RESUMO
Glucose is a universal bioenergy source; however, its role in controlling protein interactions is unappreciated, as are its actions during differentiation-associated intracellular glucose elevation. Azido-glucose click chemistry identified glucose binding to a variety of RNA binding proteins (RBPs), including the DDX21 RNA helicase, which was found to be essential for epidermal differentiation. Glucose bound the ATP-binding domain of DDX21, altering protein conformation, inhibiting helicase activity, and dissociating DDX21 dimers. Glucose elevation during differentiation was associated with DDX21 re-localization from the nucleolus to the nucleoplasm where DDX21 assembled into larger protein complexes containing RNA splicing factors. DDX21 localized to specific SCUGSDGC motif in mRNA introns in a glucose-dependent manner and promoted the splicing of key pro-differentiation genes, including GRHL3, KLF4, OVOL1, and RBPJ. These findings uncover a biochemical mechanism of action for glucose in modulating the dimerization and function of an RNA helicase essential for tissue differentiation.
Assuntos
RNA Helicases DEAD-box , Glucose , Queratinócitos , Nucléolo Celular/metabolismo , Núcleo Celular/metabolismo , RNA Helicases DEAD-box/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Glucose/metabolismo , Queratinócitos/citologia , Queratinócitos/metabolismo , HumanosRESUMO
DNA-protein interactions mediate physiologic gene regulation and may be altered by DNA variants linked to polygenic disease. To enhance the speed and signal-to-noise ratio (SNR) in the identification and quantification of proteins associated with specific DNA sequences in living cells, we developed proximal biotinylation by episomal recruitment (PROBER). PROBER uses high-copy episomes to amplify SNR, and proximity proteomics (BioID) to identify the transcription factors and additional gene regulators associated with short DNA sequences of interest. PROBER quantified both constitutive and inducible association of transcription factors and corresponding chromatin regulators to target DNA sequences and binding quantitative trait loci due to single-nucleotide variants. PROBER identified alterations in regulator associations due to cancer hotspot mutations in the hTERT promoter, indicating that these mutations increase promoter association with specific gene activators. PROBER provides an approach to rapidly identify proteins associated with specific DNA sequences and their variants in living cells.
Assuntos
Cromatina , DNA , Biotinilação , Cromatina/genética , DNA/genética , DNA/metabolismo , Plasmídeos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Aha1 is a co-chaperone of heat shock protein 90 (HSP90), and it stimulates the ATPase activity of HSP90 to promote the folding of its client proteins. By employing ascorbate peroxidase (APEX)-based proximity labeling and proteomic analysis, we identified over 30 proteins exhibiting diminished abundances in the proximity proteome of HSP90 in HEK293T cells upon genetic depletion of Aha1. Dicer1 is a top-ranked protein, and we confirmed its interactions with HSP90 and Aha1 by immunoprecipitation followed by western blot analysis. Genetic depletion of Aha1 and pharmacological inhibition of HSP90 both led to reduced levels of Dicer1 protein. Additionally, HSP90 and Aha1 bind preferentially to newly translated Dicer1. Reconstitution of Aha1-depleted cells with wild-type Aha1 substantially rescued Dicer1 protein level, and a lower level of restoration was observed for complementation with the HSP90-binding-defective Aha1-E67K, whereas an Aha1 mutant lacking the first 20 amino acids-which abolishes its chaperone activity-failed to rescue Dicer1 protein level. Moreover, knockdown of Aha1 and inhibition of HSP90 led to diminished levels of mature microRNAs (miRNAs), but not their corresponding primary miRNAs. Together, we uncovered a novel mechanism of HSP90 and Aha1 in regulating the miRNA pathway through promoting the folding of Dicer1 protein, and we also demonstrated that Aha1 modulates this process by acting as an autonomous chaperone and a co-chaperone for HSP90.
