RESUMEN

Extracellular vesicles (EVs) frequently express human leukocyte antigen class I (HLA-I) molecules. The immunopeptidomes presented on EV HLA-I are being mapped to provide key information on both specific cancer-related peptides, and for larger immunopeptidomic signatures associated with disease. Utilizing HLA-I immunoisolation and mass spectrometry, we characterised the HLA-I immunopeptidome of EVs derived from the melanoma cancer cell line, ESTDAB-026, and the plasma of 12 patients diagnosed with advanced stage melanoma, alongside 11 healthy controls. The EV HLA-I immunopeptidome derived from melanoma cells features T cell epitopes with known immunogenicity and peptides derived from known tumour associated antigens (TAAs). Both T cell epitopes with known immunogenicity and peptides derived from known TAAs were also identifiable in the melanoma patient samples. Patient stratification into two distinct groups with varying immunological profiles was also observed. The data obtained in this study suggests for the first time that the HLA-I immunopeptidome of EVs derived from blood may aid in the detection of important diagnostic or prognostic biomarkers and also provide new immunotherapy targets.

RESUMEN

LMNA-related congenital muscular dystrophy (L-CMD) is caused by mutations in the LMNA gene, encoding lamin A/C. To further understand the molecular mechanisms of L-CMD, proteomic profiling using DIA mass spectrometry was conducted on immortalized myoblasts and myotubes from controls and L-CMD donors each harbouring a different LMNA mutation (R249W, del.32 K and L380S). Compared to controls, 124 and 228 differentially abundant proteins were detected in L-CMD myoblasts and myotubes, respectively, and were associated with enriched canonical pathways including synaptogenesis and necroptosis in myoblasts, and Huntington's disease and insulin secretion in myotubes. Abnormal nuclear morphology and reduced lamin A/C and emerin abundance was evident in all L-CMD cell lines compared to controls, while nucleoplasmic aggregation of lamin A/C was restricted to del.32 K cells, and mislocalization of emerin was restricted to R249W cells. Abnormal nuclear morphology indicates loss of nuclear lamina integrity as a common feature of L-CMD, likely rendering muscle cells vulnerable to mechanically induced stress, while differences between L-CMD cell lines in emerin and lamin A localization suggests that some molecular alterations in L-CMD are mutation specific. Nonetheless, identifying common proteomic alterations and molecular pathways across all three L-CMD lines has highlighted potential targets for the development of non-mutation specific therapies.

Asunto(s)

Lamina Tipo A , Distrofias Musculares , Proteómica , Humanos , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Distrofias Musculares/genética , Distrofias Musculares/metabolismo , Distrofias Musculares/patología , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patología , Mutación , Mioblastos/metabolismo , Masculino , Línea Celular , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo

RESUMEN

Structural, functional and molecular cardiac defects have been reported in spinal muscular atrophy (SMA) patients and mouse models. Previous quantitative proteomics analyses demonstrated widespread molecular defects in the severe Taiwanese SMA mouse model. Whether such changes are conserved across different mouse models, including less severe forms of the disease, has yet to be established. Here, using the same high-resolution proteomics approach in the less-severe Smn2B/- SMA mouse model, 277 proteins were found to be differentially abundant at a symptomatic timepoint (post-natal day (P) 18), 50 of which were similarly dysregulated in severe Taiwanese SMA mice. Bioinformatics analysis linked many of the differentially abundant proteins to cardiovascular development and function, with intermediate filaments highlighted as an enriched cellular compartment in both datasets. Lamin A/C was increased in the cardiac tissue, whereas another intermediate filament protein, desmin, was reduced. The extracellular matrix (ECM) protein, elastin, was also robustly decreased in the heart of Smn2B/- mice. AAV9-SMN1-mediated gene therapy rectified low levels of survival motor neuron protein and restored desmin levels in heart tissues of Smn2B/- mice. In contrast, AAV9-SMN1 therapy failed to correct lamin A/C or elastin levels. Intermediate filament proteins and the ECM have key roles in cardiac function and their dysregulation may explain cardiac impairment in SMA, especially since mutations in genes encoding these proteins cause other diseases with cardiac aberration. Cardiac pathology may need to be considered in the long-term care of SMA patients, as it is unclear whether currently available treatments can fully rescue peripheral pathology in SMA.

