| Background:Thyroid hormone,synthesized and secreted by the thyroid gland,plays a crucial role in the normal development,differentiation and metabolism of human beings.Disturbances in thyroid homeostasis may result in several thyroid disorders such as hypothyroidism.Hypothyroidism is a disorder of the endocrine system that results from low production of thyroid hormone thyroxine(TT4)from the thyroid gland.This leads to metabolic dysfunction because thyroid hormone is an essential regulator of glucose-lipid metabolism and energy homeostasis.Hypothyroidism also leads to a rise in the concentration of thyrotropin(TSH)through the negative feedback of the hypothalamus-pituitary-thyroid axis.Primary hypothyroidism,caused by a dysfunction of the thyroid itself,is the main cause of hypothyroidism.The onset of hypothyroidism in adults is often subtle presenting with a range of nonspecific symptoms.However,severe untreated hypothyroidism may result in poor prognoses,such as heart failure,psychosis,and even coma.So far,possible measures for the treatment of hypothyroidism include improvements in symptoms and prevention of adverse event.Undoubtedly,hypothyroidism places a huge burden on the economy and greatly lowers the quality of the patient’s life.Thus,it is essential to investigate the pathogenesis and explore novel treatment strategies.Post-translational modifications(PTMs),which refer to covalent modifications introduced to amino acids of proteins either enzymatically or non-enzymatically,are key mechanisms for increasing proteomic diversity and exert crucial effects on biological function in a variety of species.PTMs modulate protein properties through proteolytic cleavage of regulatory subunits,addition of a modified group to one or more amino acids,or degradation of entire proteins,thus determining activity status,localization,turnover and interactions with other molecules.Lysine,as the most common post-translation modified amino acid residue,is critical for the formation of protein structures and regulation of protein functions.Lysine residues can be subjected to various PTMs,such as methylation,acetylation,biotinylation,ubiquitination,ubiquitin-like modifications,propionylation and butyrylation.These lysine PTMs play important roles in cellular physiology and pathology,thereby influencing almost all aspects of cell biology and pathogenesis.Lysine succinylation is one of significant posttranslational protein modifications,which can ℃cur on cytosolic,nuclear and mitochondria proteins by a nonenzymatic chemical reaction and enzymatic catalytic reaction.The former succinylation originates directly from succinyl-CoA,which can be generated from the TCA cycle,lipids,and amino acid metabolism,and the enzymatic succinylation of lysine take place by lysine succinyltransferase.Lysine succinylation has been identified and verified as an important form of PTM and is involved in a diverse array of cellular functions associated with thyroid diseases.One research has found that acetylated levels of lysine at positions 9-14 of H3 histone(H3K9-K14ac)were significantly higher in follicular adenomas,papillary thyroid carcinomas,follicular thyroid carcinomas and undifferentiated carcinomas than in normal tissues.There is one investigation report that oxidative modifications of Cys10 seemed to affect the binding of T3 to transthyretin and provided a sensitive mechanism for adjusting thyroid hormone availability.The role of lysine succinylation in thyroid diseases is still unknown,and needs further investigation.Our previous studies have found that excess intake of dietary fat induced decreased serum TT4 and FT4 concentration in parallel with elevated serum TSH concentration,as well as abnormal morphology and lipid profile change of thyroid gland,providing evidence for the correlation between lipid profiles and organ function,as well as a new prospect for understanding the pathogenesis of hypothyroidism.However,the underlying molecular mechanism remains unclear and needs further investigation.Liquid chromatography-tandem mass spectrometry(LC-MS/MS)-based proteomic analysis has emerged as a powerful tool for studying disease mechanisms due to its high throughput and accuracy.In the present study,to investigate which PTM played a role in hypothyroxinemia,we detected four types of lysine acylations by western blotting,including succinylation,crotonylation,2-hydroxybutyrylaion and malonylation.Significant change of lysine succinylaiton was observed in HFD group relative to control group.Then,we carried out label-free based quantitative analyses on the global proteome and lysine succinylome of thyroid tissues in the HFD-induced thyroid dysfunction rat model using LC-MS/MS methods.A