines through lentiviral infection of a CRISPR interference (CRISPRi) system followed by lysine malonylation measurement using mass spectrometry (MS). We detail guide RNA (gRNA) vector cloning, lentiviral infection, cell line purification, protein digestion, malonyl-lysine enrichment, desalting, and MS acquisition and analysis. For complete details on the use and execution of this protocol, please refer to Zhang et al.1 and Bons et al.2.
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Lisina Acetiltransferases , Lisina , Espectrometria de Massas , Humanos , Células K562 , Lisina/metabolismo , Espectrometria de Massas/métodos , Lisina Acetiltransferases/metabolismo , Lisina Acetiltransferases/genética , Sistemas CRISPR-Cas , Processamento de Proteína Pós-Traducional , Malonatos/metabolismo , RNA Guia de Sistemas CRISPR-Cas/metabolismoRESUMO
Dicarboxylic fatty acids are generated in the liver and kidney in a minor pathway called fatty acid ω-oxidation. The effects of consuming dicarboxylic fatty acids as an alternative source of dietary fat have not been explored. Here, we fed dodecanedioic acid, a 12-carbon dicarboxylic (DC12), to mice at 20% of daily caloric intake for nine weeks. DC12 increased metabolic rate, reduced body fat, reduced liver fat, and improved glucose tolerance. We observed DC12-specific breakdown products in liver, kidney, muscle, heart, and brain, indicating that oral DC12 escaped first-pass liver metabolism and was utilized by many tissues. In tissues expressing the "a" isoform of acyl-CoA oxidase-1 (ACOX1), a key peroxisomal fatty acid oxidation enzyme, DC12 was chain shortened to the TCA cycle intermediate succinyl-CoA. In tissues with low peroxisomal fatty acid oxidation capacity, DC12 was oxidized by mitochondria. In vitro, DC12 was catabolized even by adipose tissue and was not stored intracellularly. We conclude that DC12 and other dicarboxylic acids may be useful for combatting obesity and for treating metabolic disorders.
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The post-translational modification lysine succinylation is implicated in the regulation of various metabolic pathways. However, its biological relevance remains uncertain due to methodological difficulties in determining high-impact succinylation sites. Here, using stable isotope labelling and data-independent acquisition mass spectrometry, we quantified lysine succinylation stoichiometries in mouse livers. Despite the low overall stoichiometry of lysine succinylation, several high-stoichiometry sites were identified, especially upon deletion of the desuccinylase SIRT5. In particular, multiple high-stoichiometry lysine sites identified in argininosuccinate synthase (ASS1), a key enzyme in the urea cycle, are regulated by SIRT5. Mutation of the high-stoichiometry lysine in ASS1 to succinyl-mimetic glutamic acid significantly decreased its enzymatic activity. Metabolomics profiling confirms that SIRT5 deficiency decreases urea cycle activity in liver. Importantly, SIRT5 deficiency compromises ammonia tolerance, which can be reversed by the overexpression of wild-type, but not succinyl-mimetic, ASS1. Therefore, lysine succinylation is functionally important in ammonia metabolism.
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Lisina , Sirtuínas , Camundongos , Animais , Lisina/química , Lisina/metabolismo , Amônia , Sirtuínas/metabolismo , Camundongos Knockout , UreiaRESUMO
Poor bone quality is a major factor in skeletal fragility in elderly individuals. The molecular mechanisms that establish and maintain bone quality, independent of bone mass, are unknown but are thought to be primarily determined by osteocytes. We hypothesize that the age-related decline in bone quality results from the suppression of osteocyte perilacunar/canalicular remodeling (PLR), which maintains bone material properties. We examined bones from young and aged mice with osteocyte-intrinsic repression of TGFß signaling (TßRIIocy-/-) that suppresses PLR. The control aged bone displayed decreased TGFß signaling and PLR, but aging did not worsen the existing PLR suppression in male TßRIIocy-/- bone. This relationship impacted the behavior of collagen material at the nanoscale and tissue scale in macromechanical tests. The effects of age on bone mass, density, and mineral material behavior were independent of osteocytic TGFß. We determined that the decline in bone quality with age arises from the loss of osteocyte function and the loss of TGFß-dependent maintenance of collagen integrity.
