RESUMEN
Malignant cutaneous melanoma is one of the most common cancers in young adults. During the last decade, targeted and immunotherapies have significantly increased the overall survival of patients with malignant cutaneous melanoma. Nevertheless, disease progression is common, and a lack of predictive biomarkers of patient response to therapy hinders individualized treatment strategies. To address this issue, we performed a longitudinal study using an unbiased proteomics approach to identify and quantify proteins in plasma both before and during treatment from 109 patients treated with either targeted or immunotherapy. Linear modeling and machine learning approaches identified 43 potential prognostic and predictive biomarkers. A reverse correlation between apolipoproteins and proteins related to inflammation was observed. In the immunotherapy group, patients with low pretreatment expression of apolipoproteins and high expression of inflammation markers had shorter progression-free survival. Similarly, increased expression of LDHB during treatment elicited a significant impact on response to immunotherapy. Overall, we identified potential common and treatment-specific biomarkers in malignant cutaneous melanoma, paving the way for clinical use of these biomarkers following validation on a larger cohort. SIGNIFICANCE: This study identifies a potential biomarker panel that could improve the selection of therapy for patients with cutaneous melanoma.
Asunto(s)
Apolipoproteínas/sangre , Proteína C-Reactiva/análisis , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Inmunoterapia/métodos , Melanoma/sangre , Melanoma/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteoma/análisis , Proteína Amiloide A Sérica/análisis , Neoplasias Cutáneas/sangre , Neoplasias Cutáneas/tratamiento farmacológico , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/sangre , Femenino , Humanos , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Pronóstico , Supervivencia sin Progresión , Inhibidores de Proteínas Quinasas/farmacología , Proteómica/métodos , Adulto Joven , Melanoma Cutáneo MalignoRESUMEN
Current treatment modalities for disseminated cutaneous malignant melanoma (CMM) improve survival, however disease progression commonly ensues. In a previous study we identified afatinib and crizotinib in combination as a novel potential therapy for CMM independent of BRAF/NRAS mutation status. Herein, we elucidate the underlying mechanisms of the combination treatment effect to find biomarkers and novel targets for development of therapy that may provide clinical benefit by proteomic analysis of CMM cell lines and xenografts using mass spectrometry based analysis and reverse phase protein array. Identified candidates were validated using immunoblotting or immunofluorescence. Our analysis revealed that mTOR/Insulin signaling pathways were significantly decreased by the afatinib and crizotinib combination treatment. Both in vitro and in vivo analyses showed that the combination treatment downregulated pRPS6KB1 and pRPS6, downstream of mTOR signaling, and IRS-1 in the insulin signaling pathway, specifically ablating IRS-1 nuclear signal. Silencing of RPS6 and IRS-1 alone had a similar effect on cell death, which was further induced when IRS-1 and RPS6 were concomitantly silenced in the CMM cell lines. Silencing of IRS-1 and RPS6 resulted in reduced sensitivity towards combination treatment. Additionally, we found that IRS-1 and RPS6KB1 expression levels were increased in advanced stages of CMM clinical samples. We could demonstrate that induced resistance towards combination treatment was reversible by a drug holiday. CD171/L1CAM, mTOR and PI3K-p85 were induced in the combination resistant cells whereas AXL and EPHA2, previously identified mediators of resistance to MAPK inhibitor therapy in CMM were downregulated. We also found that CD171/L1CAM and mTOR were increased at progression in tumor biopsies from two matched cases of patients receiving targeted therapy with BRAFi. Overall, these findings provide insights into the molecular mechanisms behind the afatinib and crizotinib combination treatment effect and leverages a platform for discovering novel biomarkers and therapy regimes for CMM treatment.