Asunto(s)

Neuronas Motoras , Atrofia Muscular Espinal , Humanos , Ratones , Animales , Neuronas Motoras/metabolismo , Desmina/genética , Desmina/metabolismo , Elastina/genética , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/terapia , Atrofia Muscular Espinal/patología , Terapia Genética , Modelos Animales de Enfermedad , Proteína 1 para la Supervivencia de la Neurona Motora/genética , Proteína 1 para la Supervivencia de la Neurona Motora/metabolismo

RESUMEN

Most research to characterise the molecular consequences of spinal muscular atrophy (SMA) has focused on SMA I. Here, proteomic profiling of skin fibroblasts from severe (SMA I), intermediate (SMA II), and mild (SMA III) patients, alongside age-matched controls, was conducted using SWATH mass spectrometry analysis. Differentially expressed proteomic profiles showed limited overlap across each SMA type, and variability was greatest within SMA II fibroblasts, which was not explained by SMN2 copy number. Despite limited proteomic overlap, enriched canonical pathways common to two of three SMA severities with at least one differentially expressed protein from the third included mTOR signalling, regulation of eIF2 and eIF4 signalling, and protein ubiquitination. Network expression clustering analysis identified protein profiles that may discriminate or correlate with SMA severity. From these clusters, the differential expression of PYGB (SMA I), RAB3B (SMA II), and IMP1 and STAT1 (SMA III) was verified by Western blot. All SMA fibroblasts were transfected with an SMN-enhanced construct, but only RAB3B expression in SMA II fibroblasts demonstrated an SMN-dependent response. The diverse proteomic profiles and pathways identified here pave the way for studies to determine their utility as biomarkers for patient stratification or monitoring treatment efficacy and for the identification of severity-specific treatments.

Asunto(s)

Atrofia Muscular Espinal , Proteoma , Western Blotting , Fibroblastos/metabolismo , Humanos , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/metabolismo , Proteoma/metabolismo , Proteómica

RESUMEN

Oesophageal adenocarcinoma (OAC) is an aggressive cancer with a five-year survival of <15%. Current chemotherapeutic strategies only benefit a minority (20-30%) of patients and there are no methods available to differentiate between responders and non-responders. We performed quantitative proteomics using Sequential Window Acquisition of all THeoretical fragment-ion spectra-Mass Spectrometry (SWATH-MS) on albumin/IgG-depleted and non-depleted plasma samples from 23 patients with locally advanced OAC prior to treatment. Individuals were grouped based on tumour regression (TRG) score (TRG1/2/3 vs TRG4/5) after chemotherapy, and differentially abundant proteins were compared. Protein depletion of highly abundant proteins led to the identification of around twice as many proteins. SWATH-MS revealed significant quantitative differences in the abundance of several proteins between the two groups. These included complement c1q subunit proteins, C1QA, C1QB and C1QC, which were of higher abundance in the low TRG group. Of those that were found to be of higher abundance in the high TRG group, glutathione S-transferase pi (GSTP1) exhibited the lowest p-value and highest classification accuracy and Cohen's kappa value. Concentrations of these proteins were further examined using ELISA-based assays. This study provides quantitative information relating to differences in the plasma proteome that underpin response to chemotherapeutic treatment in oesophageal cancers. SIGNIFICANCE: Oesophageal cancers, including oesophageal adenocarcinoma (OAC) and oesophageal gastric junction cancer (OGJ), are one of the leading causes of cancer mortality worldwide. Curative therapy consists of surgery, either alone or in combination with adjuvant or neoadjuvant chemotherapy or radiation, or combination chemoradiotherapy regimens. There are currently no clinico-pathological means of predicting which patients will benefit from chemotherapeutic treatments. There is therefore an urgent need to improve oesophageal cancer disease management and treatment strategies. This work compared proteomic differences in OAC patients who responded well to chemotherapy as compared to those who did not, using quantitative proteomics prior to treatment commencement. SWATH-MS analysis of plasma (with and without albumin/IgG-depletion) from OAC patients prior to chemotherapy was performed. This approach was adopted to determine whether depletion offered a significant improvement in peptide coverage. Resultant datasets demonstrated that depletion increased peptide coverage significantly. Additionally, there was good quantitative agreement between commonly observed peptides. Data analysis was performed by adopting both univariate as well as multivariate analysis strategies. Differentially abundant proteins were identified between treatment response groups based on tumour regression grade. Such proteins included complement C1q sub-components and GSTP1. This study provides a platform for further work, utilising larger sample sets across different treatment regimens for oesophageal cancer, that will aid the development of 'treatment response prediction assays' for stratification of OAC patients prior to chemotherapy.

