RESUMO
Migrasomes, vesicular organelles generated on the retraction fibers of migrating cells, play a crucial role in migracytosis, mediating intercellular communication. The cargoes determine the functional specificity of migrasomes. Migrasomes harbor numerous intraluminal vesicles, a pivotal component of their cargoes. The mechanism underlying the transportation of these intraluminal vesicles to the migrasomes remains enigmatic. In this study, we identified that Rab10 and Caveolin-1 (CAV1) mark the intraluminal vesicles in migrasomes. Transport of Rab10-CAV1 vesicles to migrasomes required the motor protein Myosin Va and adaptor proteins RILPL2. Notably, the phosphorylation of Rab10 by the kinase LRRK2 regulated this process. Moreover, CSF-1 can be transported to migrasomes through this mechanism, subsequently fostering monocyte-macrophage differentiation in skin wound healing, which served as a proof of the physiological importance of this transporting mechanism.
Assuntos
Caveolina 1 , Movimento Celular , Proteínas rab de Ligação ao GTP , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab de Ligação ao GTP/genética , Humanos , Caveolina 1/metabolismo , Caveolina 1/genética , Macrófagos/metabolismo , Fosforilação , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Miosina Tipo V/metabolismo , Miosina Tipo V/genética , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Camundongos , Cadeias Pesadas de Miosina/metabolismo , Cadeias Pesadas de Miosina/genética , Transporte Biológico , Cicatrização/fisiologia , Organelas/metabolismoRESUMO
Single-cell proteomic analyses are of fundamental importance in order to capture biological heterogeneity within complex cell systems' heterogeneous populations. Mass spectrometry (MS)-based proteomics is a promising alternative for quantitative single-cell proteomics. Various techniques are continually evolving to address the challenges of limited sample material, detection sensitivity, and throughput constraints. In this chapter, we describe a nanoliter-scale glass-oil-air-droplet (gOAD) chip engineered for heat tolerance, which combines droplet-based microfluidics and shotgun proteomic analysis techniques to enable multistep sample pretreatment.
Assuntos
Vidro , Proteômica , Análise de Célula Única , Proteômica/métodos , Análise de Célula Única/métodos , Análise de Célula Única/instrumentação , Vidro/química , Humanos , Óleos/química , Espectrometria de Massas/métodos , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Dispositivos Lab-On-A-Chip , Ar , Proteoma/análise , Nanotecnologia/métodos , Nanotecnologia/instrumentação , Microfluídica/métodos , Microfluídica/instrumentaçãoRESUMO
Advanced-stage cutaneous T-cell lymphomas (CTCLs) are notorious for their highly aggressive behavior, resistance to conventional treatments, and poor prognosis, particularly when large-cell transformation occurs. PEG10 has been recently proposed as a potent driver for large-cell transformation in CTCL. However, the targeting of PEG10 continues to present a formidable clinical challenge that has yet to be addressed. In this study, we report an important post-translational regulatory mechanism of PEG10 in CTCL. USP9X, a deubiquitinase, interacted with and deubiquitinated PEG10, thereby stabilizing PEG10. Knockdown of USP9X or pharmacological targeting of USP9X resulted in a prominent downregulation of PEG10 and its downstream pathway in CTCL. Moreover, USP9X inhibition conferred tumor cell growth disadvantage and enhanced apoptosis in vitro, an effect that occurred in part through its regulation on PEG10. Furthermore, we demonstrated that inhibition of USP9X obviously restrained CTCL tumor growth in vivo and that high expression of USP9X is associated with poor survival in patients with CTCL. Collectively, our findings uncover USP9X as a key post-translational regulator in the stabilization of PEG10 and suggest that targeting PEG10 stabilization through USP9X inhibition may represent a promising therapeutic strategy for advanced-stage CTCL.