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Remodelação Óssea , Osteócitos , Humanos , Idoso , Masculino , Animais , Camundongos , Remodelação Óssea/fisiologia , Colágeno/farmacologia , Envelhecimento , Fator de Crescimento Transformador beta/farmacologiaRESUMO
Dietary restriction (DR) delays aging, but the mechanism remains unclear. We identified polymorphisms in mtd, the fly homolog of OXR1, which influenced lifespan and mtd expression in response to DR. Knockdown in adulthood inhibited DR-mediated lifespan extension in female flies. We found that mtd/OXR1 expression declines with age and it interacts with the retromer, which regulates trafficking of proteins and lipids. Loss of mtd/OXR1 destabilized the retromer, causing improper protein trafficking and endolysosomal defects. Overexpression of retromer genes or pharmacological restabilization with R55 rescued lifespan and neurodegeneration in mtd-deficient flies and endolysosomal defects in fibroblasts from patients with lethal loss-of-function of OXR1 variants. Multi-omic analyses in flies and humans showed that decreased Mtd/OXR1 is associated with aging and neurological diseases. mtd/OXR1 overexpression rescued age-related visual decline and tauopathy in a fly model. Hence, OXR1 plays a conserved role in preserving retromer function and is critical for neuronal health and longevity.
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Envelhecimento , Doenças do Sistema Nervoso , Humanos , Feminino , Envelhecimento/genética , Longevidade/genética , Neurônios/metabolismo , Doenças do Sistema Nervoso/metabolismo , Encéfalo/metabolismo , Restrição Calórica , Proteínas Mitocondriais/metabolismoRESUMO
Acute kidney injury (AKI) manifests as a major health concern, particularly for the elderly. Understanding AKI-related proteome changes is critical for prevention and development of novel therapeutics to recover kidney function and to mitigate the susceptibility for recurrent AKI or development of chronic kidney disease. In this study, mouse kidneys were subjected to ischemia-reperfusion injury, and the contralateral kidneys remained uninjured to enable comparison and assess injury-induced changes in the kidney proteome. A ZenoTOF 7600 mass spectrometer was optimized for data-independent acquisition (DIA) to achieve comprehensive protein identification and quantification. Short microflow gradients and the generation of a deep kidney-specific spectral library allowed for high-throughput, comprehensive protein quantification. Upon AKI, the kidney proteome was completely remodeled, and over half of the 3945 quantified protein groups changed significantly. Downregulated proteins in the injured kidney were involved in energy production, including numerous peroxisomal matrix proteins that function in fatty acid oxidation, such as ACOX1, CAT, EHHADH, ACOT4, ACOT8, and Scp2. Injured kidneys exhibited severely damaged tissues and injury markers. The comprehensive and sensitive kidney-specific DIA-MS assays feature high-throughput analytical capabilities to achieve deep coverage of the kidney proteome, and will serve as useful tools for developing novel therapeutics to remediate kidney function.
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Injúria Renal Aguda , Proteômica , Humanos , Camundongos , Animais , Idoso , Proteoma , Regulação para Baixo , RimRESUMO
X-linked dystonia-parkinsonism (XDP) is a rare neurodegenerative disease endemic to the Philippines. The genetic cause for XDP is an insertion of a SINE-VNTR-Alu (SVA)-type retrotransposon within intron 32 of TATA-binding protein associated factor 1 (TAF1) that causes an alteration of TAF1 splicing, partial intron retention, and decreased transcription. Although TAF1 is expressed in all organs, medium spiny neurons (MSNs) within the striatum are one of the cell types most affected in XDP. To define how mutations in the TAF1 gene lead to MSN vulnerability, we carried out a proteomic analysis of human XDP patient-derived neural stem cells (NSCs) and MSNs derived from induced pluripotent stem cells. NSCs and MSNs were grown in parallel and subjected to quantitative proteomic analysis in data-independent acquisition mode on the Orbitrap Eclipse Tribrid mass spectrometer. Subsequent functional enrichment analysis demonstrated that neurodegenerative disease-related pathways, such as Huntington's disease, spinocerebellar ataxia, cellular senescence, mitochondrial function and RNA binding metabolism, were highly represented. We used weighted coexpression network analysis (WGCNA) of the NSC and MSN proteomic data set to uncover disease-driving network modules. Three of the modules significantly correlated with XDP genotype when compared to the non-affected control and were enriched for DNA helicase and nuclear chromatin assembly, mitochondrial disassembly, RNA location and mRNA processing. Consistent with aberrant mRNA processing, we found splicing and intron retention of TAF1 intron 32 in XDP MSN. We also identified TAF1 as one of the top enriched transcription factors, along with YY1, ATF2, USF1 and MYC. Notably, YY1 has been implicated in genetic forms of dystonia. Overall, our proteomic data set constitutes a valuable resource to understand mechanisms relevant to TAF1 dysregulation and to identify new therapeutic targets for XDP.