Asunto(s)
Afatinib/farmacología , Crizotinib/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Melanoma/tratamiento farmacológico , Neoplasias Cutáneas/tratamiento farmacológico , Antineoplásicos/farmacología , Humanos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas B-raf/metabolismo , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/efectos de los fármacos , Melanoma Cutáneo MalignoRESUMEN
The cell cycle is a highly conserved process involving the coordinated separation of a single cell into two daughter cells. To relate transcriptional regulation across the cell cycle with oscillatory changes in protein abundance and activity, we carried out a proteome- and phospho-proteome-wide mass spectrometry profiling. We compared protein dynamics with gene transcription, revealing many transcriptionally regulated G2 mRNAs that only produce a protein shift after mitosis. Integration of CRISPR/Cas9 survivability studies further highlighted proteins essential for cell viability. Analyzing the dynamics of phosphorylation events and protein solubility dynamics over the cell cycle, we characterize predicted phospho-peptide motif distributions and predict cell cycle-dependent translocating proteins, as exemplified by the S-adenosylmethionine synthase MAT2A. Our study implicates this enzyme in translocating to the nucleus after the G1/S-checkpoint, which enables epigenetic histone methylation maintenance during DNA replication. Taken together, this data set provides a unique integrated resource with novel insights on cell cycle dynamics.
Asunto(s)
Ciclo Celular/genética , Perfilación de la Expresión Génica , Proteínas de Neoplasias/genética , Núcleo Celular/metabolismo , Células HeLa , Humanos , Proteínas de Neoplasias/metabolismo , Fosforilación , Transporte de Proteínas , Proteoma/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Fracciones Subcelulares/metabolismo , Transcriptoma/genéticaRESUMEN
The availability of proteomics resources hosting protein and peptide standards, as well as the data describing their analytical performances, will continue to enhance our current capabilities to develop targeted proteomics methods for quantitative biology. This study describes the analysis of a resource of 26,840 individually purified recombinant protein fragments corresponding to more than 16,000 human protein-coding genes. The resource was screened to identify proteotypic peptides suitable for targeted proteomics efforts, and we report LC-MS/MS assay coordinates for more than 25,000 proteotypic peptides, corresponding to more than 10,000 unique proteins. Additionally, peptide formation and digestion kinetics were, for a subset of the standards, monitored using a time-course protocol involving parallel digestion of isotope-labeled recombinant protein standards and endogenous human plasma proteins. We show that the strategy by adding isotope-labeled recombinant proteins before trypsin digestion enables short digestion protocols (≤60 min) with robust quantitative precision. In a proof-of-concept study, we quantified 23 proteins in human plasma using assay parameters defined in our study and used the standards to describe distinct clusters of individuals linked to different levels of LPA, APOE, SERPINA5, and TFRC. In summary, we describe the use and utility of a resource of recombinant proteins to identify proteotypic peptides useful for targeted proteomics assay development.
Asunto(s)
Fragmentos de Péptidos/análisis , Proteómica/métodos , Proteínas Recombinantes/análisis , Proteínas Sanguíneas/análisis , Cromatografía Liquida/métodos , Humanos , Marcaje Isotópico/métodos , Espectrometría de Masas en Tándem/métodos , Tripsina/metabolismoRESUMEN
Cyanobacteria must balance separate demands for energy generation, carbon assimilation, and biomass synthesis. We used shotgun proteomics to investigate proteome allocation strategies in the model cyanobacterium Synechocystis sp. PCC 6803 as it adapted to light and inorganic carbon (Ci) limitation. When partitioning the proteome into seven functional sectors, we find that sector sizes change linearly with growth rate. The sector encompassing ribosomes is significantly smaller than in E. coli, which may explain the lower maximum growth rate in Synechocystis. Limitation of light dramatically affects multiple proteome sectors, whereas the effect of Ci limitation is weak. Carbon assimilation proteins respond more strongly to changes in light intensity than to Ci. A coarse-grained cell economy model generally explains proteome trends. However, deviations from model predictions suggest that the large proteome sectors for carbon and light assimilation are not optimally utilized under some growth conditions and may constrain the proteome space available to ribosomes.