Assuntos
MicroRNAs , Humanos , MicroRNAs/genética , Proteômica , Células HEK293 , Ribonuclease III/genética , RNA Helicases DEAD-box/genéticaRESUMO
Osteogenesis is modulated by multiple regulatory networks. Recent studies showed that RNA modifications and their reader, writer, and eraser (RWE) proteins are involved in regulating various biological processes. Few studies, however, were conducted to investigate the functions of RNA modifications and their RWE proteins in osteogenesis. By using LC-MS/MS in parallel-reaction monitoring (PRM) mode, we performed a comprehensive quantitative assessment of 154 epitranscriptomic RWE proteins throughout the entire time course of osteogenic differentiation in H9 human embryonic stem cells (ESCs). We found that approximately half of the 127 detected RWE proteins were down-regulated during osteogenic differentiation, and they included mainly proteins involved in RNA methylation and pseudouridylation. Protein-protein interaction (PPI) network analysis unveiled significant associations between the down-regulated epitranscriptomic RWE proteins and osteogenesis-related proteins. Gene set enrichment analysis (GSEA) of publicly available RNA-seq data obtained from osteogenesis imperfecta patients suggested a potential role of METTL1 in osteogenesis through the cytokine network. Together, this is the first targeted profiling of epitranscriptomic RWE proteins during osteogenic differentiation of human ESCs, and our work unveiled potential regulatory roles of these proteins in osteogenesis. LC-MS/MS data were deposited on ProteomeXchange (PXD039249).
Assuntos
Células-Tronco Embrionárias Humanas , Osteogênese , Humanos , Osteogênese/genética , Cromatografia Líquida , Espectrometria de Massas em Tandem , Diferenciação Celular/genética , RNA/genéticaRESUMO
The small GTPase superfamily of proteins are crucial for numerous cellular processes, including early development. The roles of these proteins in osteogenic differentiation, however, remained poorly explored. In this study, we employed a high-throughput targeted proteomic method, relying on scheduled liquid chromatography-multiple-reaction monitoring (LC-MRM) coupled with synthetic stable isotope-labeled peptides, to interrogate systematically the temporal responses of the entire small GTPase proteome during the course of osteogenic differentiation of H9 human embryonic stem cells. Our results demonstrated that the method offers high quantification accuracy, reproducibility, and throughput. In addition, the quantification results revealed altered expression of a large number of small GTPases accompanied with osteogenic differentiation, especially those involved with autophagy. We also documented a previously unrecognized role of KRAS in osteogenesis, where it regulates the accumulation of extracellular matrix for mineralization through attenuating the activity of secreted matrix metalloproteinase 9 (MMP9). Together, this study represents a novel application of a state-of-the-art analytical method, i.e., targeted quantitative proteomics, for revealing the progressive reprogramming of the small GTPase proteome during osteogenic differentiation of human embryonic stem cells, and our results revealed KRAS as a new regulator for osteogenesis.
Assuntos
Proteínas Monoméricas de Ligação ao GTP , Humanos , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Osteogênese , Proteoma/metabolismo , Proteômica/métodos , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Reprodutibilidade dos Testes , Diferenciação CelularRESUMO
Viral proteins localize within subcellular compartments to subvert host machinery and promote pathogenesis. To study SARS-CoV-2 biology, we generated an atlas of 2422 human proteins vicinal to 17 SARS-CoV-2 viral proteins using proximity proteomics. This identified viral proteins at specific intracellular locations, such as association of accessary proteins with intracellular membranes, and projected SARS-CoV-2 impacts on innate immune signaling, ER-Golgi transport, and protein translation. It identified viral protein adjacency to specific host proteins whose regulatory variants are linked to COVID-19 severity, including the TRIM4 interferon signaling regulator which was found proximal to the SARS-CoV-2 M protein. Viral NSP1 protein adjacency to the EIF3 complex was associated with inhibited host protein translation whereas ORF6 localization with MAVS was associated with inhibited RIG-I 2CARD-mediated IFNB1 promoter activation. Quantitative proteomics identified candidate host targets for the NSP5 protease, with specific functional cleavage sequences in host proteins CWC22 and FANCD2. This data resource identifies host factors proximal to viral proteins in living human cells and nominates pathogenic mechanisms employed by SARS-CoV-2.
