RESUMO
Amino acids (AAs) in the d-form are involved in multiple pivotal neurological processes, although their l-enantiomers are most commonly found. Mass spectrometry-based analysis of low-abundance d-AAs has been hindered by challenging enantiomeric separation from l-AAs, low sensitivity for detection, and lack of suitable internal standards for accurate quantification. To address these critical gaps, N,N-dimethyl-l-leucine (l-DiLeu) tags are first validated as novel chiral derivatization reagents for chromatographic separation of 20 pairs of d/l-AAs, allowing the construction of a 4-plex isobaric labeling strategy for enantiomer-resolved quantification through single step tagging. Additionally, the creative design of N,N-dimethyl-d-leucine (d-DiLeu) reagents offers an alternative approach to generate analytically equivalent internal references of d-AAs using d-DiLeu-labeled l-AAs. By labeling cost-effective l-AA standards using paired d- and l-DiLeu, this approach not only enables absolute quantitation of both d-AAs and l-AAs from complex biological matrices with enhanced precision but also significantly boosts the combined signal intensities from all isobaric channels, greatly improving the detection and quantitation of low-abundance AAs, particularly d-AAs. We term this quantitative strategy CHRISTMAS, which stands for chiral pair isobaric labeling strategy for multiplexed absolute quantitation. Leveraging the ion mobility collision cross section (CCS) alignment, interferences from coeluting isomers/isobars are effectively filtered out to provide improved quantitative accuracy. From wild-type and Alzheimer's disease (AD) mouse brains, we successfully quantified 20 l-AAs and 5 d-AAs. The significant presence and differential trends of certain d-AAs compared to those of their l-counterparts provide valuable insights into the involvement of d-AAs in aging, AD progression, and neurodegeneration.
Assuntos
Aminoácidos , Proteômica , Animais , Camundongos , Aminoácidos/análise , Proteômica/métodos , Leucina/química , Aminas , Cromatografia Líquida/métodosRESUMO
N ε-lysine acetylation within the lumen of the endoplasmic reticulum is a recently characterized protein quality control system that positively selects properly folded glycoproteins in the early secretory pathway. Overexpression of the endoplasmic reticulum acetyl-CoA transporter AT-1 in mouse forebrain neurons results in increased dendritic branching, spine formation and an autistic-like phenotype that is attributed to altered glycoprotein flux through the secretory pathway. AT-1 overexpressing neurons maintain the cytosolic pool of acetyl-CoA by upregulation of SLC25A1, the mitochondrial citrate/malate antiporter and ATP citrate lyase, which converts cytosolic citrate into acetyl-CoA. All three genes have been associated with autism spectrum disorder, suggesting that aberrant cytosolic-to-endoplasmic reticulum flux of acetyl-CoA can be a mechanistic driver for the development of autism spectrum disorder. We therefore generated a SLC25A1 neuron transgenic mouse with overexpression specifically in the forebrain neurons. The mice displayed autistic-like behaviours with a jumping stereotypy. They exhibited increased steady-state levels of citrate and acetyl-CoA, disrupted white matter integrity with activated microglia and altered synaptic plasticity and morphology. Finally, quantitative proteomic and acetyl-proteomic analyses revealed differential adaptations in the hippocampus and cortex. Overall, our study reinforces the connection between aberrant cytosolic-to-endoplasmic reticulum acetyl-CoA flux and the development of an autistic-like phenotype.