Asunto(s)
Proteínas Bacterianas/metabolismo , Dióxido de Carbono/farmacología , Cianobacterias/crecimiento & desarrollo , Regulación Bacteriana de la Expresión Génica , Luz , Proteoma/análisis , Cianobacterias/efectos de los fármacos , Cianobacterias/efectos de la radiación , Proteoma/efectos de los fármacos , Proteoma/efectos de la radiaciónRESUMEN
There is a need for standardized validation methods for antibody specificity and selectivity. Recently, five alternative validation pillars were proposed to explore the specificity of research antibodies using methods with no need for prior knowledge about the protein target. Here, we show that these principles can be used in a streamlined manner for enhanced validation of research antibodies in Western blot applications. More than 6,000 antibodies were validated with at least one of these strategies involving orthogonal methods, genetic knockdown, recombinant expression, independent antibodies, and capture mass spectrometry analysis. The results show a path forward for efforts to validate antibodies in an application-specific manner suitable for both providers and users.
Asunto(s)
Anticuerpos/inmunología , Especificidad de Anticuerpos/inmunología , Ensayos Analíticos de Alto Rendimiento/métodos , Estudios de Validación como Asunto , Animales , Western Blotting/métodos , Western Blotting/normas , Ensayos Analíticos de Alto Rendimiento/normas , Humanos , Estándares de Referencia , Reproducibilidad de los ResultadosRESUMEN
Intrinsic and/or acquired resistance represents one of the great challenges in targeted cancer therapy. A deeper understanding of the molecular biology of cancer has resulted in more efficient strategies, where one or multiple drugs are adopted in novel therapies to tackle resistance. This beneficial effect of using combination treatments has also been observed in colorectal cancer patients harboring the BRAF(V600E) mutation, whereby dual inhibition of BRAF(V600E) and EGFR increases antitumor activity. Notwithstanding this success, it is not clear whether this combination treatment is the only or most effective treatment to block intrinsic resistance to BRAF inhibitors. Here, we investigate molecular responses upon single and multi-target treatments, over time, using BRAF(V600E) mutant colorectal cancer cells as a model system. Through integration of transcriptomic, proteomic and phosphoproteomics data we obtain a comprehensive overview, revealing both known and novel responses. We primarily observe widespread up-regulation of receptor tyrosine kinases and metabolic pathways upon BRAF inhibition. These findings point to mechanisms by which the drug-treated cells switch energy sources and enter a quiescent-like state as a defensive response, while additionally compensating for the MAPK pathway inhibition.
Asunto(s)
Neoplasias Colorrectales/patología , Receptores ErbB/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Biología de Sistemas/métodos , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Neoplasias Colorrectales/genética , Regulación hacia Abajo/efectos de los fármacos , Sinergismo Farmacológico , Receptores ErbB/metabolismo , Retroalimentación Fisiológica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Técnicas de Inactivación de Genes , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Modelos Biológicos , Mutación/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Proteínas Proto-Oncogénicas B-raf/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
Novel therapies are undergoing clinical trials, for example, the Hsp90 inhibitor, XL888, in combination with BRAF inhibitors for the treatment of therapy-resistant melanomas. Unfortunately, our data show that this combination elicits a heterogeneous response in a panel of melanoma cell lines including PDX-derived models. We sought to understand the mechanisms underlying the differential responses and suggest a patient stratification strategy. Thermal proteome profiling (TPP) identified the protein targets of XL888 in a pair of sensitive and unresponsive cell lines. Unbiased proteomics and phosphoproteomics analyses identified CDK2 as a driver of resistance to both BRAF and Hsp90 inhibitors and its expression is regulated by the transcription factor MITF upon XL888 treatment. The CDK2 inhibitor, dinaciclib, attenuated resistance to both classes of inhibitors and combinations thereof. Notably, we found that MITF expression correlates with CDK2 upregulation in patients; thus, dinaciclib would warrant consideration for treatment of patients unresponsive to BRAF-MEK and/or Hsp90 inhibitors and/or harboring MITF amplification/overexpression.
