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INTRODUCTION: Epidermolysis bullosa (EB) describes a family of rare genetic blistering skin disorders. Various subtypes are clinically and genetically heterogeneous, and a lethal postpartum form of EB is the generalized severe junctional EB (gs-JEB). gs-JEB is mainly caused by premature termination codon (PTC) mutations in the skin anchor protein LAMB3 (laminin subunit beta-3) gene. The ribosome in majority of translational reads of LAMB3PTC mRNA aborts protein synthesis at the PTC signal, with production of a truncated, nonfunctional protein. This leaves an endogenous readthrough mechanism needed for production of functional full-length Lamb3 protein albeit at insufficient levels. Here, we report on the development of drugs targeting ribosomal protein L35 (rpL35), a ribosomal modifier for customized increase in production of full-length Lamb3 protein from a LAMB3PTC mRNA. METHODS: Molecular docking studies were employed to identify small molecules binding to human rpL35. Molecular determinants of small molecule binding to rpL35 were further characterized by titration of the protein with these ligands as monitored by nuclear magnetic resonance (NMR) spectroscopy in solution. Changes in NMR chemical shifts were used to map the docking sites for small molecules onto the 3D structure of the rpL35. RESULTS: Molecular docking studies identified 2 FDA-approved drugs, atazanavir and artesunate, as candidate small-molecule binders of rpL35. Molecular interaction studies predicted several binding clusters for both compounds scattered throughout the rpL35 structure. NMR titration studies identified the amino acids participating in the ligand interaction. Combining docking predictions for atazanavir and artesunate with rpL35 and NMR analysis of rpL35 ligand interaction, one binding cluster located near the N-terminus of rpL35 was identified. In this region, the nonidentical binding sites for atazanavir and artesunate overlap and are accessible when rpL35 is integrated in its natural ribosomal environment. CONCLUSION: Atazanavir and artesunate were identified as candidate compounds binding to ribosomal protein rpL35 and may now be tested for their potential to trigger a rpL35 ribosomal switch to increase production of full-length Lamb3 protein from a LAMB3PTC mRNA for targeted systemic therapy in treating gs-JEB.
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Moléculas de Adhesión Celular/genética , Epidermólisis Ampollosa de la Unión/genética , ARN Mensajero/metabolismo , Proteínas Ribosómicas/metabolismo , Artesunato/química , Sulfato de Atazanavir/química , Epidermólisis Ampollosa de la Unión/patología , Humanos , Simulación del Acoplamiento Molecular , Unión Proteica/fisiología , Piel/patología , Fenómenos Fisiológicos de la Piel , KalininaRESUMEN
Oligoclonal Ig bands (OCBs) of the cerebrospinal fluid are a hallmark of multiple sclerosis (MS), a disabling inflammatory disease of the central nervous system (CNS). OCBs are locally produced by clonally expanded antigen-experienced B cells and therefore are believed to hold an important clue to the pathogenesis. However, their target antigens have remained unknown, mainly because it was thus far not possible to isolate distinct OCBs against a background of polyclonal antibodies. To overcome this obstacle, we copurified disulfide-linked Ig heavy and light chains from distinct OCBs for concurrent analysis by mass spectrometry and aligned patient-specific peptides to corresponding transcriptome databases. This method revealed the full-length sequences of matching chains from distinct OCBs, allowing for antigen searches using recombinant OCB antibodies. As validation, we demonstrate that an OCB antibody from a patient with an infectious CNS disorder, neuroborreliosis, recognized a Borrelia protein. Next, we produced six recombinant antibodies from four MS patients and identified three different autoantigens. All of them are conformational epitopes of ubiquitous intracellular proteins not specific to brain tissue. Our findings indicate that the B-cell response in MS is heterogeneous and partly directed against intracellular autoantigens released during tissue destruction. In addition to helping elucidate the role of B cells in MS, our approach allows the identification of target antigens of OCB antibodies in other neuroinflammatory diseases and the production of therapeutic antibodies in infectious CNS diseases.