Assuntos
Transtorno do Espectro Autista , Transtorno Autístico , Transportadores de Ânions Orgânicos , Acetilcoenzima A/genética , Acetilcoenzima A/metabolismo , Animais , Transtorno do Espectro Autista/genética , Transtorno Autístico/genética , Ácido Cítrico , Humanos , Camundongos , Proteínas Mitocondriais/genética , Neurônios/metabolismo , Transportadores de Ânions Orgânicos/genética , Fenótipo , ProteômicaRESUMO
Nε-lysine acetylation of nascent glycoproteins within the endoplasmic reticulum (ER) lumen regulates the efficiency of the secretory pathway. The ER acetylation machinery consists of the membrane transporter, acetyl-CoA transporter 1 (AT-1/SLC33A1), and two acetyltransferases, ATase1/NAT8B and ATase2/NAT8. Dysfunctional ER acetylation is associated with severe neurological diseases with duplication of AT-1/SLC33A1 being associated with autism spectrum disorder, intellectual disability, and dysmorphism. Neuron-specific AT-1 over-expression in the mouse alters neuron morphology and function, causing an autism-like phenotype, indicating that ER acetylation plays a key role in neurophysiology. As such, characterizing the molecular mechanisms that regulate the acetylation machinery could reveal critical information about its biology. By using structure-biochemistry approaches, we discovered that ATase1 and ATase2 share enzymatic properties but differ in that ATase1 is post-translationally regulated via acetylation. Furthermore, gene expression studies revealed that the promoters of AT-1, ATase1, and ATase2 contain functional binding sites for the neuron-related transcription factors cAMP response element-binding protein and the immediate-early genes c-FOS and c-JUN, and that ATase1 and ATase2 exhibit additional modes of transcriptional regulation relevant to aging and Alzheimer's disease. In vivo rodent gene expression experiments revealed that Atase2 is specifically induced following activity-dependent events. Finally, over-expression of either ATase1 or ATase2 was sufficient to increase the engagement of the secretory pathway in PC12 cells. Our results indicate important regulatory roles for ATase1 and ATase2 in neuron function with induction of ATase2 expression potentially serving as a critical event that adjusts the efficiency of the secretory pathway for activity-dependent neuronal functions.
Assuntos
Acetiltransferases/metabolismo , Retículo Endoplasmático/metabolismo , Plasticidade Neuronal/fisiologia , Neurônios/metabolismo , Via Secretória/fisiologia , Acetilação , Animais , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células PC12 , Processamento de Proteína Pós-Traducional , Ratos , Ratos Endogâmicos F344 , Transcrição GênicaRESUMO
Protein post-translational modifications (PTMs) take many shapes, have many effects and are necessary for cellular homeostasis. One of these PTMs, Nε-lysine acetylation, was thought to occur only in the mitochondria, cytosol and nucleus, but this paradigm was challenged in the past decade with the discovery of lysine acetylation in the lumen of the endoplasmic reticulum (ER). This process is governed by the ER acetylation machinery: the cytosol:ER-lumen acetyl-CoA transporter AT-1 (also known as SLC33A1), and the ER-resident lysine acetyltransferases ATase1 and ATase2 (also known as NAT8B and NAT8, respectively). This Review summarizes the more recent biochemical, cellular and mouse model studies that underscore the importance of the ER acetylation process in maintaining protein homeostasis and autophagy within the secretory pathway, and its impact on developmental and age-associated diseases.
Assuntos
Autofagia/fisiologia , Retículo Endoplasmático/metabolismo , Lisina/metabolismo , Proteostase/fisiologia , Animais , Humanos , CamundongosRESUMO
N-linked glycosylation, featuring various glycoforms, is one of the most common and complex protein post-translational modifications (PTMs) controlling protein structures and biological functions. It has been revealed that abnormal changes of protein N-glycosylation patterns are associated with many diseases. Hence, unraveling the disease-related alteration of glycosylation, especially the glycoforms, is crucial and beneficial to improving our understanding about the pathogenic mechanisms of various diseases. In past decades, given the capability of in situ mapping of biomolecules and their region-specific localizations, matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) has been widely applied to the discovery of potential biomarkers for many diseases. In this study, we coupled a novel subatmospheric pressure (SubAP)/MALDI source with a Q Exactive HF hybrid quadrupole-orbitrap mass spectrometer for in situ imaging of N-linked glycans from formalin-fixed paraffin-embedded (FFPE) tissue sections. The utility of this new platform for N-glycan imaging analysis was demonstrated with a variety of FFPE tissue sections. A total of 55 N-glycans were successfully characterized and visualized from a FFPE mouse brain section. Furthermore, 29 N-glycans with different spatial distribution patterns could be identified from a FFPE mouse ovarian cancer tissue section. High-mannose N-glycans exhibited elevated expression levels in the tumor region, indicating the potential association of this type of N-glycans with tumor progression.