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Distonia , Distúrbios Distônicos , Doenças Neurodegenerativas , Transtornos Parkinsonianos , Humanos , Distonia/genética , Distonia/metabolismo , Doenças Neurodegenerativas/metabolismo , Proteômica , Fator de Transcrição TFIID/genética , Distúrbios Distônicos/genética , Distúrbios Distônicos/metabolismo , Neurônios/metabolismo , RNA Mensageiro/metabolismo , Transtornos Parkinsonianos/genética , Transtornos Parkinsonianos/metabolismoRESUMO
SIGNIFICANCE STATEMENT: In this study, we demonstrate that a common, low-cost compound known as octanedioic acid (DC 8 ) can protect mice from kidney damage typically caused by ischemia-reperfusion injury or the chemotherapy drug cisplatin. This compound seems to enhance peroxisomal activity, which is responsible for breaking down fats, without adversely affecting mitochondrial function. DC 8 is not only affordable and easy to administer but also effective. These encouraging findings suggest that DC 8 could potentially be used to assist patients who are at risk of experiencing this type of kidney damage. BACKGROUND: Proximal tubules are rich in peroxisomes, which are damaged during AKI. Previous studies demonstrated that increasing peroxisomal fatty acid oxidation (FAO) is renoprotective, but no therapy has emerged to leverage this mechanism. METHODS: Mice were fed with either a control diet or a diet enriched with dicarboxylic acids, which are peroxisome-specific FAO substrates, then subjected to either ischemia-reperfusion injury-AKI or cisplatin-AKI models. Biochemical, histologic, genetic, and proteomic analyses were performed. RESULTS: Both octanedioic acid (DC 8 ) and dodecanedioic acid (DC 12 ) prevented the rise of AKI markers in mice that were exposed to renal injury. Proteomics analysis demonstrated that DC 8 preserved the peroxisomal and mitochondrial proteomes while inducing extensive remodeling of the lysine succinylome. This latter finding indicates that DC 8 is chain shortened to the anaplerotic substrate succinate and that peroxisomal FAO was increased by DC 8 . CONCLUSIONS: DC 8 supplementation protects kidney mitochondria and peroxisomes and increases peroxisomal FAO, thereby protecting against AKI.
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Injúria Renal Aguda , Ácidos Dicarboxílicos , Suplementos Nutricionais , Traumatismo por Reperfusão , Animais , Humanos , Camundongos , Injúria Renal Aguda/prevenção & controle , Injúria Renal Aguda/patologia , Cisplatino , Ácidos Dicarboxílicos/administração & dosagem , Ácidos Graxos , Proteômica , Traumatismo por Reperfusão/prevenção & controle , Traumatismo por Reperfusão/patologiaRESUMO
The ovarian microenvironment becomes fibrotic and stiff with age, in part due to increased collagen and decreased hyaluronan. However, the extracellular matrix (ECM) is a complex network of hundreds of proteins, glycoproteins, and glycans which are highly tissue specific and undergo pronounced changes with age. To obtain an unbiased and comprehensive profile of age-associated alterations to the murine ovarian proteome and ECM, we used a label-free quantitative proteomic methodology. We validated conditions to enrich for the ECM prior to proteomic analysis. Following analysis by data-independent acquisition (DIA) and quantitative data processing, we observed that both native and ECM-enriched ovaries clustered separately based on age, indicating distinct age-dependent proteomic signatures. We identified a total of 4,721 proteins from both native and ECM-enriched ovaries, of which 383 proteins were significantly altered with advanced age, including 58 ECM proteins. Several ECM proteins upregulated with age have been associated with fibrosis in other organs, but to date their roles in ovarian fibrosis are unknown. Pathways regulating DNA metabolism and translation were downregulated with age, whereas pathways involved in ECM remodeling and immune response were upregulated. Interestingly, immune-related pathways were upregulated with age even in ECM-enriched ovaries, suggesting a novel interplay between the ECM and the immune system. Moreover, we identified putative markers of unique immune cell populations present in the ovary with age. These findings provide evidence from a proteomic perspective that the aging ovary provides a fibroinflammatory milieu, and our study suggests target proteins which may drive these age-associated phenotypes for future investigation.