Asunto(s)
Compuestos de Azabiciclo/farmacología , Quinasa 2 Dependiente de la Ciclina/metabolismo , Resistencia a Antineoplásicos , Imidazoles/farmacología , Melanoma/metabolismo , Factor de Transcripción Asociado a Microftalmía/metabolismo , Oximas/farmacología , Ácidos Ftálicos/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Óxidos N-Cíclicos , Quinasa 2 Dependiente de la Ciclina/genética , Resistencia a Antineoplásicos/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Humanos , Indolizinas , Melanoma/tratamiento farmacológico , Melanoma/genética , Factor de Transcripción Asociado a Microftalmía/genética , Fosfoproteínas/metabolismo , Proteómica , Compuestos de Piridinio/farmacología , Regulación hacia ArribaRESUMEN
Glioma-initiating cells (GIC) are considered the underlying cause of recurrences of aggressive glioblastomas, replenishing the tumor population and undermining the efficacy of conventional chemotherapy. Here we report the discovery that inhibiting T-type voltage-gated Ca2+ and KCa channels can effectively induce selective cell death of GIC and increase host survival in an orthotopic mouse model of human glioma. At present, the precise cellular pathways affected by the drugs affecting these channels are unknown. However, using cell-based assays and integrated proteomics, phosphoproteomics, and transcriptomics analyses, we identified the downstream signaling events these drugs affect. Changes in plasma membrane depolarization and elevated intracellular Na+, which compromised Na+-dependent nutrient transport, were documented. Deficits in nutrient deficit acted in turn to trigger the unfolded protein response and the amino acid response, leading ultimately to nutrient starvation and GIC cell death. Our results suggest new therapeutic targets to attack aggressive gliomas. Cancer Res; 77(7); 1741-52. ©2017 AACR.
Asunto(s)
Aminoácidos/metabolismo , Neoplasias Encefálicas/tratamiento farmacológico , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo T/fisiología , Glioma/tratamiento farmacológico , Canales de Potasio Calcio-Activados/antagonistas & inhibidores , Respuesta de Proteína Desplegada/efectos de los fármacos , Animales , Transporte Biológico , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Muerte Celular , Línea Celular Tumoral , Dihidropiridinas/farmacología , Glioma/metabolismo , Glioma/patología , Humanos , Ratones , Micotoxinas/farmacología , Células Madre Neoplásicas/patología , Proteómica , Sodio/metabolismoRESUMEN
No effective targeted therapy is currently available for NRAS mutant melanoma. Experimental MEK inhibition is rather toxic and has only limited efficacy in clinical trials. At least in part, this is caused by the emergence of drug resistance, which is commonly seen for single agent treatment and shortens clinical responses. Therefore, there is a dire need to identify effective companion drug targets for NRAS mutant melanoma. Here, we show that at concentrations where single drugs had little effect, ROCK inhibitors GSK269962A or Fasudil, in combination with either MEK inhibitor GSK1120212 (Trametinib) or ERK inhibitor SCH772984 cooperatively caused proliferation inhibition and cell death in vitro. Simultaneous inhibition of MEK and ROCK caused induction of BimEL , PARP, and Puma, and hence apoptosis. In vivo, MEK and ROCK inhibition suppressed growth of established tumors. Our findings warrant clinical investigation of the effectiveness of combinatorial targeting of MAPK/ERK and ROCK in NRAS mutant melanoma.