Assuntos
Encéfalo/metabolismo , Formaldeído/química , Neoplasias Ovarianas/metabolismo , Polissacarídeos/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Feminino , Glicosilação , Humanos , Camundongos , Neoplasias Ovarianas/patologia , Fixação de TecidosRESUMO
The aberrant accumulation of toxic protein aggregates is a key feature of many neurodegenerative diseases, including Huntington's disease, amyotrophic lateral sclerosis and Alzheimer's disease. As such, improving normal proteostatic mechanisms is an active target for biomedical research. Although they share common pathological features, protein aggregates form in different subcellular locations. Nε-lysine acetylation in the lumen of the endoplasmic reticulum has recently emerged as a new mechanism to regulate the induction of autophagy. The endoplasmic reticulum acetylation machinery includes AT-1/SLC33A1, a membrane transporter that translocates acetyl-CoA from the cytosol into the endoplasmic reticulum lumen, and ATase1 and ATase2, two acetyltransferases that acetylate endoplasmic reticulum cargo proteins. Here, we used a mutant form of α-synuclein to show that inhibition of the endoplasmic reticulum acetylation machinery specifically improves autophagy-mediated disposal of toxic protein aggregates that form within the secretory pathway, but not those that form in the cytosol. Consequently, haploinsufficiency of AT-1/SLC33A1 in the mouse rescued Alzheimer's disease, but not Huntington's disease or amyotrophic lateral sclerosis. In fact, intracellular toxic protein aggregates in Alzheimer's disease form within the secretory pathway while in Huntington's disease and amyotrophic lateral sclerosis they form in different cellular compartments. Furthermore, biochemical inhibition of ATase1 and ATase2 was also able to rescue the Alzheimer's disease phenotype in a mouse model of the disease. Specifically, we observed reduced levels of soluble amyloid-ß aggregates, reduced amyloid-ß pathology, reduced phosphorylation of tau, improved synaptic plasticity, and increased lifespan of the animals. In conclusion, our results indicate that Nε-lysine acetylation in the endoplasmic reticulum lumen regulates normal proteostasis of the secretory pathway; they also support therapies targeting endoplasmic reticulum acetyltransferases, ATase1 and ATase2, for a subset of chronic degenerative diseases.
Assuntos
Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Proteínas de Membrana Transportadoras/biossíntese , Biossíntese de Proteínas/fisiologia , Via Secretória/fisiologia , Doença de Alzheimer/prevenção & controle , Animais , Células CHO , Linhagem Celular Tumoral , Cricetinae , Cricetulus , Humanos , Camundongos , Camundongos Transgênicos , Processamento de Proteína Pós-Traducional/fisiologiaRESUMO
The import of acetyl-CoA into the ER lumen by AT-1/SLC33A1 is essential for the N(ε)-lysine acetylation of ER-resident and ER-transiting proteins. A point-mutation (S113R) in AT-1 has been associated with a familial form of spastic paraplegia. Here, we report that AT-1S113R is unable to form homodimers in the ER membrane and is devoid of acetyl-CoA transport activity. The reduced influx of acetyl-CoA into the ER lumen results in reduced acetylation of ER proteins and an aberrant form of autophagy. Mice homozygous for the mutation display early developmental arrest. In contrast, heterozygous animals develop to full term, but display neurodegeneration and propensity to infections, inflammation, and cancer. The immune and cancer phenotypes are contingent on the presence of pathogens in the colony, whereas the nervous system phenotype is not. In conclusion, our results reveal a previously unknown aspect of acetyl-CoA metabolism that affects the immune and nervous systems and the risk for malignancies.
Assuntos
Acetilcoenzima A/metabolismo , Retículo Endoplasmático/metabolismo , Infecções/metabolismo , Inflamação/metabolismo , Neoplasias/metabolismo , Degeneração Neural/metabolismo , Acetilação , Animais , Infecções/genética , Inflamação/genética , Camundongos , Camundongos Transgênicos , Neoplasias/genética , Degeneração Neural/patologiaRESUMO
The endoplasmic reticulum (ER) has two membrane-bound acetyltransferases responsible for the endoluminal N(ϵ)-lysine acetylation of ER-transiting and -resident proteins. Mutations that impair the ER-based acetylation machinery are associated with developmental defects and a familial form of spastic paraplegia. Deficient ER acetylation in the mouse leads to defects of the immune and nervous system. Here, we report that both ATase1 and ATase2 form homo- and heterodimers and associate with members of the oligosaccharyltransferase (OST) complex. In contrast to the OST, the ATases only modify correctly folded polypetides. Collectively, our studies suggest that one of the functions of the ATases is to work in concert with the OST and "select" correctly folded from unfolded/misfolded transiting polypeptides.