Assuntos
COVID-19/metabolismo , Interações Hospedeiro-Parasita/fisiologia , SARS-CoV-2/metabolismo , Proteínas Virais/metabolismo , Humanos , Biossíntese de Proteínas/fisiologia , Proteoma/metabolismoRESUMO
Radiation therapy benefits more than 50% of all cancer patients and cures 40% of them, where ionizing radiation (IR) deposits energy to cells and tissues, thereby eliciting DNA damage and resulting in cell death. Small GTPases are a superfamily of proteins that play critical roles in cell signaling. Several small GTPases, including RAC1, RHOB, and RALA, were previously shown to modulate radioresistance in cancer cells. However, there is no systematic proteomic study on small GTPases that regulate radioresistance in cancer cells. Herein, we applied a high-throughput scheduled multiple-reaction monitoring (MRM) method, along with the use of synthetic stable isotope-labeled (SIL) peptides, to identify differentially expressed small GTPase proteins in two pairs of breast cancer cell lines, MDA-MB-231 and MCF7, and their corresponding radioresistant cell lines. We identified 7 commonly altered small GTPase proteins with over 1.5-fold changes in the two pairs of cell lines. We also discovered ARFRP1 as a novel regulator of radioresistance, where its downregulation promotes radioresistance in breast cancer cells. Together, this represents the first comprehensive investigation about the differential expression of the small GTPase proteome associated with the development of radioresistance in breast cancer cells. Our work also uncovered ARFRP1 as a new target for enhancing radiation sensitivity in breast cancer.
Assuntos
Neoplasias da Mama , Proteínas Monoméricas de Ligação ao GTP , Humanos , Feminino , Proteômica/métodos , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Neoplasias da Mama/metabolismo , Células MCF-7 , Tolerância a Radiação/genética , Linhagem Celular TumoralRESUMO
As optimum temperature is essential for all living organisms, heat shock represents a challenging problem for their survival. Therefore, cellular response to heat shock is among the most extensively investigated stress response pathways; however, how the human proteome responds to heat shock has not been comprehensively investigated. In this study, we employed stable isotope labeling by amino acids in cell culture (SILAC), together with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, to fulfill an in-depth analysis of the alterations in the human proteome in M14 human melanoma cells in response to heat shock stress. We found that, after heat shock, 284 and 278 out of the 4319 quantified proteins were with substantially diminished and elevated expressions, respectively. We also examined the alterations in human kinome after heat shock by using our recently developed targeted proteomic method relying on parallel-reaction monitoring. Our results showed that the expression levels of 11 and 22 kinase proteins were increased and decreased, respectively, by at least 1.5-fold upon heat shock. By interrogating publicly available RNA-seq and m6A sequencing data, we observed that the elevated expression of more than 30 proteins, including CHEK1 and CCND3 kinases, could occur via an m6A-mediated mechanism. Furthermore, our results from single-base elongation and ligation-based quantitative polymerase chain reaction (qPCR) amplification (SELECT) and luciferase reporter assays revealed that heat shock gave rise to elevated m6A levels at A280 and A286 sites in the 5'-untranslated region of HSPH1 mRNA, thereby leading to increased translation of HSPH1 protein. Together, our discovery and targeted proteomic methods revealed the reprogramming of human proteome and kinome upon heat shock stress and provided insights into cellular responses toward heat shock stress.
Assuntos
Proteômica , Espectrometria de Massas em Tandem , Adenosina/análogos & derivados , Cromatografia Líquida , Resposta ao Choque Térmico , Humanos , Proteoma/genéticaRESUMO
Small GTPases are essential signaling molecules for regulating glucose uptake in adipose tissues upon insulin stimulation, and this regulation maintains an appropriate range of glycemia. The involvement of small GTPases in adipogenesis, however, has not been systemically investigated. In this study, we applied a high-throughput scheduled multiple-reaction monitoring (MRM) method, along with the use of synthetic stable isotope-labeled peptides, to identify differentially expressed small GTPase proteins during adipogenesis of cultured murine cells. We were able to quantify the relative levels of expression of 55 and 49 small GTPases accompanied by adipogenic differentiation in 3T3-L1 and C3H10T1/2 cells, respectively. When compared with analysis conducted in the data-dependent acquisition (DDA) mode, the MRM-based proteomic platform substantially increased the coverage of the small GTPase proteome. Western blot analysis further corroborated the MRM quantification results for selected small GTPases. Interestingly, overall a significant number of small GTPases were down-regulated during adipogenesis. Among them, the expression levels of Rab32 protein were consistently lower in differentiated adipocytes than the corresponding undifferentiated precursors in both cell lines. Overexpression of Rab32 in 3T3-L1 and C3H10T1/2 cells prior to adipogenesis induction suppressed their differentiation. Together, this is the first comprehensive analysis of the alterations in small GTPase proteome during adipogenesis, and we reveal a previously unrecognized role of Rab32 in adipogenic differentiation.