series of bioinformatics analyses were conducted to explore the underlying molecular mechanisms of hypothyroxinemia,and lysine succinylation’s involvement.Objectives:1.We aimed to explore the association between lysine succinylation and hypothyroidism2.To investigate the relationship between lysine succinylation and hypothyroidism3.Whether there is a correlation between mitochondrial function and the level of mitochondrial protein succinylation modification;4.Explore the potential molecular mechanisms of hypothyroidism and evaluate potential diagnostic biomarkers and therapeutic targets.Methods:1.Twenty-six male SD rats at 6-week-old were maintained at a constant temperature and humidity and were rendered a 12h:12h light-darkness cycle.The rats were randomly and equally divided into the CD control group(n=13)and the HFD study group(n=13).The animals were weighed weekly and fed for 24 weeks.At the 24th week of feeding,all rats were fasted for 12 hours before sacrifice.All experiment prot℃ ols were approved by the Animal Ethics Committee of Shandong Provincial Hospital,Shandong University.2.Thyroid tissue collection of rats:Thyroid tissue was removed from the hypothyroidism experimental rats that were sacrificed,and mixed thyroid tissue samples from 4,4 and 5 rats were taken as 3 biological replicates,respectively.3.Analysis of serum thyroid function parameters:Serum TT4,FT4 and TSH concentrations were measured at the end of the collection experiment.Blood collection by inferior vena cava puncture.Serum TT4,FT4 and TSH were determined by ELISA.All procedures follow the instructions provided by the manufacturer.4.Quantitative detection and analysis of total proteome and succinylated proteome4.1 Total protein extraction:The sample was grinded by liquid nitrogen into cell powder and then transferred to a 5-mL centrifuge tube.After that,four volumes of lysis buffer(8 M urea,1%Protease Inhibitor Cocktail)was added to.the cell powder,followed by sonication three times on ice using a high intensity ultrasonic processor.The remaining debris was removed by centrifugation at 12,000 g at 4℃for 10 min.Finally,the supernatant was collected and the protein concentration was determined with BCA kit according to the manufacturer’s instructions.4.2 Trypsin Digestion:For digestion,the protein solution was reduced with 5 mM dithiothreitol for 30 min at 56℃ and alkylated with 11 mM iodoacetamide for 15 min at room temperature in darkness.The protein sample was then diluted by adding 100 mM NH4HCO3 to urea concentration less than 2M.Finally,trypsin was added at 1:50 trypsin-to-protein mass ratio for the first digestion overnight and 1:100 trypsin-to-protein mass ratio for a second 4 h-digestion.4.3 Pan antibody-based PTM enrichment:To enrich succinylated modified peptides,tryptic peptides dissolved in NETN buffer were incubated with pre-washed antibody beads at 4℃ overnight with gentle shaking-Then the beads were washed four times with NETN buffer and twice with H2O.The bound peptides were eluted from the beads with 0.1%trifluoroacetic acid.Finally,the eluted fractions were combined and vacuum-dried.For LC-MS/MS analysis,the resulting peptides were desalted with C18 ZipTips(Millipore)according to the manufacturer’s instructions.4.4 HPLC Fractionation(optional):The tryptic peptides were fractionated into fractions by high pH reverse-phase HPLC using Thermo Betasil C18 column(5μm particles,10 mm ID,250 mm length).Briefly,peptides were first separated with a gradient of 8%to 32%acetonitrile(pH 9.0)over 60 min into 60 fractions.Then,the peptides were combined into 4 fractions and dried by vacuum centrifuging.4.5 LC-MS/MS Analysis:The tryptic peptides were dissolved in 0.1%formic acid(solvent A),directly loaded onto a home-made reversed-phase analytical column(15-cm length,75 μm i.d.).The gradient was comprised of an increase from 6%to 23%solvent B(0.1%formic acid in 98%acetonitrile)over 26 min,23%to 35%in 8 min and climbing to 80%in 3 min then holding at 80%for the last 3 min,all at a constant flow rate of 400nL/min on an EASY-nLC 1000 UPLC system.The peptides were subjected to NSI source followed by tandem mass spectrometry(MS/MS)in Q ExactiveTM Plus(Thermo)coupled online to the UPLC.The electrospray voltage applied was 2.0 kV.The m/z scan range was 350 to 1800 for full scan,and intact peptides were detected in the Orbitrap at a resolution of 70,000.Peptides were then selected for MS/MS using NCE setting as 28 and the fragments were detected in the Orbitrap at a resolution of 17,500.A data-dependent procedure that alternated between one MS scan followed by 20 MS/MS scans with 15.0s dynamic exclusion.Automatic gain control(AGC)was set at 5E4.The maximum injection time was