Asunto(s)
Apoptosis/efectos de los fármacos , GTP Fosfohidrolasas/genética , Melanoma/patología , Proteínas de la Membrana/genética , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Mutación/genética , Quinasas Asociadas a rho/antagonistas & inhibidores , Línea Celular Tumoral , Humanos , Melanoma/enzimología , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteoma/metabolismo , Quinasas Asociadas a rho/metabolismoRESUMEN
How proteins are trafficked, folded, and assembled into functional units in the cell envelope of Gram-negative bacteria is of significant interest. A number of chaperones have been identified, however, the molecular roles of these chaperones are often enigmatic because it has been challenging to assign substrates. Recently we discovered a novel periplasmic chaperone, called YfgM, which associates with PpiD and the SecYEG translocon and operates in a network that contains Skp and SurA. The aim of the study presented here was to identify putative substrates of YfgM. We reasoned that substrates would be incorrectly folded or trafficked when YfgM was absent from the cell, and thus more prone to proteolysis (the loss-of-function rationale). We therefore used a comparative proteomic approach to identify cell envelope proteins that were lower in abundance in a strain lacking yfgM, and strains lacking yfgM together with either skp or surA. Sixteen putative substrates were identified. The list contained nine inner membrane proteins (CusS, EvgS, MalF, OsmC, TdcB, TdcC, WrbA, YfhB, and YtfH) and seven periplasmic proteins (HdeA, HdeB, AnsB, Ggt, MalE, YcgK, and YnjE), but it did not include any lipoproteins or outer membrane proteins. Significantly, AnsB (an asparaginase) and HdeB (a protein involved in the acid stress response), were lower in abundance in all three strains lacking yfgM. For both genes, we ruled out the possibility that they were transcriptionally down-regulated, so it is highly likely that the corresponding proteins are misfolded/mistargeted and turned-over in the absence of YfgM. For HdeB we validated this conclusion in a pulse-chase experiment. The identification of HdeB and other cell envelope proteins as potential substrates will be a valuable resource for follow-up experiments that aim to delineate molecular the function of YfgM.
Asunto(s)
Proteínas de Escherichia coli/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de la Membrana/metabolismo , Chaperonas Moleculares/genética , ProteómicaRESUMEN
Treatment of BRAF mutant melanomas with specific BRAF inhibitors leads to tumor remission. However, most patients eventually relapse due to drug resistance. Therefore, we designed an integrated strategy using (phospho)proteomic and functional genomic platforms to identify drug targets whose inhibition sensitizes melanoma cells to BRAF inhibition. We found many proteins to be induced upon PLX4720 (BRAF inhibitor) treatment that are known to be involved in BRAF inhibitor resistance, including FOXD3 and ErbB3. Several proteins were down-regulated, including Rnd3, a negative regulator of ROCK1 kinase. For our genomic approach, we performed two parallel shRNA screens using a kinome library to identify genes whose inhibition sensitizes to BRAF or ERK inhibitor treatment. By integrating our functional genomic and (phospho)proteomic data, we identified ROCK1 as a potential drug target for BRAF mutant melanoma. ROCK1 silencing increased melanoma cell elimination when combined with BRAF or ERK inhibitor treatment. Translating this to a preclinical setting, a ROCK inhibitor showed augmented melanoma cell death upon BRAF or ERK inhibition in vitro. These data merit exploration of ROCK1 as a target in combination with current BRAF mutant melanoma therapies.
Asunto(s)
Melanoma/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Quinasas Asociadas a rho/metabolismo , Línea Celular Tumoral , Cromatografía Liquida , Regulación hacia Abajo , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Humanos , Indazoles/farmacología , Indoles/farmacología , Terapia Molecular Dirigida , Mutación , Piperazinas/farmacología , Proteómica , Proteínas Proto-Oncogénicas B-raf/genética , Interferencia de ARN , ARN Interferente Pequeño/genética , Receptor ErbB-3/genética , Receptor ErbB-3/metabolismo , Sulfonamidas/farmacología , Espectrometría de Masas en Tándem , Vemurafenib , Quinasas Asociadas a rho/genéticaRESUMEN
Enterococcus faecalis is a gram-positive bacterium that is part of the indigenous microbiotica of humans and animals as well as an opportunistic pathogen. In this study, we have fractionated the membrane proteome of E. faecalis and identified many of its constituents by mass spectrometry. We present blue native-/SDS-PAGE reference maps that contain 102 proteins. These proteins are important for cellular homeostasis, virulence, and antibiotic intervention. Intriguingly, many proteins with no known function were also identified, indicating that there are substantial gaps in the knowledge of this organism's biology. On a more limited scale, we also provide insight into the composition of membrane protein complexes. This study is a first step toward elucidating the membrane proteome of E. faecalis, which is critical for a better understanding of how this bacterium interacts with a host and with the extracellular milieu.