Assuntos
GTP Fosfo-Hidrolases/análise , Proteômica , Células 3T3-L1 , Adipócitos/metabolismo , Adipogenia , Animais , Diferenciação Celular , Células Cultivadas , Cromatografia Líquida , GTP Fosfo-Hidrolases/metabolismo , Ensaios de Triagem em Larga Escala , Camundongos , Espectrometria de Massas em TandemRESUMO
N6-Methyladenosine (m6A) in messenger RNA (mRNA) regulates its stability, splicing, and translation efficiency. Here, we explored how the expression levels of small GTPase proteins are regulated by m6A modulators. We employed a high-throughput scheduled multiple-reaction monitoring (MRM)-based targeted proteomic approach to quantify systemically the changes in expression of small GTPase proteins in cells upon genetic ablation of METTL3 (the catalytic subunit of the major m6A methyltransferase complex), m6A demethylases (ALKBH5 and FTO), or m6A reader proteins (YTHDF1, YTHDF2, and YTHDF3). Depletions of METTL3 and ALKBH5 resulted in substantially diminished and augmented expression, respectively, of a subset of small GTPase proteins, including RHOB and RHOC. Our results also revealed that the stability of RHOB mRNA is significantly increased in cells depleted of METTL3, suggesting an m6A-elicited destabilization of this mRNA. Those small GTPases that are targeted by METTL3 and/or ALKBH5 also displayed higher discrepancies between protein and mRNA expression in paired primary/metastatic melanoma or colorectal cancer cells than those that are not. Together, this is the first comprehensive analysis of the alterations in small GTPase proteome regulated by epitranscriptomic modulators of m6A, and our study suggests the potential of an alternative therapeutic approach to target the currently "undruggable" small GTPases.
Assuntos
Adenosina/análogos & derivados , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Proteoma/metabolismo , Adenosina/metabolismo , Células Cultivadas , Cromatografia Líquida , Células HEK293 , Humanos , Proteínas Monoméricas de Ligação ao GTP/análise , Proteínas Monoméricas de Ligação ao GTP/genética , Proteoma/análise , Proteoma/genética , Espectrometria de Massas em TandemRESUMO
Cytoadherence of Trichomonas vaginalis to human vaginal epithelial cells (hVECs) was previously shown to involve surface lipoglycans and several reputed adhesins on the parasite. Herein, we report some new observations on the host-parasite interactions of adherent versus nonadherent T. vaginalis isolates to hVECs. The binding of the TH17 adherent isolate to hVECs exhibited an initial discrete phase followed by an aggregation phase inhibited by lactose. T. vaginalis infection immediately induced surface expression of galectin-1 and -3, with extracellular amounts in the spent medium initially decreasing and then increasing thereafter over the next 60 min. Extracellular galectin-1 and -3 were detected on the parasite surface but only the TH17 adherent isolate could uptake galectin-3 via the lysosomes. Only the adherent isolate could morphologically transform from the round-up flagellate with numerous transient protrusions into a flat amoeboid form on contact with the solid surface. Cytochalasin D challenge revealed that actin organization was essential to parasite morphogenesis and cytoadherence. Real-time microscopy showed that parasite exploring and anchoring on hVECs via the axostyle may be required for initial cytoadherence. Together, the parasite cytoskeleton behaviors may collaborate with cell surface adhesion molecules for cytoadherence. The nonadherent isolate migrated faster than the adherent isolate, with motility transiently increasing in the presence of hVECs. Meanwhile, differential histone acetylation was detected between the two isolates. Also, TH17 without Mycoplasma symbiosis suggests that symbiont might not determine TH17 innate cytoadherence. Our findings regarding distinctive host-parasite interactions of the isolates may provide novel insights into T. vaginalis infection.
Assuntos
Trichomonas vaginalis , Feminino , Humanos , Galectina 1 , Interações Hospedeiro-Parasita , Adesão Celular , Células Epiteliais/parasitologia , Moléculas de Adesão CelularRESUMO
Mutations in the SOD1 (superoxide dismutase 1) gene are associated with amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease. By employing ascorbate peroxidase-based proximity labeling, coupled with LC-MS/MS analysis, we uncovered 43 and 24 proteins exhibiting higher abundance in the proximity proteomes of SOD1G85R and SOD1G93A, respectively, than that of wild-type SOD1. Immunoprecipitation followed by western blot analysis indicated the preferential binding of one of these proteins, exportin 5 (XPO5), toward the two mutants of SOD1 over the wild-type counterpart. In line with the established function of XPO5 in pre-miRNA transport, we observed diminished nucleocytoplasmic transport of pre-miRNAs in cells with ectopic expression of the two SOD1 mutants over those expressing the wild-type protein. On the other hand, RT-qPCR results revealed significant elevations in mature miRNA in cells expressing the two SOD1 mutants, which are attributed to the diminished inhibitory effect of XPO5 on Dicer-mediated cleavage of pre-miRNA to mature miRNA. Together, our chemoproteomic approach led to the revelation of a novel mechanism through which ALS-associated mutants of SOD1 perturb miRNA biogenesis, that is, through aberrant binding toward XPO5.