set as 200 ms and 100 ms for peptides and succinylated peptides,respectively.4.6 Proteome and succinylome quantification analysis:The acquired MS/MS data were processed and analyzed with the Maxquant search engine(v.1.5.2.8).Tandem mass spectra were searched against the UniProt Rat database(29,795 sequences)concatenated with protein sequences of common contaminants(such as hemoglobin,keratin,and lactoglobulin)and a reverse decoy database.Trypsin/P was specified as a cleavage enzyme allowing up to two missing cleavages as well as five modifications per peptide.The mass error was set as 20 ppm in the first search and 5 ppm in the main search for precursor ions and 0.02 Da for fragment ions.Carbamidomethyl on cysteine was specified as a fixed modification,whereas acetylation on the protein N-terminal and oxidation on methionine were specified as variable modifications.For the succinylome analysis,succinylation on lysine was also set as variable modifications.The false discovery rate(FDR)thresholds for the identification of PTM levels,peptides and proteins were adjusted to 0.01.The minimum length of peptide was set as 7 amino acid residues.The quantitative values of each sample in three replicates were obtained by LFQ intensity.The first step is to calculate the differential concentration of the protein between the two samples.First,calculate the average value of the quantitative values of each sample in multiple replicates,and then calculate the ratio of the average values between the two samples.The ratio is used as the final quantitation.For normalization to succinylated peptides,the naked intensities of succinylated peptides were first measured,and then were divided by corresponding protein intensities。4.7 Mass spectrometry quality control test:The mass error is centered at 0 and concentrated in the range of less than 10 PPM,indicating that the mass error meets the requirements.Secondly,the length of most peptides is distributed between 8-20 amino acid residues,which conforms to the law of trypsin digestion of peptides,indicating that the sample preparation meets the standards.4.8 Gene ontology(GO)analyses were derived from the UniProt-GOA database(http://www.ebi.ac.uk/GOA/),and GO annotation(http://geneontology.org/)to classify all identified proteins into three categories:biological process,cellular component and molecular function.A cutoff of absolute fold change ≥1.5 was employed to identify the differentially expressed proteins.The functional pathways of all quantified proteins or succinylated proteins were annotated by performing the Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis(http://www.genome.jp/kegg/).The functional enrichment analyses were carried out to reveal the differentially expressed proteins enriched in all identified proteins and succinylated proteins.When performing the bioinformatics analysis,a two-tailed Fisher’s exact test was performed and a corrected p value<0.05 was considered significant.4.9 Differential proteins and succinylated proteins PPI analysis:To better understand the function of lysine succinylation and hypothyroidism pathology,the differential proteins and succinylated proteins were subjected to a protein-protein interaction (PPI)network analysis using the STRING database(Vision 11.0).STRING defines a metric called ’confidence score’ to define interaction confidence;we fetched all interactions that had a confidence score ≥0.7(high confidence).A network of protein-protein interactions was generated,and clustered with Markov cluster(MCL)algorithm,which was then visualized using the Cytoscape program(Vision 3.7).5.In vitro culture of human normal thyroid epithelial cell line Nthy-ORI3-1(ECACC,Wiltshire,UK):in RPMI-1640,cultures containing 10%fetal bovine serum(fetal bovine serum,penicillin(100 IU/mL),streptomycin(100 IU/mL),and L-glutamine(2 mM)were cultured at 37℃ in a humidified incubator containing 5%CO2.6.Measurement of mitochondrial OCR:The measurement of OCR was performed using an XF96 Analyzer(Seahorse Bioscience,USA)according to the manufacturer’s instructions.In brief,Upproximately 7×103 cells per well were seeded onto the Seahorse XF96 cell culture microplate(Seahorse Bioscience,USA)and cultured for 24 hours.After the administration of PA(0.2 mM)and NAM(10 mM),the cells were cultured for another 24 hours.Then,the microplate was incubated in the low-buffered and nonbicarbonated assay medium(XF base medium with 2 mM glutamine,1 mM sodium pyruvate and 25 mM glucose)in a non-CO2 incubator at 37℃ for 1 hours.Then,OCRs were measured in an XFe 96 extracellular flux analyzer(Seahorse Bioscience)for 3 periods with 3 min of mixing in each cycle.The results were normalized to the corresponding total protein