Assuntos
Acetiltransferases/química , Retículo Endoplasmático/enzimologia , Hexosiltransferases/química , Proteínas de Membrana/química , Peptídeos/química , Acetilação , Animais , Bacillus anthracis/enzimologia , Bacillus subtilis/enzimologia , Sequência de Bases , Escherichia coli/enzimologia , Glicoproteínas/química , Humanos , Lisina/química , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Desnaturação Proteica , Dobramento de Proteína , Multimerização Proteica , Estrutura Terciária de Proteína , Salmonella enteritidis/enzimologiaRESUMO
The N(ε)-amino group of lysine residues can be transiently modified by the addition of an acetyl group. Recognized functions of N(ε)-lysine acetylation include regulation of activity, molecular stabilization and conformational assembly of a protein. For more than forty years lysine acetylation was thought to occur only in the cytosol and nucleus. Targets included cytoskeletal-associated proteins as well as transcription factors, histone proteins and proteins involved in DNA recombination and repair. However, in 2007 we reported that a type I membrane protein involved in the pathogenesis of Alzheimer's disease was transiently acetylated on the ε amino group of seven lysine residues while transiting along the secretory pathway. Surprisingly, the acetylation occurred in the lumen of the endoplasmic reticulum (ER) forcing us to reconsider old paradigms. Indeed, if lysine acetylation can occur in the lumen of the ER, then all the essential biochemical elements of the reaction must be available in the lumen of the organelle. Follow-up studies revealed the existence of ER-based acetyl-CoA:lysine acetyltransferases as well as a membrane transporter that translocates acetyl-CoA from the cytosol into the ER lumen. Large-scale proteomics showed that the list of substrates of the ER-based acetylation machinery includes both transiting and resident proteins. Finally, genetic studies revealed that this machinery is tightly linked to human diseases. Here, we describe these exciting findings as well as recent biochemical and cellular advances, and discuss possible impact on both human physiology and pathology.
Assuntos
Retículo Endoplasmático/metabolismo , Lisina/metabolismo , Processamento de Proteína Pós-Traducional , Resposta a Proteínas não Dobradas , Acetilação , Animais , HumanosRESUMO
The endoplasmic reticulum acetylation machinery has emerged as a new branch of the larger endoplasmic reticulum quality control system. It regulates the selection of correctly folded polypeptides as well as reticulophagy-mediated removal of toxic protein aggregates with the former being a particularly important aspect of the proteostatic functions of endoplasmic reticulum acetylation. Essential to this function is the Nε-lysine acetyltransferase activity of acetyltransferase 1 and acetyltransferase 2, which regulates the induction of endoplasmic reticulum-specific autophagy through the acetylation of the autophagy-related protein 9A. Here, we used three mouse models of Charcot-Marie-Tooth disease, peripheral myelin protein 22/Tr-J, C3-peripheral myelin protein 22 and myelin protein zero/ttrr, to study spatial and translational selectivity of endoplasmic reticulum acetyltransferase inhibitors. The results show that inhibition of the endoplasmic reticulum acetyltransferases selectively targets misfolding/pro-aggregating events occurring in the lumen of the organelle. Therefore, they establish acetyltransferase 1 and acetyltransferase 2 as the first proven targets for disease-causing proteotoxic states that initiate within the lumen of the endoplasmic reticulum/secretory pathway.