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A large proportion of patients with chronic pain experience co-morbid anxiety. The medial prefrontal cortex (mPFC) is proposed to underlie this comorbidity, but the molecular and neuronal mechanisms are not fully understood. Here, we reported that impaired neuronal macroautophagy in the prelimbic cortical (PrL) subregion of the mPFC paralleled the occurrence of anxiety-like behaviors in rats with chronic spared nerve injury (SNI). Intriguingly, such macroautophagy impairment was mainly observed in a FOS/c-Fos+ neuronal subpopulation in the PrL. Chemogenetic inactivation of this comorbid anxiety-related neuronal ensemble relieved pain-induced anxiety-like behaviors. Rescuing macroautophagy impairment in this neuronal ensemble relieved chronic pain-associated anxiety and mechanical allodynia and restored synaptic homeostasis at the molecular level. By contrast, artificial disruption of macroautophagy induced early-onset co-morbid anxiety in neuropathic rats, but not general anxiety in normal rats. Taken together, our work identifies causal linkage between PrL neuronal macroautophagy dysfunction and comorbid anxiety in neuropathic pain and provides novel insights into the role of PrL by differentiating its contribution in pain-induced comorbid anxiety from its modulation over general anxiety-like behaviors.Abbreviation: AAV: adeno-associated viruses; ACC: anterior cingulate cortex; ATG5: autophagy related 5; ATG7: autophagy related 7; ATG12: autophagy related 12; CAMK2/CaMKII: calcium/calmodulin-dependent protein kinase II; CNO: clozapine-N-oxide; CQ: chloroquine; DIA: data independent acquisition; DIO: double floxed inverse orf; DLG4/PSD-95: discs large MAGUK scaffold protein 4; Dox: doxycycline; GABA: γ-aminobutyric acid; GFP: green fluorescent protein; GO: gene ontology; Gi: inhibitory guanine nucleotide-binding proteins; HsCHRM4/M4D: human cholinergic receptor muscarinic 4; HsSYN: human synapsin; KEGG: Kyoto encyclopedia of genes and genomes; LAMP1: lysosomal-associated membrane protein 1; LC3-II: PE conjugated microtubule-associated protein 1 light chain3; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; mPFC: medial prefrontal cortex; P2A: 2A self-cleaving peptide; PPI: protein-protein interaction networks; PrL: prelimbic cortex; RBFOX3/NeuN: RNA binding protein, fox-1 homolog (C. elegans) 3; rtTA: reverse tetracycline-transactivator; SDS-PAGE: sodium dodecylsulfate-polyacrylamide gel electrophoresis; SHANK3: SH3 and multiple ankyrin repeat domains 3; SLC1A1/EAAC1: solute carrier family 1 (neuronal/epithelial high affinity glutamate transporter, systemXag), member 1; SNAP23: synaptosomal-associated protein 23; SNI:spared nerve injury; SQSTM1/p62: sequestosome 1; SYT3: synaptotagmin 3; TRE: tetracycline-responsive element; TRE3G: third-generation tetracycline-responsive element.
Assuntos
Ansiedade , Macroautofagia , Neuralgia , Neurônios , Córtex Pré-Frontal , Animais , Neuralgia/metabolismo , Córtex Pré-Frontal/metabolismo , Ratos , Neurônios/metabolismo , Masculino , Macroautofagia/fisiologia , Ratos Sprague-Dawley , Comportamento Animal , Dor Crônica/metabolismo , Autofagia/fisiologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , HiperalgesiaRESUMO
Alternative splicing allows a small number of human genes to encode large amounts of proteoforms that play essential roles in normal and disease physiology. Some low-abundance proteoforms may remain undiscovered due to limited detection and analysis capabilities. Peptides coencoded by novel exons and annotated exons separated by introns are called novel junction peptides, which are the key to identifying novel proteoforms. Traditional de novo sequencing does not take into account the specificity in the composition of the novel junction peptide and is therefore not as accurate. We first developed a novel de novo sequencing algorithm, CNovo, which outperformed the mainstream PEAKS and Novor in all six test sets. We then built on CNovo to develop a semi-de novo sequencing algorithm, SpliceNovo, specifically for identifying novel junction peptides. SpliceNovo identifies junction peptides with much higher accuracy than CNovo, CJunction, PEAKS, and Novor. Of course, it is also possible to replace the built-in CNovo in SpliceNovo with other more accurate de novo sequencing algorithms to further improve its performance. We also successfully identified and validated two novel proteoforms of the human EIF4G1 and ELAVL1 genes by SpliceNovo. Our results significantly improve the ability to discover novel proteoforms through de novo sequencing.