Asunto(s)

Adenocarcinoma , Neoplasias Esofágicas , Neoplasias Gástricas , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/patología , Albúminas , Proteínas Sanguíneas/uso terapéutico , Complemento C1q/uso terapéutico , Neoplasias Esofágicas/tratamiento farmacológico , Neoplasias Esofágicas/patología , Humanos , Inmunoglobulina G , Proteómica/métodos , Neoplasias Gástricas/patología , Resultado del Tratamiento

RESUMEN

The enzyme m1A22-tRNA methyltransferase (TrmK) catalyzes the transfer of a methyl group to the N1 of adenine 22 in bacterial tRNAs. TrmK is essential for Staphylococcus aureus survival during infection but has no homolog in mammals, making it a promising target for antibiotic development. Here, we characterize the structure and function of S. aureus TrmK (SaTrmK) using X-ray crystallography, binding assays, and molecular dynamics simulations. We report crystal structures for the SaTrmK apoenzyme as well as in complexes with methyl donor SAM and co-product product SAH. Isothermal titration calorimetry showed that SAM binds to the enzyme with favorable but modest enthalpic and entropic contributions, whereas SAH binding leads to an entropic penalty compensated for by a large favorable enthalpic contribution. Molecular dynamics simulations point to specific motions of the C-terminal domain being altered by SAM binding, which might have implications for tRNA recruitment. In addition, activity assays for SaTrmK-catalyzed methylation of A22 mutants of tRNALeu demonstrate that the adenine at position 22 is absolutely essential. In silico screening of compounds suggested the multifunctional organic toxin plumbagin as a potential inhibitor of TrmK, which was confirmed by activity measurements. Furthermore, LC-MS data indicated the protein was covalently modified by one equivalent of the inhibitor, and proteolytic digestion coupled with LC-MS identified Cys92 in the vicinity of the SAM-binding site as the sole residue modified. These results identify a cryptic binding pocket of SaTrmK, laying a foundation for future structure-based drug discovery.

Asunto(s)

Proteínas Bacterianas , Staphylococcus aureus , ARNt Metiltransferasas , Adenina , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Cristalografía por Rayos X , Conformación Proteica , ARN de Transferencia/metabolismo , S-Adenosilmetionina/metabolismo , Staphylococcus aureus/enzimología , ARNt Metiltransferasas/química , ARNt Metiltransferasas/metabolismo

RESUMEN

The recent success of monoclonal antibody checkpoint inhibitor therapies that enhance the ability of CD8+ T cells to detect cancer-related antigenic peptides has refocused the need to fully understand the repertoire of peptides being presented to the immune system. Whilst the peptide ligandome presented by cell surface human leucocyte antigen class I (HLA-I) molecules on cancer cells has been studied extensively, the ligandome of extracellular vesicles (EVs) remains poorly defined. Here, we report the HLA-I ligandome of both the cell surface and EVs from eight breast cancer cell lines (MCF7, MDA-MB-231, MDA-MB-361, MDA-MB-415, MDA-MB-453, HCC 1806, HCC 1395, and HCC 1954), and additionally the melanoma cell line ESTDAB-056 and the multiple myeloma line RPMI 8226. Utilizing HLA-I immunoisolation and mass spectrometry, we detected a total of 6574 peptides from the cell surface and 2461 peptides from the EVs of the cell lines studied. Within the EV HLA-I ligandome, we identified 150 peptides derived from tumour associated antigenic proteins, of which 19 peptides have been shown to elicit T-cell responses in previous studies. Our data thus show the prevalence of clinically relevant tumour-associated antigenic peptides in the HLA-I ligandome presented on EV.