concentration per well..7.Immunoprecipitation:Cells were cultured for 24 hours.After the administration of PA(0.2 mM)and NAM(10 mM),the cells were cultured for another 24 hours.Cells were scraped off from plates and cell lysates were centrifuged at 12000 g for 15 minutes.Supernatants were collected and protein concentration was measured by BCA kit.500ug of total protein was used for IP.8.Western Blotting:In order to verify succinylation levels of key protein,equal amounts of proteins from IP product from different samples were subjected to 8%SDS-PAGE,followed by electrotransfer from the gel to polyvinylidene difluoride membranes(Millipore).The membrane was blocked with 5%(w/v)skim milk in TBST and incubated overnight at 4℃with the pan anti-succinyl lysine antibody,anti-sirt5 antibody,antibody IDH2,anti-GAPDH antibody.Immune complexes were detected using Amersham Imager 600.The same membrane was re-incubated with anti-GAPDH antibodies.The GAPDH protein was used as a loading control for total proteins.Results:1.Serum TT4,FT4 and TSH were measured to observe thyroid function.The concentrations of TT4 and FT4 in HFD group decreased(p<0.01),increased TSH concentration(p<0.01).The results showed that the rat model of hypothyroidism was successful.2.We performed quantitative analysis on lysine succinylome in thyroids of rats with hypothyroxinemia which was induced through the administration of a high-fat diet.A total of 3,869 proteins were identified,among which 2,982 proteins were quantifiable.A total of 129 proteins were quantified as differentially expressed proteins between the two groups,including 69 up-regulated and 60 down-regulated proteins.Differentially expressed proteins were filtered with a fold-change threshold>1.5(p-value<0.05)for upregulation and a ratio<1/1.5(p-value<0.05)for downregulation in the thyroid of the rats with HFD relative to the control.3.Comparing rat thyroid proteome data with Protein Atlas publica database,some hallmark proteins in thyroid tissue can be found in our detected results.thyroglobulin(Tg)is the most abundant among all proteins in MS detected,which acts as a substrate for the synthesis of the thyroid hormones thyroxine(T4)and triiodothyronine(T3).Thyroid peroxidase is also high level expressed,which is is involved in the pathway thyroid hormone biosynthesis.Other specific proteins like iodotyrosine deiodinase 1,Calcitonin gene-related peptide 2 in Protein Atlas thyroid database exist in our protein detected table too.These proved our mass spectrum proteomics qualification and quantification is credible.4.Enrichment analysis of the differentially expressed proteins.Among cellular components,the expression of proteins localized to the ribosome and ribosome subunit increased,while the expression of proteins localized to the mitochondrion and ATPase complex significantly decreased.Among the molecular functions,the structural constituents of ribosomes and structural molecule activity were upregulated,while the ATPase activity was downregulated.In the biological process category,the upregulated proteins were markedly enriched in several metabolic processes.5.The KEGG pathway enrichment analyses were also performed to further investigate the functions of these differentially expressed proteins.Consistent with the results of GO analyses,the results show that the ribosome pathway was the most prominent enriched pathway for upregulated proteins.Meanwhile,downregulated proteins were observed to be enriched in the thyroid hormone signaling pathway and thyroid hormone synthesis,indicating that these pathways may play essential roles in the development of hypothyroxinemia.6.Label-free based quantitative lysine succinylome analysis was performed using antibody-based affinity enrichment,followed by the LC-MS/MS analysis.685 succinylation sites in 250 proteins were identified,among which 621 succinylation sites on 229 proteins were quantified and normalized to the proteome data.172 succinylation sites corresponding to 104 proteins showed different succinylation levels in three repeated experiments(7 up-regulated succinylated sites on 5 proteins,165 downregulated succinylated sites on 99 proteins,HFD group compared with CD group).Differentially expressed succinylated proteins were filtered with a fold-change threshold>1.5(p-value<0.05)for upregulation and a ratio<1/1.5(p-value<0.05)for downregulation in the thyroid of the rats with HFD relative to the control.7.Compared with CD group,most lysine succinylation on different proteins undergo down-regulated change in HFD group,moreover,these succinylated proteins are located in mitochondria,including ATP synthase complex,isocitrate dehydrogenase(IDH2).However,several lysine sites succinylation