Assuntos
Algoritmos , Peptídeos , Humanos , Peptídeos/genética , Peptídeos/química , Análise de Sequência , Éxons , Íntrons , Análise de Sequência de Proteína/métodosRESUMO
Sperm-induced Ca2+ rise is critical for driving oocyte activation and subsequent embryonic development, but little is known about how lasting Ca2+ oscillations are regulated. Here it is shown that NLRP14, a maternal effect factor, is essential for keeping Ca2+ oscillations and early embryonic development. Few embryos lacking maternal NLRP14 can develop beyond the 2-cell stage. The impaired developmental potential of Nlrp14-deficient oocytes is mainly caused by disrupted cytoplasmic function and calcium homeostasis due to altered mitochondrial distribution, morphology, and activity since the calcium oscillations and development of Nlrp14-deficient oocytes can be rescued by substitution of whole cytoplasm by spindle transfer. Proteomics analysis reveal that cytoplasmic UHRF1 (ubiquitin-like, containing PHD and RING finger domains 1) is significantly decreased in Nlrp14-deficient oocytes, and Uhrf1-deficient oocytes also show disrupted calcium homeostasis and developmental arrest. Strikingly, it is found that the mitochondrial Na+ /Ca2+ exchanger (NCLX) encoded by Slc8b1 is significantly decreased in the Nlrp14mNull oocyte. Mechanistically, NLRP14 interacts with the NCLX intrinsically disordered regions (IDRs) domain and maintain its stability by regulating the K27-linked ubiquitination. Thus, the study reveals NLRP14 as a crucial player in calcium homeostasis that is important for early embryonic development.
Assuntos
Cálcio , Nucleosídeo-Trifosfatase , Sêmen , Humanos , Masculino , Cálcio/metabolismo , Homeostase/fisiologia , Oócitos/metabolismo , Sêmen/metabolismo , Trocador de Sódio e Cálcio/genética , Trocador de Sódio e Cálcio/metabolismo , Ubiquitinação , Animais , Camundongos , Nucleosídeo-Trifosfatase/metabolismoRESUMO
MET inhibitors have shown promising efficacy for MET-dysregulated non-small cell lung cancer (NSCLC). However, quite a few patients cannot benefit from it due to the lack of powerful biomarkers. This study aims to explore the potential role of plasma proteomics-derived biomarkers for patients treated with MET inhibitors using mass spectrometry. We analyzed the plasma proteomics from patients with MET dysregulation (including MET amplification and MET overexpression) treated with MET inhibitors. Thirty-three MET-dysregulated NSCLC patients with longitudinal 89 plasma samples were included. We classified patients into the PD group and non-PD group based on clinical response. The baseline proteomic profiles of patients in the PD group were distinct from those in the non-PD group. Through protein screening, we found that a four-protein signature (MYH9, GNB1, ALOX12B, HSD17B4) could predict the efficacy of patients treated with MET inhibitors, with an area under the curve (AUC) of 0.93, better than conventional fluorescence in situ hybridization (FISH) or immunohistochemistry (IHC) tests. In addition, combining the four-protein signature with FISH or IHC test could also reach higher predictive performance. Further, the combined signature could predict progression-free survival for MET-dysregulated NSCLC (p < 0.001). We also validated the performance of the four-protein signature in another cohort of plasma using an enzyme-linked immunosorbent assay. In conclusion, the four plasma protein signature (MYH9, GNB1, ALOX12B, and HSD17B4 proteins) might play a substitutable or complementary role to conventional MET FISH or IHC tests. This exploration will help select patients who may benefit from MET inhibitors.