Asunto(s)

Carcinoma Hepatocelular , Vesículas Extracelulares , Neoplasias Hepáticas , Antígenos de Neoplasias , Linfocitos T CD8-positivos , Carcinoma Hepatocelular/metabolismo , Línea Celular , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Ligandos , Neoplasias Hepáticas/metabolismo , Péptidos

RESUMEN

There is a strong correlation between aging and onset of idiopathic Parkinson's disease, but little is known about whether cellular changes occur during normal aging that may explain this association. Here, proteomic and bioinformatic analysis was conducted on the substantia nigra (SN) of rats at four stages of life to identify and quantify protein changes throughout aging. This analysis revealed that proteins associated with cell adhesion, protein aggregation and oxidation-reduction are dysregulated as early as middle age in rats. Glial fibrillary acidic protein (GFAP) was identified as a network hub connecting the greatest number of proteins altered during aging. Furthermore, the isoform of GFAP expressed in the SN varied throughout life. However, the expression levels of the rate-limiting enzyme for dopamine production, tyrosine hydroxylase (TH), were maintained even in the oldest animals, despite a reduction in the number of dopamine neurons in the SN pars compact(SNc) as aging progressed. This age-related increase in TH expression per neuron would likely to increase the vulnerability of neurons, since increased dopamine production would be an additional source of oxidative stress. This, in turn, would place a high demand on support systems from local astrocytes, which themselves show protein changes that could affect their functionality. Taken together, this study highlights key processes that are altered with age in the rat SN, each of which converges upon GFAP. These findings offer insight into the relationship between aging and increased challenges to neuronal viability, and indicate an important role for glial cells in the aging process.

Asunto(s)

Envejecimiento/metabolismo , Neuronas Dopaminérgicas/metabolismo , Proteína Ácida Fibrilar de la Glía/metabolismo , Enfermedad de Parkinson/metabolismo , Sustancia Negra/metabolismo , Animales , Astrocitos/metabolismo , Femenino , Masculino , Proteómica , Ratas , Ratas Sprague-Dawley

RESUMEN

Commensal bacteria must colonize host mucosal surfaces to exert health-promoting properties, and bind to gastrointestinal tract (GIT) mucins via their cell surface adhesins. Considerable effort has been directed towards discovery of pathogen adhesins and their ligands to develop anti-infective strategies; however, little is known about the lectin-like adhesins and associated carbohydrate ligands in commensals. In this study, an in silico approach was used to detect surface exposed adhesins in the human commensal Lactobacillus paracasei subsp. paracasei, a promising probiotic commonly used in dairy product fermentation that presents anti-microbial activity. Of the 13 adhesin candidates, 3 sortase-dependent pili clusters were identified in this strain and expression of the adhesin candidate genes was confirmed in vitro. Mass spectrometry analysis confirmed the presence of surface adhesin elongation factor Tu and the chaperonin GroEL, but not pili expression. Whole cells were subsequently incubated on microarrays featuring a panel of GIT mucins from nine different mammalian species and two human-derived cell lines and a library of carbohydrate structures. Binding profiles were compared to those of two known pili-producing lactobacilli, L. johnsonii and L. rhamnosus and all Lactobacillus species displayed overlapping but distinct signatures, which may indicate different abilities for regiospecific GIT colonization. In addition, L. paracasei whole cells favoured binding to α-(2 â†’ 3)-linked sialic acid and α-(1 â†’ 2)-linked fucose-containing carbohydrate structures including blood groups A, B and O and Lewis antigens x, y and b. This study furthers our understanding of host-commensal cross-talk by identifying potential adhesins and specific GIT mucin and carbohydrate ligands and provides insight into the selection of colonization sites by commensals in the GIT.

Asunto(s)

Adhesinas Bacterianas/química , Carbohidratos/química , Microbioma Gastrointestinal , Glicómica/métodos , Lacticaseibacillus paracasei , Animales , Adhesión Bacteriana , Simulación por Computador , Fucosa/química , Humanos , Lactobacillus , Lactobacillus johnsonii , Lacticaseibacillus rhamnosus , Ligandos , Técnicas de Sonda Molecular , Probióticos , Unión Proteica , ARN Bacteriano/aislamiento & purificación , Propiedades de Superficie

RESUMEN

Cardiac pathology is emerging as a prominent systemic feature of spinal muscular atrophy (SMA), but little is known about the underlying molecular pathways. Using quantitative proteomics analysis, we demonstrate widespread molecular defects in heart tissue from the Taiwanese mouse model of severe SMA. We identify increased levels of lamin A/C as a robust molecular phenotype in the heart of SMA mice and show that lamin A/C dysregulation is also apparent in SMA patient fibroblast cells and other tissues from SMA mice. Lamin A/C expression was regulated in vitro by knockdown of the E1 ubiquitination factor ubiquitin-like modifier activating enzyme 1, a key downstream mediator of SMN-dependent disease pathways, converging on ß-catenin signaling. Increased levels of lamin A are known to increase the rigidity of nuclei, inevitably disrupting contractile activity in cardiomyocytes. The increased lamin A/C levels in the hearts of SMA mice therefore provide a likely mechanism explaining morphological and functional cardiac defects, leading to blood pooling. Therapeutic strategies directed at lamin A/C may therefore offer a new approach to target cardiac pathology in SMA.