Asunto(s)
Proteínas Bacterianas/análisis , Membrana Celular/química , Enterococcus faecalis/química , Proteoma/análisis , Electroforesis en Gel de Poliacrilamida , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
The cell envelope of Escherichia coli is an essential structure that modulates exchanges between the cell and the extra-cellular milieu. Previous proteomic analyses have suggested that it contains a significant number of proteins with no annotated function. To gain insight into these proteins and the general organization of the cell envelope proteome, we have carried out a systematic analysis of native membrane protein complexes. We have identified 30 membrane protein complexes (6 of which are novel) and present reference maps that can be used for cell envelope profiling. In one instance, we identified a protein with no annotated function (YfgM) in a complex with a well-characterized periplasmic chaperone (PpiD). Using the guilt by association principle, we suggest that YfgM is also part of the periplasmic chaperone network. The approach we present circumvents the need for engineering of tags and protein overexpression. It is applicable for the analysis of membrane protein complexes in any organism and will be particularly useful for less-characterized organisms where conventional strategies that require protein engineering (i.e., 2-hybrid based approaches and TAP-tagging) are not feasible.
Asunto(s)
Proteínas de Escherichia coli/análisis , Escherichia coli/química , Proteínas de la Membrana/análisis , Chaperonas Moleculares/análisis , Complejos Multiproteicos/química , Cromatografía por Intercambio Iónico/métodos , Electroforesis en Gel Bidimensional/métodos , Proteínas de Escherichia coli/clasificación , Proteínas de Escherichia coli/aislamiento & purificación , Espectrometría de Masas/métodos , Proteínas de la Membrana/clasificación , Proteínas de la Membrana/aislamiento & purificación , Chaperonas Moleculares/clasificación , Chaperonas Moleculares/aislamiento & purificación , Peso Molecular , Complejos Multiproteicos/aislamiento & purificación , Filogenia , Proteoma/análisis , Proteómica/métodosRESUMEN
Complementary collision-induced/electron capture dissociation Fourier-transform ion cyclotron resonance mass spectrometry was used to fully sequence the protein P2 myelin basic protein. It is an antigenic fatty-acid-binding protein that can induce experimental autoimmune neuritis: an animal model of Guillain-Barré syndrome, a disorder similar in etiology to multiple sclerosis. Neither the primary structure of the porcine variant, nor the fatty acids bound by the protein have been well established to date. A 1.8-A crystal structure shows but a bound ligand could not be unequivocally identified. A protocol for ligand extraction from protein crystals has been developed with subsequent gas chromatography MS analysis allowing determination that oleic, stearic, and palmitic fatty acids are associated with the protein. The results provide unique and general evidence of the utility of mass spectrometry for characterizing proteins from natural sources and generating biochemical information that may facilitate attempts to elucidate the causes for disorders such as demyelination.