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Phosphopeptides presented by major histocompatibility complex (MHC) class I have been regarded as a pivotal type of cancer neoantigens that are recognized by T cells. The structural basis of single-phosphorylated peptide presentation has been well studied. Diphosphorylation with one interval between two sites is one of the prevalent forms of multisite-phosphorylated peptides. Herein, we determined the molecular basis of presentation of two P4/P6 double pS-containing peptides by HLA-B27 and compared them with unmodified and single-phosphorylated peptide complexes. These data clarified not only the HLA allele-specific presentation of phosphopeptides by MHC class I molecules but also the cooperativity of peptide conformation within P4 and P6 phosphorylation sites. The phosphorylation of P6 site can influence the binding mode of P4 phosphorylated site to HLA-B27. And we found the diphospho-dependent attenuated effect of peptide binding affinity. This study provides insights into the MHC presentation features of diphosphopeptides, which is different from monophosphopeptides.
RESUMO
Autolysosomes contain components from autophagosomes and lysosomes. The contents inside the autolysosomal lumen are degraded during autophagy, while the fate of autophagosomal components on the autolysosomal membrane remains unknown. Here we report that the autophagosomal membrane components are not degraded, but recycled from autolysosomes through a process coined in this study as autophagosomal components recycling (ACR). We further identified a multiprotein complex composed of SNX4, SNX5 and SNX17 essential for ACR, which we termed 'recycler'. In this, SNX4 and SNX5 form a heterodimer that recognizes autophagosomal membrane proteins and is required for generating membrane curvature on autolysosomes, both via their BAR domains, to mediate the cargo sorting process. SNX17 interacts with both the dynein-dynactin complex and the SNX4-SNX5 dimer to facilitate the retrieval of autophagosomal membrane components. Our discovery of ACR and identification of the recycler reveal an important retrieval and recycling pathway on autolysosomes.
Assuntos
Autofagossomos , Lisossomos , Autofagossomos/metabolismo , Autofagia , Dineínas/metabolismo , Lisossomos/metabolismo , Transporte ProteicoRESUMO
Coronavirus disease 2019 (COVID-19) has caused huge deaths and economic losses worldwide in the current pandemic. The main protease (Mpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is thought to be an ideal drug target for treating COVID-19. Leupeptin, a broad-spectrum covalent inhibitor of serine, cysteine, and threonine proteases, showed inhibitory activity against Mpro, with a 50% inhibitory concentration (IC50) value of 127.2 µM in vitro in our study here. In addition, leupeptin can also inhibit SARS-CoV-2 in Vero cells, with 50% effective concentration (EC50) values of 42.34 µM. More importantly, various strains of streptomyces that have a broad symbiotic relationship with medicinal plants can produce leupeptin and leupeptin analogs to regulate autogenous proteases. Fingerprinting and structure elucidation using high-performance liquid chromatography (HPLC) and high-resolution mass spectrometry (HRMS), respectively, further proved that the Qing-Fei-Pai-Du (QFPD) decoction, a traditional Chinese medicine (TCM) formula for the effective treatment of COVID-19 during the period of the Wuhan outbreak, contains leupeptin. All these results indicate that leupeptin at least contributes to the antiviral activity of the QFPD decoction against SARS-CoV-2. This also reminds us to pay attention to the microbiomes in TCM herbs as streptomyces in the soil might produce leupeptin that will later infiltrate the medicinal plant. We propose that plants, microbiome, and microbial metabolites form an ecosystem for the effective components of TCM herbs. IMPORTANCE A TCM formula has played an important role in the treatment of COVID-19 in China. However, the mechanism of TCM action is still unclear. In this study, we identified leupeptin, a metabolite produced by plant-symbiotic actinomyces (PSA), which showed antiviral activity in both cell culture and enzyme assays. Moreover, leupeptin found in the QFPD decoction was confirmed by both HPLC fingerprinting and HRMS. These results suggest that leupeptin likely contributes to the antiviral activity of the QFPD decoction against SARS-CoV-2. This result gives us important insight into further studies of the PSA metabolite and medicinal plant ecosystem for future TCM modernization research.