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Ovário , Proteômica , Feminino , Animais , Camundongos , Ovário/metabolismo , Proteômica/métodos , Matriz Extracelular/metabolismo , Proteínas da Matriz Extracelular/metabolismo , FibroseRESUMO
Dysregulation of cell signaling in chondrocytes and in bone cells, such as osteocytes, osteoblasts, osteoclasts, and an elevated burden of senescent cells in cartilage and bone, are implicated in osteoarthritis (OA). Mass spectrometric analyses provides a crucial molecular tool-kit to understand complex signaling relationships in age-related diseases, such as OA. Here we introduce a novel mass spectrometric workflow to promote proteomic studies of bone. This workflow uses highly specialized steps, including extensive overnight demineralization, pulverization, and incubation for 72 h in 6 M guanidine hydrochloride and EDTA, followed by proteolytic digestion. Analysis on a high-resolution Orbitrap Eclipse and Orbitrap Exploris 480 mass spectrometer using Data-Independent Acquisition (DIA) provides deep coverage of the bone proteome, and preserves post-translational modifications, such as hydroxyproline. A spectral library-free quantification strategy, directDIA, identified and quantified over 2,000 protein groups (with ≥ 2 unique peptides) from calcium-rich bone matrices. Key components identified were proteins of the extracellular matrix (ECM), bone-specific proteins (e.g., secreted protein acidic and cysteine rich, SPARC, and bone sialoprotein 2, IBSP), and signaling proteins (e.g., transforming growth factor beta-2, TGFB2), and lysyl oxidase homolog 2 (LOXL2), an important protein in collagen crosslinking. Post-translational modifications (PTMs) were identified without the need for specific enrichment. This includes collagen hydroxyproline modifications, chemical modifications for collagen self-assembly and network formation. Multiple senescence factors were identified, such as complement component 3 (C3) protein of the complement system and many matrix metalloproteinases, that might be monitored during age-related bone disease progression. Our innovative workflow yields in-depth protein coverage and quantification strategies to discover underlying biological mechanisms of bone aging and to provide tools to monitor therapeutic interventions. These novel tools to monitor the bone proteome open novel horizons to investigate bone-specific diseases, many of which are age-related.
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Osteoartrite , Proteoma , Humanos , Proteoma/análise , Proteômica/métodos , Hidroxiprolina , Osso e Ossos/metabolismo , Osteoartrite/metabolismo , ColágenoRESUMO
Protein post-translational modifications (PTMs) are crucial and dynamic players in a large variety of cellular processes and signaling. Proteomic technologies have emerged as the method of choice to profile PTMs. However, these analyses remain challenging due to potential low PTM stoichiometry, the presence of multiple PTMs per proteolytic peptide, PTM site localization of isobaric peptides, and neutral losses. Collision-induced dissociation (CID) is commonly used to characterize PTMs, but the application of collision energy can lead to neutral losses and incomplete peptide sequencing for labile PTM groups. In this study, we assessed the performance of an alternative fragmentation, electron activated dissociation (EAD), to characterize, site localize, and quantify peptides with labile modifications in comparison to CID, both operated on a recently introduced fast-scanning quadrupole-time-of-flight (QqTOF) mass spectrometer. We analyzed biologically relevant phosphorylated, succinylated, malonylated, and acetylated synthetic peptides using targeted parallel reaction monitoring (PRM or MRMHR) assays. We report that electron-based fragmentation preserves the malonyl group from neutral losses. The novel tunable EAD kinetic energy maintained labile modification integrity and provided better peptide sequence coverage with strong PTM-site localization fragment ions. Activation of a novel trap-and-release technology significantly improves the duty cycle and provided significant MS/MS sensitivity gains by an average of 6-11-fold for EAD analyses. Evaluation of the quantitative EAD PRM workflows revealed high reproducibility with coefficients of variation of â¼2-7%, as well as very good linearity and quantification accuracy. This novel workflow combining EAD and trap-and-release technology provides high sensitivity, alternative fragmentation information to achieve confident PTM characterization and quantification.