Asunto(s)
Proteínas de Unión a Ácidos Grasos/química , Ácidos Grasos/química , Espectrometría de Masas/métodos , Proteína P2 de Mielina/química , Secuencia de Aminoácidos , Animales , Cristalización , Proteínas de Unión a Ácidos Grasos/genética , Proteínas de Unión a Ácidos Grasos/metabolismo , Ácidos Grasos/metabolismo , Humanos , Conformación Molecular , Datos de Secuencia Molecular , Proteína P2 de Mielina/genética , Proteína P2 de Mielina/metabolismo , Unión Proteica , Alineación de Secuencia , PorcinosRESUMEN
There is a group of proteins that are encoded by a single gene, expressed as a single precursor protein and dually targeted to both mitochondria and chloroplasts using an ambiguous targeting peptide. Sequence analysis of 43 dual targeted proteins in comparison with 385 mitochondrial proteins and 567 chloroplast proteins of Arabidopsis thaliana revealed an overall significant increase in phenylalanines, leucines, and serines and a decrease in acidic amino acids and glycine in dual targeting peptides (dTPs). The N-terminal portion of dTPs has significantly more serines than mTPs. The number of arginines is similar to those in mTPs, but almost twice as high as those in cTPs. We have investigated targeting determinants of the dual targeting peptide of Thr-tRNA synthetase (ThrRS-dTP) studying organellar import of N- and C-terminal deletion constructs of ThrRS-dTP coupled to GFP. These results show that the 23 amino acid long N-terminal portion of ThrRS-dTP is crucial but not sufficient for the organellar import. The C-terminal deletions revealed that the shortest peptide that was capable of conferring dual targeting was 60 amino acids long. We have purified the ThrRS-dTP(2-60) to homogeneity after its expression as a fusion construct with GST followed by CNBr cleavage and ion exchange chromatography. The purified ThrRS-dTP(2-60) inhibited import of pF1beta into mitochondria and of pSSU into chloroplasts at microM concentrations showing that dual and organelle-specific proteins use the same organellar import pathways. Furthermore, the CD spectra of ThrRS-dTP(2-60) indicated that the peptide has the propensity for forming alpha-helical structure in membrane mimetic environments; however, the membrane charge was not important for the amount of induced helical structure. This is the first study in which a dual targeting peptide has been purified and investigated by biochemical and biophysical means.
Asunto(s)
Aminoacil-ARNt Sintetasas/genética , Mitocondrias/genética , Secuencia de Aminoácidos , Aminoacil-ARNt Sintetasas/química , Aminoacil-ARNt Sintetasas/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Cloroplastos/enzimología , Cloroplastos/genética , Dicroismo Circular , Regulación de la Expresión Génica de las Plantas , Mitocondrias/enzimología , Datos de Secuencia Molecular , Aminoacil-ARN de Transferencia/química , Aminoacil-ARN de Transferencia/genética , Aminoacil-ARN de Transferencia/metabolismo , Alineación de Secuencia , Eliminación de Secuencia , Homología de Secuencia de Aminoácido , Espectrofotometría UltravioletaRESUMEN
Quaternary protoberberine alkaloids belong to a pharmaceutically important class of isoquinoline alkaloids associated with bactericidal, fungicidal, insecticidal and antiviral activities. As traditional medicine gains wider acceptance, quick and robust analytical methods for the screening and analysis of plants containing these compounds attract considerable interest. Thin-layer chromatography (TLC) combined with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a powerful technique but suffers from dilution of the TLC bands resulting in decreased sensitivity and masking of signals in the low-mass region both due to addition of matrix. This study integrates for the first time conventional silica gel TLC and laser desorption ionization mass spectrometry (LDI-MS) thus eliminating the need for any external matrix. Successful separation of berberine (R(f) = 0.56) and palmatine (R(f) = 0.46) from Berberis barandana including their identification by MS are demonstrated. Furthermore, a robust electrospray ionization (ESI)-MS method utilizing residual sample from TLC for quantification of berberine applying selected reaction monitoring and standard addition method is presented. The amount of berberine in the plant root prepared for the study was determined to be 0.70% (w/w).