Assuntos
Tratamento Farmacológico da COVID-19 , Leupeptinas/uso terapêutico , Medicina Tradicional Chinesa/métodos , Animais , Chlorocebus aethiops , Ecossistema , Humanos , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/patogenicidade , Células VeroRESUMO
In eukaryotes, alternative pre-mRNA splicing allows a single gene to encode different protein isoforms that function in many biological processes, and they are used as biomarkers or therapeutic targets for diseases. Although protein isoforms in the human genome are well annotated, we speculate that some low-abundance protein isoforms may still be under-annotated because most genes have a primary coding product and alternative protein isoforms tend to be under-expressed. A peptide coencoded by a novel exon and an annotated exon separated by an intron is known as a novel junction peptide. In the absence of known transcripts and homologous proteins, traditional whole-genome six-frame translation-based proteogenomics cannot identify novel junction peptides, and it cannot capture novel alternative splice sites. In this article, we first propose a strategy and tool for identifying novel junction peptides, called CJunction, which we then integrate into a proteogenomics process specifically designed for novel protein isoform discovery and apply to the analysis of a deep-coverage HeLa mass spectrometry data set with identifier PXD004452 in ProteomeXchange. We succeeded in identifying and validating three novel protein isoforms of two functionally important genes, NHSL1 (causative gene of Nance-Horan syndrome) and EEF1B2 (translation elongation factor), which validate our hypothesis. These novel protein isoforms have significant sequence differences from the annotated gene-coding products introduced by the novel N-terminal, suggesting that they may play importantly different functions.
Assuntos
Processamento Alternativo , Fatores de Troca do Nucleotídeo Guanina/genética , Fator 1 de Elongação de Peptídeos/genética , Proteínas , Proteogenômica , Genoma Humano , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Espectrometria de Massas , Fator 1 de Elongação de Peptídeos/metabolismo , Peptídeos/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas/genética , Proteínas/metabolismo , Proteogenômica/métodosRESUMO
The persistence of leukemia stem cells (LSCs) is one of the leading causes of chemoresistance in acute myeloid leukemia (AML). To explore the factors important in LSC-mediated resistance, we use mass spectrometry to screen the factors related to LSC chemoresistance and defined IFN-γ-inducible lysosomal thiol reductase (GILT) as a candidate. We found that the GILT expression was upregulated in chemoresistant CD34+ AML cells. Loss of function studies demonstrated that silencing of GILT in AML cells sensitized them to Ara-C treatment both in vitro and in vivo. Further mechanistic findings revealed that the ROS-mediated mitochondrial damage plays a pivotal role in inducing apoptosis of GILT-inhibited AML cells after Ara-C treatment. The inactivation of PI3K/Akt/ nuclear factor erythroid 2-related factor 2 (NRF2) pathway, causing reduced generation of antioxidants such as SOD2 and leading to a shifted ratio of GSH/GSSG to the oxidized form, contributed to the over-physiological oxidative status in the absence of GILT. The prognostic value of GILT was also validated in AML patients. Taken together, our work demonstrated that the inhibition of GILT increases AML chemo-sensitivity through elevating ROS level and induce oxidative mitochondrial damage-mediated apoptosis, and inhibition of the PI3K/Akt/NRF2 pathway enhances the intracellular oxidative state by disrupting redox homeostasis, providing a potentially effective way to overcome chemoresistance of AML.
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RNA-binding proteins (RBPs) have essential functions during germline and early embryo development. However, current methods are unable to identify the in vivo targets of a RBP in these low-abundance cells. Here, by coupling RBP-mediated reverse transcription termination with linear amplification of complementary DNA ends and sequencing, we present the LACE-seq method for identifying RBP-regulated RNA networks at or near the single-oocyte level. We determined the binding sites and regulatory mechanisms for several RBPs, including Argonaute 2 (Ago2), Mili, Ddx4 and Ptbp1, in mature mouse oocytes. Unexpectedly, transcriptomics and proteomics analysis of Ago2-/- oocytes revealed that Ago2 interacts with endogenous small interfering RNAs (endo-siRNAs) to repress mRNA translation globally. Furthermore, the Ago2 and endo-siRNA complexes fine-tune the transcriptome by slicing long terminal repeat retrotransposon-derived chimeric transcripts. The precise mapping of RBP-binding sites in low-input cells opens the door to studying the roles of RBPs in embryonic development and reproductive diseases.