RESUMO
Dysregulated glycerophospholipid (GP) metabolism in the brain is associated with the progression of neurodegenerative diseases including Alzheimer's disease (AD). Routine liquid chromatography-mass spectrometry (LC-MS)-based large-scale lipidomic methods often fail to elucidate subtle yet important structural features such as sn-position, hindering the precise interrogation of GP molecules. Leveraging high-resolution demultiplexing (HRdm) ion mobility spectrometry (IMS), we develop a four-dimensional (4D) lipidomic strategy to resolve GP sn-position isomers. We further construct a comprehensive experimental 4D GP database of 498 GPs identified from the mouse brain and an in-depth extended 4D library of 2500 GPs predicted by machine learning, enabling automated profiling of GPs with detailed acyl chain sn-position assignment. Analyzing three mouse brain regions (hippocampus, cerebellum, and cortex), we successfully identify a total of 592 GPs including 130 pairs of sn-position isomers. Further temporal GPs analysis in the three functional brain regions illustrates their metabolic alterations in AD progression.
Assuntos
Doença de Alzheimer , Encéfalo , Glicerofosfolipídeos , Lipidômica , Animais , Doença de Alzheimer/metabolismo , Lipidômica/métodos , Glicerofosfolipídeos/metabolismo , Camundongos , Encéfalo/metabolismo , Espectrometria de Mobilidade Iônica/métodos , Masculino , Cerebelo/metabolismo , Modelos Animais de Doenças , Cromatografia Líquida/métodos , Camundongos Endogâmicos C57BL , Hipocampo/metabolismoRESUMO
Age is the greatest risk factor for Alzheimer's disease (AD) as well as for other disorders that increase the risk of AD such as diabetes and obesity. There is growing interest in determining if interventions that promote metabolic health can prevent or delay AD. Acarbose is an anti-diabetic drug that not only improves glucose homeostasis, but also extends the lifespan of wild-type mice. Here, we test the hypothesis that acarbose will not only preserve metabolic health, but also slow or prevent AD pathology and cognitive deficits in 3xTg mice, a model of AD, fed either a Control diet or a high-fat, high-sucrose Western diet (WD). We find that acarbose decreases the body weight and adiposity of WD-fed 3xTg mice, increasing energy expenditure while also stimulating food consumption, and improves glycemic control. Both male and female WD-fed 3xTg mice have worsened cognitive deficits than Control-fed mice, and these deficits are ameliorated by acarbose treatment. Molecular and histological analysis of tau and amyloid pathology identified sex-specific effects of acarbose which are uncoupled from the dramatic improvements in cognition, suggesting that the benefits of acarbose on AD are largely driven by improved metabolic health. In conclusion, our results suggest that acarbose may be a promising intervention to prevent, delay, or even treat AD, especially in individuals consuming a Western diet.
RESUMO
Despite the efforts to identify fluid biomarkers to improve diagnosis of Frontotemporal dementia (FTD), only a few candidates have been described in recent years. In a previous study, we identified three circulating miRNAs (miR-92a-3p, miR-320a and miR-320b) differentially expressed in FTD patients with respect to healthy controls and/or Alzheimer's disease (AD) patients. Now, we investigated whether those changes could be due to miRNAs contained in neuron-derived extracellular vesicles (NDEVs). We also evaluated miRNAs content in total plasma EVs and in CSF samples. The analysis of plasma NDEVs carried out on 40 subjects including controls (n = 13), FTD (n = 13) and AD (n = 14) patients, showed that both miR-92a-3p and miR-320a levels were triplicated in the FTD group if compared with CT and AD patients. Increased levels of the same miRNAs were found also in CSF derived from FTD group compared to CTs. No differences were observed in expression levels of miR-320b among the three groups. Worthy of note, all miRNAs analysed were increased in an FTD cell model, MAPT IVS10 + 16 neurons. Our results suggest that miR-92a and miR-320a in NDEVs could be proposed as FTD biomarkers.
RESUMO
Dietary protein is a critical regulator of metabolic health and aging. Low protein diets are associated with healthy aging in humans, and dietary protein restriction extends the lifespan and healthspan of mice. In this study, we examined the effect of protein restriction (PR) on metabolic health and the development and progression of Alzheimer's disease (AD) in the 3xTg mouse model of AD. Here, we show that PR promotes leanness and glycemic control in 3xTg mice, specifically rescuing the glucose intolerance of 3xTg females. PR induces sex-specific alterations in circulating and brain metabolites, downregulating sphingolipid subclasses in 3xTg females. PR also reduces AD pathology and mTORC1 activity, increases autophagy, and improves the cognition of 3xTg mice. Finally, PR improves the survival of 3xTg mice. Our results suggest that PR or pharmaceutical interventions that mimic the effects of this diet may hold promise as a treatment for AD.