Assuntos
Esclerose Lateral Amiotrófica , MicroRNAs , Doenças Neurodegenerativas , Humanos , Animais , Camundongos , Superóxido Dismutase-1/genética , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Cromatografia Líquida , Superóxido Dismutase/metabolismo , Espectrometria de Massas em Tandem , Mutação , MicroRNAs/genética , Carioferinas/genética , Camundongos TransgênicosRESUMO
Viral proteins localize within subcellular compartments to subvert host machinery and promote pathogenesis. To study SARS-CoV-2 biology, we generated an atlas of 2422 human proteins vicinal to 17 SARS-CoV-2 viral proteins using proximity proteomics. This identified viral proteins at specific intracellular locations, such as association of accessary proteins with intracellular membranes, and projected SARS-CoV-2 impacts on innate immune signaling, ER-Golgi transport, and protein translation. It identified viral protein adjacency to specific host proteins whose regulatory variants are linked to COVID-19 severity, including the TRIM4 interferon signaling regulator which was found proximal to the SARS-CoV-2 M protein. Viral NSP1 protein adjacency to the EIF3 complex was associated with inhibited host protein translation whereas ORF6 localization with MAVS was associated with inhibited RIG-I 2CARD-mediated IFNB1 promoter activation. Quantitative proteomics identified candidate host targets for the NSP5 protease, with specific functional cleavage sequences in host proteins CWC22 and FANCD2. This data resource identifies host factors proximal to viral proteins in living human cells and nominates pathogenic mechanisms employed by SARS-CoV-2. AUTHOR SUMMARY: SARS-CoV-2 is the latest pathogenic coronavirus to emerge as a public health threat. We create a database of proximal host proteins to 17 SARS-CoV-2 viral proteins. We validate that NSP1 is proximal to the EIF3 translation initiation complex and is a potent inhibitor of translation. We also identify ORF6 antagonism of RNA-mediate innate immune signaling. We produce a database of potential host targets of the viral protease NSP5, and create a fluorescence-based assay to screen cleavage of peptide sequences. We believe that this data will be useful for identifying roles for many of the uncharacterized SARS-CoV-2 proteins and provide insights into the pathogenicity of new or emerging coronaviruses.
RESUMO
The ATP-sensitive potassium channel (KATP) functions as a metabo-electric transducer in regulating insulin secretion from pancreatic ß-cells. The pancreatic KATP channel is composed of a pore-forming inwardly-rectifying potassium channel, Kir6.2, and a regulatory subunit, sulphonylurea receptor 1 (SUR1). Loss-of-function mutations in either subunit often lead to the development of persistent hyperinsulinemic hypoglycemia of infancy (PHHI). PHHI is a rare genetic disease and most patients present with immediate onset within the first few days after birth. In this study, we report an unusual form of PHHI, in which the index patient developed hyperinsulinemic hypoglycemia after 1 year of age. The patient failed to respond to routine medication for PHHI and underwent a complete pancreatectomy. Genotyping of the index patient and his immediate family members showed that the patient and other family members with hypoglycemic episodes carried a heterozygous novel mutation in KCNJ11 (C83T), which encodes Kir6.2 (A28V). Electrophysiological and cell biological experiments revealed that A28V hKir6.2 is a dominant-negative, loss-of-function mutation and that KATP channels carrying this mutation failed to reach the cell surface. De novo protein structure prediction indicated that this A28V mutation reoriented the ER retention motif located at the C-terminal of the hKir6.2, and this result may explain the trafficking defect caused by this point mutation. Our study is the first report of a novel form of late-onset PHHI that is caused by a dominant mutation in KCNJ11 and exhibits a defect in proper surface expression of Kir6.2.