Asunto(s)
Alcaloides de Berberina/aislamiento & purificación , Berberina/aislamiento & purificación , Berberis/química , Cromatografía en Capa Delgada/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Berberina/química , Alcaloides de Berberina/química , Extractos Vegetales/química , Raíces de Plantas/química , Sensibilidad y EspecificidadRESUMEN
Eukaryotic cells have evolved quality control mechanisms to degrade aberrant mRNA molecules and prevent the synthesis of defective proteins that could be deleterious for the cell. The exosome, a protein complex with ribonuclease activity, is a key player in quality control. An early quality checkpoint takes place cotranscriptionally but little is known about the molecular mechanisms by which the exosome is recruited to the transcribed genes. Here we study the core exosome subunit Rrp4 in two insect model systems, Chironomus and Drosophila. We show that a significant fraction of Rrp4 is associated with the nascent pre-mRNPs and that a specific mRNA-binding protein, Hrp59/hnRNP M, interacts in vivo with multiple exosome subunits. Depletion of Hrp59 by RNA interference reduces the levels of Rrp4 at transcription sites, which suggests that Hrp59 is needed for the exosome to stably interact with nascent pre-mRNPs. Our results lead to a revised mechanistic model for cotranscriptional quality control in which the exosome is constantly recruited to newly synthesized RNAs through direct interactions with specific hnRNP proteins.
Asunto(s)
Exosomas/metabolismo , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Precursores de Proteínas/metabolismo , Ribonucleoproteínas/metabolismo , Animales , Western Blotting , Línea Celular , Células Cultivadas , Chironomidae/citología , Chironomidae/genética , Chironomidae/metabolismo , Cromosomas/genética , Cromosomas/metabolismo , Cromosomas/ultraestructura , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citología , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Exosomas/ultraestructura , Ribonucleoproteínas Nucleares Heterogéneas/genética , Inmunoprecipitación , Microscopía Confocal , Microscopía Inmunoelectrónica , Unión Proteica , Precursores de Proteínas/genética , Interferencia de ARN , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteínas/genética , Transcripción GenéticaRESUMEN
The cyanobacterial plasma membrane is an essential cell barrier with functions such as the control of taxis, nutrient uptake and secretion. These functions are carried out by integral membrane proteins, which are difficult to identify using standard proteomic methods. In this study, integral proteins were enriched from purified plasma membranes of Synechocystis sp. PCC 6803 using urea wash followed by protein resolution in 1D SDS/PAGE. In total, 51 proteins were identified by peptide mass fingerprinting using MALDI-TOF MS. More than half of the proteins were predicted to be integral with 1-12 transmembrane helices. The majority of the proteins had not been identified previously, and include members of metalloproteases, chemotaxis proteins, secretion proteins, as well as type 2 NAD(P)H dehydrogenase and glycosyltransferase. The obtained results serve as a useful reference for further investigations of the address codes for targeting of integral membrane proteins in cyanobacteria.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de la Membrana/metabolismo , Proteómica/métodos , Synechocystis/metabolismo , Proteínas Bacterianas/química , Membrana Celular/metabolismo , Electroforesis en Gel de Poliacrilamida , Glicosiltransferasas/química , Glicosiltransferasas/metabolismo , Proteínas de la Membrana/química , Proteínas Quimiotácticas Aceptoras de Metilo , NADPH Deshidrogenasa/química , NADPH Deshidrogenasa/metabolismo , Péptido Hidrolasas/química , Péptido Hidrolasas/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
BACKGROUND: The development of MALDI Imaging Mass Spectrometry is a promising technique in the investigation of biological molecular repertoire. We have pursued an analytical assessment of this technique in its application to proteome analysis. METHODS: A specific statistical method of analysis has been developed to enable data processing in the absence of internal standards, by defining similarity scores. RESULTS: The investigated linear mode MALDI-TOF set-up allows to obtain data variations comprised within the 30% of variation when assaying tissues samples from the same animal, while the 60% of variation was highlighted in the inter-mice series assaying syngenic animals. CONCLUSIONS: This analytical assessment represents the first step of a process that should validate the utilisation of this technique in the clinical practice.