Asunto(s)

Lamina Tipo A/metabolismo , Atrofia Muscular Espinal/metabolismo , Miocardio/patología , Animales , Modelos Animales de Enfermedad , Humanos , Lamina Tipo A/genética , Masculino , Ratones , Ratones Transgénicos , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/patología , Miocardio/metabolismo

RESUMEN

Dendritic cells are key immune cells that respond to pathogens and co-ordinate many innate and adaptive immune responses. Quantitative mass spectrometry using Sequential Window Acquisition of all THeoretical fragment-ion spectra-Mass Spectrometry (SWATH-MS) was performed here to determine the global alterations in monocyte-derived dendritic cells (moDCs) in response to stimulation with lipopolysaccharide (LPS). A moDC library of 4,666 proteins was generated and proteins were quantified at 0, 6 and 24 h post-LPS stimulation using SWATH-MS. At 6 h and 24 h post-LPS exposure, the relative abundance of 227 and 282 proteins was statistically significantly altered (p-value ≤ 0.05), respectively. Functional annotation of proteins exhibiting significant changes in expression between the various time points led to the identification of clusters of proteins implicated in distinct cellular processes including interferon and interleukin signalling, endocytosis, the ER-phagosome pathway and antigen-presentation. In SWATH-MS major histocompatibility complex (MHC) class I proteins were highly upregulated at 24 h, whilst MHC class II proteins exhibited comparatively fewer changes over this period. This study provides new detailed insight into the global proteomic changes that occur in moDCs during antigen processing and presentation and further demonstrates the potential of SWATH-MS for the quantitative study of proteins involved in cellular processes.

Asunto(s)

Células Dendríticas/efectos de los fármacos , Lipopolisacáridos/farmacología , Monocitos/efectos de los fármacos , Proteómica , Citocinas/metabolismo , Células Dendríticas/metabolismo , Endocitosis , Humanos , Espectrometría de Masas , Monocitos/citología , Monocitos/metabolismo , Fagocitosis

RESUMEN

Age-related macular degeneration (AMD) is associated with the formation of sub-retinal pigment epithelial (RPE) deposits that block circulatory exchange with the retina. The factors that contribute to deposit formation are not well understood. Recently, we identified the presence of spherular hydroxyapatite (HAP) structures within sub-RPE deposits to which several AMD-associated proteins were bound. This suggested that protein binding to HAP represents a potential mechanism for the retention of proteins in the sub-RPE space. Here we performed quantitative proteomics using Sequential Window Acquisition of all THeoretical fragment-ion spectra-Mass Spectrometry (SWATH-MS) on plasma samples from 23 patients with late-stage neovascular AMD following HAP-binding. Individuals were genotyped for the high risk CFH variant (T1277C) and binding to HAP was compared between wild type and risk variants. From a library of 242 HAP binding plasma proteins (1% false discovery rate), SWATH-MS revealed significant quantitative differences in the abundance of 32 HAP-binding proteins (p < 0.05) between the two homozygous groups. The concentrations of six proteins (FHR1, FHR3, APOC4, C4A, C4B and PZP) in the HAP eluted fractions and whole plasma were further analysed using ELISA and their presence in sections from human cadaver eyes was examined using immunofluorescence. All six proteins were found to be present in the RPE/choroid interface, and four of these (FHR1, FHR3, APOC4 and PZP) were associated with spherules in sub-RPE space. This study provides qualitative and quantitative information relating to the degree by which plasma proteins may contribute to sub-RPE deposit formation through binding to HAP spherules and how genetic differences might contribute to deposit formation.