Assuntos
Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Oócitos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Sítios de Ligação , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Células HeLa , Ribonucleoproteínas Nucleares Heterogêneas/genética , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Humanos , Células K562 , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Knockout , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/genética , RNA-Seq , TranscriptomaRESUMO
Cigarette smoking, the leading cause of chronic obstructive pulmonary disease (COPD), has been implicated as a risk factor for severe disease in patients infected with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here we show that mice with lung epithelial cell-specific loss of function of Miz1, which we identified as a negative regulator of nuclear factor κB (NF-κB) signaling, spontaneously develop progressive age-related changes resembling COPD. Furthermore, loss of Miz1 up-regulates the expression of Ace2, the receptor for SARS-CoV-2. Concomitant partial loss of NF-κB/RelA prevented the development of COPD-like phenotype in Miz1-deficient mice. Miz1 protein levels are reduced in the lungs from patients with COPD, and in the lungs of mice exposed to chronic cigarette smoke. Our data suggest that Miz1 down-regulation-induced sustained activation of NF-κB-dependent inflammation in the lung epithelium is sufficient to induce progressive lung and airway destruction that recapitulates features of COPD, with implications for COVID-19.
Assuntos
Células Epiteliais/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Pulmão/metabolismo , Peptidil Dipeptidase A/metabolismo , Fenótipo , Proteínas Inibidoras de STAT Ativados/genética , Doença Pulmonar Obstrutiva Crônica/genética , Ubiquitina-Proteína Ligases/genética , Regulação para Cima/genética , Enzima de Conversão de Angiotensina 2 , Animais , Betacoronavirus , COVID-19 , Infecções por Coronavirus/metabolismo , Infecções por Coronavirus/virologia , Técnicas de Inativação de Genes , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pandemias , Pneumonia Viral/metabolismo , Pneumonia Viral/virologia , Proteínas Inibidoras de STAT Ativados/metabolismo , Doença Pulmonar Obstrutiva Crônica/etiologia , Doença Pulmonar Obstrutiva Crônica/metabolismo , SARS-CoV-2 , Transdução de Sinais/genética , Fumar/efeitos adversos , Fator de Transcrição RelA/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
As members of the membrane-bound O-acyltransferase (MBOAT) enzyme family, acyl-coenzyme A:cholesterol acyltransferases (ACATs) catalyse the transfer of an acyl group from acyl-coenzyme A to cholesterol to generate cholesteryl ester, the primary form in which cholesterol is stored in cells and transported in plasma1. ACATs have gained attention as potential drug targets for the treatment of diseases such as atherosclerosis, Alzheimer's disease and cancer2-7. Here we present the cryo-electron microscopy structure of human ACAT1 as a dimer of dimers. Each protomer consists of nine transmembrane segments, which enclose a cytosolic tunnel and a transmembrane tunnel that converge at the predicted catalytic site. Evidence from structure-guided mutational analyses suggests that acyl-coenzyme A enters the active site through the cytosolic tunnel, whereas cholesterol may enter from the side through the transmembrane tunnel. This structural and biochemical characterization helps to rationalize the preference of ACAT1 for unsaturated acyl chains, and provides insight into the catalytic mechanism of enzymes within the MBOAT family8.