Assuntos
Doença de Alzheimer , Encéfalo , Dieta com Restrição de Proteínas , Modelos Animais de Doenças , Progressão da Doença , Camundongos Transgênicos , Animais , Doença de Alzheimer/patologia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/genética , Feminino , Masculino , Camundongos , Encéfalo/metabolismo , Encéfalo/patologia , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Autofagia , Intolerância à Glucose/metabolismo , Esfingolipídeos/metabolismo , Cognição , Camundongos Endogâmicos C57BLRESUMO
In addition to the nucleus, cytosol, and mitochondrial lumen, N(ε)-lysine acetylation also occurs in the lumen of the endoplasmic reticulum (ER). However, the impact of such an event on ER functions is still unknown. Here, we analyzed the "ER acetyl-lysine proteome" by nano-LC-MS/MS and discovered that a large number of ER-resident and -transiting proteins undergo N(ε)-lysine acetylation in the lumen of the organelle. The list of ER-resident proteins includes chaperones and enzymes involved with post-translational modification and folding. Grouping of all acetylated proteins into major functional categories suggests that the ER-based acetylation machinery regulates very diverse biological events. As such, it is predicted to play a fundamental role in human physiology as well as human pathology.
Assuntos
Retículo Endoplasmático/metabolismo , Lisina/metabolismo , Proteínas de Membrana/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Proteômica/métodos , Acetilcoenzima A/metabolismo , Acetilação , Neoplasias Encefálicas , Calreticulina/química , Calreticulina/genética , Calreticulina/metabolismo , Linhagem Celular Tumoral , Chaperona BiP do Retículo Endoplasmático , Glioma , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Plasmídeos , Dobramento de ProteínaRESUMO
One of the main functions of the unfolded protein response is to ensure disposal of large protein aggregates that accumulate in the lumen of the endoplasmic reticulum (ER) whereas avoiding, at least under nonlethal levels of ER stress, cell death. When tightly controlled, autophagy-dependent ER-associated degradation (ERAD(II)) allows the cell to recover from the transient accumulation of protein aggregates; however, when unchecked, it can be detrimental and cause autophagic cell death/type 2 cell death. Here we show that IRE1/XBP1 controls the induction of autophagy/ERAD(II) during the unfolded protein response by activating the ER membrane transporter SLC33A1/AT-1, which ensures continuous supply of acetyl-CoA into the lumen of the ER. Failure to induce AT-1 leads to widespread autophagic cell death. Mechanistically, the regulation of the autophagic process involves N(ε)-lysine acetylation of Atg9A.
Assuntos
Autofagia/fisiologia , Proteínas de Ligação a DNA/metabolismo , Estresse do Retículo Endoplasmático/fisiologia , Degradação Associada com o Retículo Endoplasmático/fisiologia , Endorribonucleases/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição/metabolismo , Acetilcoenzima A/genética , Acetilcoenzima A/metabolismo , Acetilação , Animais , Proteínas Relacionadas à Autofagia , Células CHO , Cricetinae , Cricetulus , Proteínas de Ligação a DNA/genética , Endorribonucleases/genética , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana Transportadoras/genética , Proteínas Serina-Treonina Quinases/genética , Fatores de Transcrição de Fator Regulador X , Fatores de Transcrição/genética , Proteínas de Transporte Vesicular , Proteína 1 de Ligação a X-BoxRESUMO
The cellular levels of ß-site APP cleaving enzyme 1 (BACE1), the rate-limiting enzyme for the generation of the Alzheimer disease (AD) amyloid ß-peptide (Aß), are tightly regulated by two ER-based acetyl-CoA:lysine acetyltransferases, ATase1 and ATase2. Here we report that both acetyltransferases are expressed in neurons and glial cells, and are up-regulated in the brain of AD patients. We also report the identification of first and second generation compounds that inhibit ATase1/ATase2 and down-regulate the expression levels as well as activity of BACE1. The mechanism of action involves competitive and non-competitive inhibition as well as generation of unstable intermediates of the ATases that undergo degradation.