Asunto(s)

Proteínas Sanguíneas/metabolismo , Durapatita/metabolismo , Degeneración Macular Húmeda/sangre , Anciano , Anciano de 80 o más Años , Secuencia de Aminoácidos , Proteínas Sanguíneas/genética , Factor H de Complemento/genética , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente Indirecta , Técnicas de Genotipaje , Humanos , Espectrometría de Masas , Datos de Secuencia Molecular , Unión Proteica , Proteómica , Degeneración Macular Húmeda/genética

RESUMEN

Cyclic ribosomally derived peptides possess diverse bioactivities and are currently of major interest in drug development. However, it can be chemically challenging to synthesize these molecules, hindering the diversification and testing of cyclic peptide leads. Enzymes used in vitro offer a solution to this; however peptide macrocyclization remains the bottleneck. PCY1, involved in the biosynthesis of plant orbitides, belongs to the class of prolyl oligopeptidases and natively displays substrate promiscuity. PCY1 is a promising candidate for in vitro utilization, but its substrates require an 11 to 16 residue C-terminal recognition tail. We have characterized PCY1 both kinetically and structurally with multiple substrate complexes revealing the molecular basis of recognition and catalysis. Using these insights, we have identified a three residue C-terminal extension that replaces the natural recognition tail permitting PCY1 to operate on synthetic substrates. We demonstrate that PCY1 can macrocyclize a variety of substrates with this short tail, including unnatural amino acids and nonamino acids, highlighting PCY1's potential in biocatalysis.

Asunto(s)

Descubrimiento de Drogas , Péptidos Cíclicos/metabolismo , Plantas/enzimología , Biocatálisis , Compuestos Macrocíclicos/síntesis química , Prolil Oligopeptidasas , Serina Endopeptidasas/metabolismo , Especificidad por Sustrato

RESUMEN

Extracellular vesicles (EVs) are released by most cell types and have been associated with multiple immunomodulatory functions. MHC class I molecules have crucial roles in antigen presentation and in eliciting immune responses and are known to be incorporated into EVs. However, the MHC class I immunopeptidome of EVs has not been established. Here, using a small-scale immunoisolation of the antigen serotypes HLA-A*02:01 and HLA-B*27:05 expressed on the Epstein-Barr virus-transformed B cell line Jesthom and MS of the eluted peptides from both cells and EVs, we identified 516 peptides that bind either HLA-A*02:01 or HLA-B*27:05. Of importance, the predicted serotype-binding affinities and peptide-anchor motifs did not significantly differ between the peptide pools isolated from cells or EVs, indicating that during EV biogenesis, no obvious editing of the MHC class I immunopeptidome occurs. These results, for the first time, establish EVs as a source of MHC class I peptides that can be used for the study of the immunopeptidome and in the discovery of potential neoantigens for immunotherapies.

Asunto(s)

Antígenos/química , Linfocitos B/química , Antígeno HLA-A2/química , Antígeno HLA-B27/química , Péptidos/química , Antígenos/inmunología , Linfocitos B/inmunología , Línea Celular Transformada , Antígeno HLA-A2/inmunología , Antígeno HLA-B27/inmunología , Humanos , Péptidos/inmunología

RESUMEN

The complexity of signalling pathways was boosted at the origin of the vertebrates, when two rounds of whole genome duplication (2R-WGD) occurred. Those genes and proteins that have survived from the 2R-WGD-termed 2R-ohnologues-belong to families of two to four members, and are enriched in signalling components relevant to cancer. Here, we find that while only approximately 30% of human transcript-coding genes are 2R-ohnologues, they carry 42-60% of the gene mutations in 30 different cancer types. Across a subset of cancer datasets, including melanoma, breast, lung adenocarcinoma, liver and medulloblastoma, we identified 673 2R-ohnologue families in which one gene carries mutations at multiple positions, while sister genes in the same family are relatively mutation free. Strikingly, in 315 of the 322 2R-ohnologue families displaying such a skew in multiple cancers, the same gene carries the heaviest mutation load in each cancer, and usually the second-ranked gene is also the same in each cancer. Our findings inspire the hypothesis that in certain cancers, heterogeneous combinations of genetic changes impair parts of the 2R-WGD signalling networks and force information flow through a limited set of oncogenic pathways in which specific non-mutated 2R-ohnologues serve as effectors. The non-mutated 2R-ohnologues are therefore potential therapeutic targets. These include proteins linked to growth factor signalling, neurotransmission and ion channels.