Assuntos
Biocatálise , Microscopia Crioeletrônica , Esterol O-Aciltransferase/química , Esterol O-Aciltransferase/metabolismo , Domínio Catalítico , Humanos , Modelos Moleculares , Multimerização Proteica , Esterol O-Aciltransferase/ultraestrutura , Especificidade por SubstratoRESUMO
BACKGROUND AND AIMS: Acetaminophen (APAP) overdose induces severe liver injury and hepatic failure. While the activation of c-Jun NH2 -terminal kinase (JNK) has been implicated as a mechanism in APAP-induced liver injury, the hepatic defense system controlled by nuclear factor erythroid 2-related factor 2 (Nrf2) plays a central role in the mitigation of APAP toxicity. However, the link between the two signaling pathways in APAP-induced liver injury (AILI) remains unclear. APPROACH AND RESULTS: In this study, we demonstrated that the activation of JNK in mouse liver following exposure to APAP was correlated with the phosphorylation of Nrf2 and down-regulation of the antioxidant response element (ARE)-driven genes, NAD(P)H:quinone dehydrogenase 1, glutathione S-transferase α3, glutathione S-transferase M1, glutathione S-transferase M5, and aldo-keto reductase 1C. The JNK inhibitor, SP600125, or knockdown of JNK by infection of adenovirus expressing JNK small interfering RNA, ameliorated the APAP induced liver toxicity, and inhibited the phosphorylation of Nrf2 and down-regulation of detoxifying enzymes by stabilizing the transcription factor. Mechanistically, JNK antagonized Nrf2- and ARE-driven gene expression in a Kelch-like ECH-associated protein 1-independent manner. Biochemical analysis revealed that phosphorylated JNK (P-JNK) directly interacted with the Nrf2-ECH homology (Neh) 1 domain of Nrf2 and phosphorylated the serine-aspartate-serine motif 1 (SDS1) region in the Neh6 domain of Nrf2. CONCLUSIONS: Mass spectrometric analysis identified serine 335 in the SDS1 region of mNrf2 as the major phosphorylation site for modulation of Nrf2 ubiquitylation by P-JNK. This study demonstrates that Nrf2 is a target of P-JNK in AILI. Our finding may provide a strategy for the treatment of AILI.
Assuntos
Acetaminofen/toxicidade , Analgésicos não Narcóticos/toxicidade , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Citoproteção/efeitos dos fármacos , Fígado/efeitos dos fármacos , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Animais , Antracenos/farmacologia , Doença Hepática Induzida por Substâncias e Drogas/genética , Doença Hepática Induzida por Substâncias e Drogas/patologia , Citoproteção/genética , Modelos Animais de Doenças , Regulação para Baixo , Inibidores Enzimáticos/farmacologia , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fator 2 Relacionado a NF-E2 , Fosforilação/efeitos dos fármacos , Domínios Proteicos , UbiquitinaçãoRESUMO
Single cell proteomic analysis provides crucial information on cellular heterogeneity in biological systems. Herein, we describe a nanoliter-scale oil-air-droplet (OAD) chip for achieving multistep complex sample pretreatment and injection for single cell proteomic analysis in the shotgun mode. By using miniaturized stationary droplet microreaction and manipulation techniques, our system allows all sample pretreatment and injection procedures to be performed in a nanoliter-scale droplet with minimum sample loss and a high sample injection efficiency (>99%), thus substantially increasing the analytical sensitivity for single cell samples. We applied the present system in the proteomic analysis of 100 ± 10, 50 ± 5, 10, and 1 HeLa cell(s), and protein IDs of 1360, 612, 192, and 51 were identified, respectively. The OAD chip-based system was further applied in single mouse oocyte analysis, with 355 protein IDs identified at the single oocyte level, which demonstrated its special advantages of high enrichment of sequence coverage, hydrophobic proteins, and enzymatic digestion efficiency over the traditional in-tube system.
Assuntos
Nanopartículas/química , Óleos/química , Oócitos/química , Peptídeos/análise , Proteômica , Análise de Célula Única , Ar , Animais , Células HeLa , Humanos , Camundongos , Oócitos/citologia , Tamanho da Partícula , Propriedades de SuperfícieRESUMO
Acetylation is increasingly recognized as one of the major post-translational mechanisms for the regulation of multiple cellular functions in mammalian cells. Acetyltransferase p300, which acetylates histone and non-histone proteins, has been intensively studied in its role in cell growth and metabolism. However, the mechanism underlying the activation of p300 in cells remains largely unknown. Here, we identify the homeostatic sensor mTORC1 as a direct activator of p300. Activated mTORC1 interacts with p300 and phosphorylates p300 at 4 serine residues in the C-terminal domain. Mechanistically, phosphorylation of p300 by mTORC1 prevents the catalytic HAT domain from binding to the RING domain, thereby eliminating intra-molecular inhibition. Functionally, mTORC1-dependent phosphorylation of p300 suppresses cell-starvation-induced autophagy and activates cell lipogenesis. These results uncover p300 as a direct target of mTORC1 and suggest that the mTORC1-p300 pathway plays a pivotal role in cell metabolism by coordinately controlling cell anabolism and catabolism.