are up-regulated on thyroglobulin.8.Enrichment analysis of the differentially changed succinylated proteins:Among cellular components,the downregulated succinylated proteins mainly exhibited the tricarboxylic acid cycle(TCA cycle)enzyme complex.The most enriched molecular function was ligase activity.In the biological process category,proteins related to the system process that responded to the lipids were enriched among the upregulated succinylated proteins,while proteins related to the TCA metabolic process,cellular respiration,TCA cycle,citrate metabolic process,energy derivation by oxidation of organic compounds and aerobic respiration were enriched among the downregulated succinylated proteins.The KEGG pathway enrichment analysis indicated that the citrate cycle(TCA cycle)was the most enriched pathway among the downregulated succinylated proteins.9.Differential proteins and succinylated proteins PPI analysis:Differential protein-protein net analysis reveals that ribosome proteins interaction is highly clustered,and the proteins of this PPI net cluster is characterized with up-regulated co-expression.The differential succinylated proteins interacting net show three function clusters:citrate cycle(TCA cycle),ATP synthase complex,valine,leucine and isoleucine degradation,and all of them are down-regulated succinylation.10.Results of cellular mitochondrial oxygen consumption rate(OCR):In order to further investigate the role of fatty acids in mitochondrial function and to avoid common factors in vivo,OCR representing mitochondrial function level in normal thyroid epithelial cells was measured.After 24 h treatment with palmitic acid,mitochondrial OCR associated with basal respiration,ATP production,and maximal respiration was significantly passivated(p<0.05).The addition of palmitic acid and desuccinylase inhibitor to human thyroid epithelial cells reversed the above changes(p<0.05).11.Verification experiment of representative protein succinylation level:To determine the changes of protein succinylation in human normal thyroid epithelial cell lines,protein immunoprecipitation analysis was performed,and the isocitrate dehydrogenase 2(IDH2)succinylation level was detected by western blotting.IDH2 is considered to be a key enzyme in the Krebs cycle.Nthy-ori3-1 cells were treated with either a deacetylation inhibitor(NAM),palmitic acid(PA),or both NAM and PA.The results showed that IDH2 was succinylated and PA inhibited its succinylation.Although there was no significant change in the succinylation level of IDH2 under NAM treatment,both PA and NAM treatment restored the succinylation level of IDH2 compared with that only with PA treatment.Conclusions:1.By inducing rats with a high-fat diet,we successfully established an animal model of hypothyroidism(hypothyroidism)in culture.2.We used the quantitative proteomics strategy and the succinylated peptide antibody-based enrichment strategy to study the succinylation in thyroid tissues,identified 129 differentially expressed proteins and 172 differentially expressed succinylation sites,and found that the modification level of succinylation sites was significantly down-regulated rather than up-regulated.3.Through bioinformatics in-depth mining analysis,we analyzed the significant difference in the modification level of succinylation.Proteins significantly changed in succinylation level are mainly located in mitochondria and related to mitochondrial function.4.Through the detection of OCR representing the mitochondrial function level in normal thyroid epithelial cells,it was found that the mitochondrial OCR associated with basic respiration,ATP production and maximum respiration was significantly passivated after 24 h treatment with palmitic acid(p<0.05).The addition of palmitic acid and desuccinylase inhibitor to human thyroid epithelial cells reversed the above changes(p<0.05).5.By immunoprecipitation and Western blot experiments,the expression level of citric acid dehydrogenase 2(IDH2),a key protein in mitochondria,was basically unchanged after cell culture with palmitate,while the level of IDH2 succinylation modification was significantly reduced,which was consistent with the results of the succinylation group in our hypoxia rat animal model.6.Based on the results of OCR experimental data,immunoprecipitation and Western blot experiments to verify the level of succinylation of key mitochondrial proteins IDH2,the function of mitochondria is positively correlated with the level of succinylation of key proteins.The down-regulated lysine succinylated proteins are mainly located in the mitochondria and co-occur with the decrease of mitochondrial activity. |
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