Asunto(s)

Genoma Humano , Neoplasias/genética , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Bases de Datos Genéticas , Evolución Molecular , Duplicación de Gen , Humanos , Mutación , Neoplasias/metabolismo , Neoplasias/patología , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , ARN Mensajero/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Proteínas ras/genética , Proteínas ras/metabolismo

RESUMEN

How does signalling via PI3K-PKB (AKT)-mTORC1-p70S6K and ERK-p90RSK mediate wide-ranging physiological responses to insulin? Quantitative proteomics and biochemical experiments are revealing that these signalling pathways induce the phosphorylation of large and overlapping sets of proteins, which are then captured by phosphoprotein-binding proteins named 14-3-3s. The 14-3-3s are dimers that dock onto dual-phosphorylated sites in a configuration with special signalling and mechanical properties. They interact with the Rab GTPase-activating proteins AS160 and TBC1D1 to regulate glucose uptake into target tissues in response to insulin and energy stress. Dynamic patterns in the 14-3-3-binding phosphoproteome are providing new insights into how insulin triggers coherent shifts in metabolism that are integrated with other cellular response systems.

Asunto(s)

Proteínas 14-3-3/metabolismo , Insulina/metabolismo , Fosfoproteínas/metabolismo , Proteínas 14-3-3/fisiología , Animales , Humanos , Insulina/fisiología , Fosfoproteínas/fisiología , Fosforilación , Proteoma/metabolismo , Proteoma/fisiología

RESUMEN

Hundreds of candidate 14-3-3-binding (phospho)proteins have been reported in publications that describe one interaction at a time, as well as high-throughput 14-3-3-affinity and mass spectrometry-based studies. Here, we transcribed these data into a common format, deposited the collated data from low-throughput studies in MINT (http://mint.bio.uniroma2.it/mint), and compared the low- and high-throughput data in VisANT graphs that are easy to analyze and extend. Exploring the graphs prompted questions about technical and biological specificity, which were addressed experimentally, resulting in identification of phosphorylated 14-3-3-binding sites in the mitochondrial import sequence of the iron-sulfur cluster assembly enzyme (ISCU), cytoplasmic domains of the mitochondrial fission factor (MFF), and endoplasmic reticulum-tethered receptor expression-enhancing protein 4 (REEP4), RNA regulator SMAUG2, and cytoskeletal regulatory proteins, namely debrin-like protein (DBNL) and kinesin light chain (KLC) isoforms. Therefore, 14-3-3s undergo physiological interactions with proteins that are destined for diverse subcellular locations. Graphing and validating interactions underpins efforts to use 14-3-3-phosphoproteomics to identify mechanisms and biomarkers for signaling pathways in health and disease.

Asunto(s)

Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Mitocondrias/metabolismo , Fosfoproteínas/análisis , Fosfoproteínas/metabolismo , Animales , Sitios de Unión/genética , Biomarcadores/metabolismo , Bases de Datos de Proteínas , Células HEK293 , Humanos , Espectrometría de Masas , Ratones , Mitocondrias/genética , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transducción de Señal

RESUMEN

The structural analysis of macromolecular functional protein assemblies by contemporary high resolution structural biology techniques (such as nuclear magnetic resonance, X-ray crystallography, and electron microscopy) is often still challenging. The potential of a rather new method to generate structural information, native mass spectrometry, in combination with ion mobility mass spectrometry (IM-MS), is highlighted here. IM-MS allows the assessment of gas phase ion collision cross sections of protein complex ions, which can be related to overall shapes/volumes of protein assemblies, and thus be used to monitor changes in structure. Here we applied IM-MS to study several (intermediate) chaperonin complexes that can be present during substrate folding. Our results reveal that the protein assemblies retain their solution phase structural properties in the gas phase, addressing a long-standing issue in mass spectrometry. All IM-MS data on the chaperonins point toward the burial of genuine substrates inside the GroEL cavity being retained in the gas phase. Additionally, the overall dimensions of the ternary complexes between GroEL, a substrate, and cochaperonin were found to be similar to the dimensions of the empty GroEL-GroES complex. We also investigated the effect of reducing the charge, obtained in the electrospray process, of the protein complex on the global shape of the chaperonin. At decreased charge, the protein complex was found to be more compact, possibly occupying a lower number of conformational states, enabling an improved ion mobility separation. Charge state reduction was found not to affect the relative differences observed in collision cross sections for the chaperonin assemblies.