Assuntos
Acetiltransferases/metabolismo , Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/biossíntese , Peptídeos beta-Amiloides/metabolismo , Ácido Aspártico Endopeptidases/biossíntese , Regulação Enzimológica da Expressão Gênica , Neuroglia/metabolismo , Neurônios/metabolismo , Acetiltransferases/antagonistas & inibidores , Acetiltransferases/genética , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Secretases da Proteína Precursora do Amiloide/genética , Peptídeos beta-Amiloides/genética , Animais , Ácido Aspártico Endopeptidases/genética , Células CHO , Cricetinae , Cricetulus , Inibidores Enzimáticos/farmacologia , Humanos , Camundongos , Camundongos Transgênicos , Neuroglia/patologia , Neurônios/patologia , Células PC12 , Ratos , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
BACKGROUND: Key cellular metabolites reflecting the immediate activity of metabolic enzymes as well as the functional metabolic state of intracellular organelles can act as powerful signal regulators to ensure the activation of homeostatic responses. The citrate/acetyl-CoA pathway, initially recognized for its role in intermediate metabolism, has emerged as a fundamental branch of this nutrient-sensing homeostatic response. Emerging studies indicate that fluctuations in acetyl-CoA availability within different cellular organelles and compartments provides substrate-level regulation of many biological functions. A fundamental aspect of these regulatory functions involves Nε-lysine acetylation. SCOPE OF REVIEW: Here, we will examine the emerging regulatory functions of the citrate/acetyl-CoA pathway and the specific role of the endoplasmic reticulum (ER) acetylation machinery in the maintenance of intracellular crosstalk and homeostasis. These functions will be analyzed in the context of associated human diseases and specific mouse models of dysfunctional ER acetylation and citrate/acetyl-CoA flux. A primary objective of this review is to highlight the complex yet integrated response of compartment- and organelle-specific Nε-lysine acetylation to the intracellular availability and flux of acetyl-CoA, linking this important post-translational modification to cellular metabolism. MAJOR CONCLUSIONS: The ER acetylation machinery regulates the proteostatic functions of the organelle as well as the metabolic crosstalk between different intracellular organelles and compartments. This crosstalk enables the cell to impart adaptive responses within the ER and the secretory pathway. However, it also enables the ER to impart adaptive responses within different cellular organelles and compartments. Defects in the homeostatic balance of acetyl-CoA flux and ER acetylation reflect different but converging disease states in humans as well as converging phenotypes in relevant mouse models. In conclusion, citrate and acetyl-CoA should not only be seen as metabolic substrates of intermediate metabolism but also as signaling molecules that direct functional adaptation of the cell to both intracellular and extracellular messages. Future discoveries in CoA biology and acetylation are likely to yield novel therapeutic approaches.
Assuntos
Ácido Cítrico , Lisina , Camundongos , Animais , Humanos , Acetilcoenzima A/metabolismo , Lisina/metabolismo , Ácido Cítrico/metabolismo , Acetilação , Processamento de Proteína Pós-Traducional , Retículo Endoplasmático/metabolismo , Citratos/metabolismoRESUMO
Single-cell (SC) analysis provides unique insight into individual cell dynamics and cell-to-cell heterogeneity. Here, we utilize trapped ion mobility separation coupled with dual-polarity ionization mass spectrometry imaging (MSI) to enable high-throughput in situ profiling of the SC lipidome. Multimodal SC imaging, in which dual-polarity-mode MSI is used to perform serial data acquisition runs on individual cells, significantly enhanced SC lipidome coverage. High-spatial resolution SC-MSI identifies both inter- and intracellular lipid heterogeneity; this heterogeneity is further explicated by Uniform Manifold Approximation and Projection and machine learning-driven classifications. We characterize SC lipidome alteration in response to stearoyl-CoA desaturase 1 inhibition and, additionally, identify cell-layer specific lipid distribution patterns in mouse cerebellar cortex. This integrated multimodal SC-MSI technology enables high-resolution spatial mapping of intercellular and cell-to-cell lipidome heterogeneity, SC lipidome remodeling induced by pharmacological intervention, and region-specific lipid diversity within tissue.