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Elétrons , Espectrometria de Massas em Tandem , Reprodutibilidade dos Testes , Proteômica/métodos , Proteínas/química , Processamento de Proteína Pós-Traducional , Peptídeos/químicaRESUMO
During cellular senescence, persistent growth arrest and changes in protein expression programs are accompanied by a senescence-associated secretory phenotype (SASP). In this study, we detected the upregulation of the SASP-related protein dipeptidyl peptidase 4 (DDP4) in human primary lung cells rendered senescent by exposure to ionizing radiation. DPP4 is an exopeptidase that plays a crucial role in the cleavage of various proteins, resulting in the loss of N-terminal dipeptides and proinflammatory effects. Interestingly, our data revealed an association between severe coronavirus disease 2019 (COVID-19) and DDP4, namely that DPP4 levels increased in the plasma of patients with COVID-19 and were correlated with age and disease progression. Although we could not determine the direct effect of DDP4 on viral replication, mechanistic studies in cell culture revealed a negative impact on the expression of the tight junction protein zonula occludens-1 (ZO-1), which contributes to epithelial barrier function. Mass spectrometry analysis indicated that DPP4 overexpressing cells exhibited a decrease in ZO-1 and increased expression of pro-inflammatory cytokines and chemokines. By investigating the effect of DPP4 on the barrier function of human primary cells, we detected an increase in ZO-1 using DPP4 inhibitors. These results provide an important contribution to our understanding of DPP4 in the context of senescence, suggesting that DPP4 plays a major role as part of the SASP. Our results provide evidence that cellular senescence, a hallmark of aging, has an important impact on respiratory infections.
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Esophageal adenocarcinoma arises from Barrett's esophagus, a precancerous metaplastic replacement of squamous by columnar epithelium in response to chronic inflammation. Multi-omics profiling, integrating single-cell transcriptomics, extracellular matrix proteomics, tissue-mechanics and spatial proteomics of 64 samples from 12 patients' paths of progression from squamous epithelium through metaplasia, dysplasia to adenocarcinoma, revealed shared and patient-specific progression characteristics. The classic metaplastic replacement of epithelial cells was paralleled by metaplastic changes in stromal cells, ECM and tissue stiffness. Strikingly, this change in tissue state at metaplasia was already accompanied by appearance of fibroblasts with characteristics of carcinoma-associated fibroblasts and of an NK cell-associated immunosuppressive microenvironment. Thus, Barrett's esophagus progresses as a coordinated multi-component system, supporting treatment paradigms that go beyond targeting cancerous cells to incorporating stromal reprogramming.
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Non-alcoholic fatty liver disease (NAFLD) is a liver manifestation of metabolic syndrome, and is estimated to affect one billion individuals worldwide. An increased intake of a high-fat diet (HFD) and sugar-sweetened beverages are risk-factors for NAFLD development, but how their combined intake promotes progression to a more severe form of liver injury is unknown. Here we show that fructose metabolism via ketohexokinase (KHK) C isoform leads to unresolved endoplasmic reticulum (ER) stress when coupled with a HFD intake. Conversely, a liver-specific knockdown of KHK in mice consuming fructose on a HFD is adequate to improve the NAFLD activity score and exert a profound effect on the hepatic transcriptome. Overexpression of KHK-C in cultured hepatocytes is sufficient to induce ER stress in fructose free media. Upregulation of KHK-C is also observed in mice with genetically induced obesity or metabolic dysfunction, whereas KHK knockdown in these mice improves metabolic function. Additionally, in over 100 inbred strains of male or female mice hepatic KHK expression correlates positively with adiposity, insulin resistance, and liver triglycerides. Similarly, in 241 human subjects and their controls, hepatic Khk expression is upregulated in early, but not late stages of NAFLD. In summary, we describe a novel role of KHK-C in triggering ER stress, which offers a mechanistic understanding of how the combined intake of fructose and a HFD propagates the development of metabolic complications.