Asunto(s)

Chaperoninas/química , Hierro/química , Calibración , Chaperoninas/metabolismo , Gases/química , Hierro/metabolismo , Espectrometría de Masas , Modelos Moleculares , Unión Proteica , Conformación Proteica

RESUMEN

Here we combined tandem affinity purification with several mass-spectrometry-based approaches to gain more insight into the composition and structure of the yeast nuclear-cytoplasmic exosome protein complex. The yeast exosome fulfills several different functions in RNA metabolism and can be localized in both the cytoplasm and the nucleus. These two exosome complexes differ in protein composition, although they share several constituents. We focused on these differences in composition by selecting a nuclear-specific exosome protein (Rrp6) and a cytoplasmic-specific protein (Ski7) as the tandem-affinity-purification-tagged affinity bait protein. First, we investigated both these purified exosome assemblies by macromolecular mass spectrometry (MS) to determine the stability and mass of the intact protein complexes and to obtain information on composition and core constituents. We used tandem MS on these intact protein complexes to further probe the composition and to obtain insight into the peripheral nature of some of the constituents. Finally, we combine stable isotope labeling with MS to quantitate differences in exosome composition and posttranslational modifications. We identified a few phosphorylation sites that are differentially regulated between the cytoplasmic exosome and the nuclear exosome. From all of these data, we conclude that the yeast nuclear exosome and the cytoplasmic exosome share a common stable core complex, but are decorated with quite a few differing peripheral proteins. We show that the nuclear exosome selectively copurifies with the alpha/beta importin heterodimer, which is known to be involved in the transport of proteins across the nuclear membrane.

Asunto(s)

Núcleo Celular/metabolismo , Exosomas/metabolismo , Saccharomyces cerevisiae/metabolismo , Espectrometría de Masas en Tándem , Secuencia de Aminoácidos , Marcaje Isotópico , Datos de Secuencia Molecular , Fosforilación , Proteómica , Proteínas de Saccharomyces cerevisiae/análisis , Proteínas de Saccharomyces cerevisiae/química

RESUMEN

Hydroquinone 1,2-dioxygenase (HQDO), an enzyme involved in the catabolism of 4-hydroxyacetophenone in Pseudomonas fluorescens ACB, was purified to apparent homogeneity. Ligandation with 4-hydroxybenzoate prevented the enzyme from irreversible inactivation. HQDO was activated by iron(II) ions and catalyzed the ring fission of a wide range of hydroquinones to the corresponding 4-hydroxymuconic semialdehydes. HQDO was inactivated by 2,2'-dipyridyl, o-phenanthroline, and hydrogen peroxide and inhibited by phenolic compounds. The inhibition with 4-hydroxybenzoate (K(i) = 14 microM) was competitive with hydroquinone. Online size-exclusion chromatography-mass spectrometry revealed that HQDO is an alpha2beta2 heterotetramer of 112.4 kDa, which is composed of an alpha-subunit of 17.8 kDa and a beta-subunit of 38.3 kDa. Each beta-subunit binds one molecule of 4-hydroxybenzoate and one iron(II) ion. N-terminal sequencing and peptide mapping and sequencing based on matrix-assisted laser desorption ionization--two-stage time of flight analysis established that the HQDO subunits are encoded by neighboring open reading frames (hapC and hapD) of a gene cluster, implicated to be involved in 4-hydroxyacetophenone degradation. HQDO is a novel member of the family of nonheme-iron(II)-dependent dioxygenases. The enzyme shows insignificant sequence identity with known dioxygenases.

Asunto(s)

Hidroquinonas/metabolismo , Oxigenasas/aislamiento & purificación , Oxigenasas/metabolismo , Pseudomonas fluorescens/enzimología , Pseudomonas fluorescens/metabolismo , 2,2'-Dipiridil/farmacología , Acetofenonas/metabolismo , Secuencia de Aminoácidos , Cromatografía en Gel , ADN Bacteriano/genética , Activadores de Enzimas/farmacología , Inhibidores Enzimáticos/farmacología , Estabilidad de Enzimas , Ácidos Grasos Insaturados/metabolismo , Peróxido de Hidrógeno/farmacología , Hierro/farmacología , Espectrometría de Masas , Datos de Secuencia Molecular , Peso Molecular , Familia de Multigenes , Sistemas de Lectura Abierta , Oxigenasas/química , Oxigenasas/genética , Parabenos/metabolismo , Fenantrolinas/farmacología , Subunidades de Proteína/química , Pseudomonas fluorescens/genética , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Temperatura