Assuntos
Autofagia , Lipogênese , Complexos Multiproteicos/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Fatores de Transcrição de p300-CBP/metabolismo , Animais , Células HEK293 , Células HeLa , Células Hep G2 , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Complexos Multiproteicos/genética , Fosforilação/genética , Domínios Proteicos , Serina-Treonina Quinases TOR/genética , Fatores de Transcrição de p300-CBP/genéticaRESUMO
ShuFengJieDu capsule (SFJDC), a traditional Chinese medicine (TCM) that contains eight medicinal herbs, has been extensively utilized for the treatment of acute lung injury (ALI) and respiratory infections for more than 30 years in China. SFJDC has also been listed in the official guidelines of the China Food and Drug Administration (CFDA) due to its stable clinical manifestations. However, the underlying mechanism of SFJDC during ALI repair remains unclear. In the present study, we explored the protective and therapeutic mechanisms of SFJDC in a rat model by performing qualitative and label-free quantitative proteomics studies. After establishing lipopolysaccharide (LPS)-induced ALI rat models, we profiled macrophage cells isolated from freshly resected rat lung tissues derived from ALI models and ALI rat lung tissue sections using a high performance liquid chromatography-mass spectrometry (HPLC-MS/MS) shotgun proteomics approach to identify changes in the expression levels of proteins of interest. On the basis of our proteomics results and the results of a protein dysregulation analysis of ALI rat lung tissues and rat lung macrophages, AKT1 was selected as a putative key factor that may play an important role in mediating the effects of SFJDC treatment during ALI progression. Follow-up validation studies demonstrated that AKT1 expression effectively regulates various ALI-related molecules, and Gene Ontology analysis indicated that SFJDC-treated ALI rat macrophages were influenced by AKT1-based networks. Gain- and loss-of-function analyses following lentivirus-AKT1 or lentivirus-si-AKT1 infection in macrophages also indicated that AKT1 was essential for the development of ALI due to its ability to regulate oxidative stress, apoptosis, or inflammatory responses. In summary, SFJDC effectively modulated anti-inflammatory and immunomodulation activity during ALI, potentially due to AKT1 regulation during ALI progression. New insights into SFJDC mechanisms may facilitate the development of novel pharmaceutical strategies to control the expression of inflammatory factors.
Assuntos
Lesão Pulmonar Aguda/tratamento farmacológico , Medicamentos de Ervas Chinesas/uso terapêutico , Proteômica/métodos , Lesão Pulmonar Aguda/induzido quimicamente , Animais , Anti-Inflamatórios , Cromatografia Líquida de Alta Pressão , Medicamentos de Ervas Chinesas/farmacologia , Imunomodulação , Lipopolissacarídeos , Pulmão , Macrófagos Alveolares , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/fisiologia , Ratos , Espectrometria de Massas em Tandem , TranscriptomaRESUMO
The class III phosphoinositide 3-kinase VPS34 plays a key role in the regulation of vesicular trafficking and macroautophagy. So far, we know little about the molecular mechanism of VPS34 activation besides its interaction with regulatory proteins to form complexes. Here, we report that VPS34 is specifically acetylated by the acetyltransferase p300, and p300-mediated acetylation represses VPS34 activity. Acetylation at K771 directly diminishes the affinity of VPS34 for its substrate PI, while acetylation at K29 hinders the VPS34-Beclin 1 core complex formation. Inactivation of p300 induces VPS34 deacetylation, PI3P production, and autophagy, even in AMPK-/-, TSC2-/-, or ULK1-/- cells. In fasting mice, liver autophagy correlates well with p300 inactivation/VPS34 deacetylation, which facilitates the clearance of lipid droplets in hepatocytes. Thus, p300-dependent VPS34 acetylation/deacetylation is the physiological key to VPS34 activation, which controls the initiation of canonical autophagy and of non-canonical autophagy in which the upstream kinases of VPS34 can be bypassed.