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Hepatopatia Gordurosa não Alcoólica , Animais , Feminino , Humanos , Masculino , Camundongos , Dieta Hiperlipídica/efeitos adversos , Frutoquinases/genética , Frutoquinases/metabolismo , Frutose/farmacologia , Lipogênese/fisiologia , Fígado/metabolismo , Modelos Genéticos , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/metabolismo , Obesidade/metabolismoRESUMO
Acute kidney injury (AKI) manifests as a major health concern, particularly for the elderly. Understanding AKI-related proteome changes is critical for prevention and development of novel therapeutics to recover kidney function and to mitigate the susceptibility for recurrent AKI or development of chronic kidney disease. In this study, mouse kidneys were subjected to ischemia-reperfusion injury, and the contralateral kidneys remained uninjured to enable comparison and assess injury-induced changes in the kidney proteome. A fast-acquisition rate ZenoTOF 7600 mass spectrometer was introduced for data-independent acquisition (DIA) for comprehensive protein identification and quantification. Short microflow gradients and the generation of a deep kidney-specific spectral library allowed for high-throughput, comprehensive protein quantification. Upon AKI, the kidney proteome was completely remodeled, and over half of the 3,945 quantified protein groups changed significantly. Downregulated proteins in the injured kidney were involved in energy production, including numerous peroxisomal matrix proteins that function in fatty acid oxidation, such as ACOX1, CAT, EHHADH, ACOT4, ACOT8, and Scp2. Injured mice exhibited severely declined health. The comprehensive and sensitive kidney-specific DIA assays highlighted here feature high-throughput analytical capabilities to achieve deep coverage of the kidney proteome and will serve as useful tools for developing novel therapeutics to remediate kidney function.
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The posttranslational modification lysine malonylation is found in many proteins, including histones. However, it remains unclear whether histone malonylation is regulated or functionally relevant. Here, we report that availability of malonyl-co-enzyme A (malonyl-CoA), an endogenous malonyl donor, affects lysine malonylation, and that the deacylase SIRT5 selectively reduces malonylation of histones. To determine if histone malonylation is enzymatically catalyzed, we knocked down each of the 22 lysine acetyltransferases (KATs) to test their malonyltransferase potential. KAT2A knockdown in particular reduced histone malonylation levels. By mass spectrometry, H2B_K5 was highly malonylated and regulated by SIRT5 in mouse brain and liver. Acetyl-CoA carboxylase (ACC), the malonyl-CoA producing enzyme, was partly localized in the nucleolus, and histone malonylation increased nucleolar area and ribosomal RNA expression. Levels of global lysine malonylation and ACC expression were higher in older mouse brains than younger mice. These experiments highlight the role of histone malonylation in ribosomal gene expression.
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Huntington's disease (HD) is a neurodegenerative disease caused by a CAG repeat expansion in the Huntingtin (HTT) gene. The resulting polyglutamine (polyQ) tract alters the function of the HTT protein. Although HTT is expressed in different tissues, the medium-spiny projection neurons (MSNs) in the striatum are particularly vulnerable in HD. Thus, we sought to define the proteome of human HD patient-derived MSNs. We differentiated HD72-induced pluripotent stem cells and isogenic controls into MSNs and carried out quantitative proteomic analysis. Using data-dependent acquisitions with FAIMS for label-free quantification on the Orbitrap Lumos mass spectrometer, we identified 6323 proteins with at least two unique peptides. Of these, 901 proteins were altered significantly more in the HD72-MSNs than in isogenic controls. Functional enrichment analysis of upregulated proteins demonstrated extracellular matrix and DNA signaling (DNA replication pathway, double-strand break repair, G1/S transition) with the highest significance. Conversely, processes associated with the downregulated proteins included neurogenesis-axogenesis, the brain-derived neurotrophic factor-signaling pathway, Ephrin-A:EphA pathway, regulation of synaptic plasticity, triglyceride homeostasis cholesterol, plasmid lipoprotein particle immune response, interferon-γ signaling, immune system major histocompatibility complex, lipid metabolism, and cellular response to stimulus. Moreover, proteins involved in the formation and maintenance of axons, dendrites, and synapses (e.g., septin protein members) were dysregulated in HD72-MSNs. Importantly, lipid metabolism pathways were altered, and using quantitative image analysis, we found that lipid droplets accumulated in the HD72-MSN, suggesting a deficit in the turnover of lipids possibly through lipophagy. Our proteomics analysis of HD72-MSNs identified relevant pathways that are altered in MSNs and confirm current and new therapeutic targets for HD.