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Autoantígenos/inmunología , Esclerosis Múltiple/inmunología , Bandas Oligoclonales/inmunología , Borrelia/inmunología , Células HEK293 , Humanos , Neuroborreliosis de Lyme/inmunologíaRESUMEN
Neoadjuvant chemoradiation (nCRT) is an established procedure in stage union internationale contre le cancer (UICC) II/III rectal carcinomas. Around 53% of the tumours present with good tumor regression after nCRT, and 8%-15% are complete responders. Reliable selection markers would allow the identification of poor or non-responders prior to therapy. Tumor biopsies were harvested from 20 patients with rectal carcinomas, and stored in liquid nitrogen prior to therapy after obtaining patients' informed consent (Erlangen-No.3784). Patients received standardized nCRT with 5-Fluoruracil (nCRT I) or 5-Fluoruracil ± Oxaliplatin (nCRT II) according to the CAO/ARO/AIO-04 protocol. After surgery, regression grading (Dworak) of the tumors was performed during histopathological examination of the specimens. Tumors were classified as poor (Dworak 1 + 2) or good (Dworak 3 + 4) responders. Laser capture microdissection (LCM) for tumor enrichment was performed on preoperative biopsies. Differences in expressed proteins between poor and good responders to nCRT I and II were identified by proteomic analysis (Isotope Coded Protein Label, ICPL™) and selected markers were validated by immunohistochemistry. Tumors of 10 patients were classified as histopathologically poor (Dworak 1 or 2) and the other 10 tumor samples as histopathologically good (Dworak 3 or 4) responders to nCRT after surgery. Sufficient material in good quality was harvested for ICPL analysis by LCM from all biopsies. We identified 140 differentially regulated proteins regarding the selection criteria and the response to nCRT. Fourteen of these proteins were synchronously up-regulated at least 1.5-fold after nCRT I or nCRT II (e.g., FLNB, TKT, PKM2, SERINB1, IGHG2). Thirty-five proteins showed a complete reciprocal regulation (up or down) after nCRT I or nCRT II and the rest was regulated either according to nCRT I or II. The protein expression of regulated proteins such as PLEC1, TKT, HADHA and TAGLN was validated successfully by immunohistochemistry. ICPL is a valid method to identify differentially expressed proteins in rectal carcinoma tissue between poor vs. good responders to nCRT. The identified protein markers may act as selection criteria for nCRT in the future, but our preliminary findings must be reproduced and validated in a prospective cohort.
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Proteoma , Proteómica , Neoplasias del Recto/metabolismo , Neoplasias del Recto/mortalidad , Biomarcadores , Biopsia , Quimioradioterapia , Humanos , Inmunohistoquímica , Terapia Neoadyuvante , Pronóstico , Proteómica/métodos , Neoplasias del Recto/diagnóstico , Neoplasias del Recto/terapia , Resultado del TratamientoRESUMEN
Caulobacter crescentus is an oligotrophic bacterium that lives in dilute organic environments such as soil and freshwater. This bacterium represents an interesting model for cellular differentiation and regulation because daughter cells after division have different forms: one is motile while the other is non-motile and can adhere to surfaces. Interestingly, the known genome of C. crescentus does not contain genes predicted to code for outer membrane porins of the OmpF/C general diffusion type present in enteric bacteria or those coding for specific porins selective for classes of substrates. Instead, genes coding for 67 TonB-dependent outer membrane receptors have been identified, suggesting that active transport of specific nutrients may be the norm. Here, we report that high channel-forming activity was observed with crude outer membrane extracts of C. crescentus in lipid bilayer experiments, indicating that the outer membrane of C. crescentus contained an ion-permeable channel with a single-channel conductance of about 120 pS in 1M KCl. The channel-forming protein with an apparent molecular mass of about 20 kDa was purified to homogeneity. Partial protein sequencing of the protein indicated it was a member of the OmpW family of outer membrane proteins from Gram-negative bacteria. This channel was not observed in reconstitution experiments with crude outer membrane extracts of an OmpW deficient C. crescentus mutant. Biophysical analysis of the C. crescentus OmpW suggested that it has features that are special for general diffusion porins of Gram-negative outer membranes because it was not a wide aqueous channel. Furthermore, OmpW of C. crescentus seems to be different to known OmpW porins and has a preference for ions, in particular cations. A putative model for OmpW of C. crescentus was built on the basis of the known 3D-structures of OmpW of Escherichia coli and OprG of Pseudomonas aeruginosa using homology modeling. A comparison of the two known structures with the model of OmpW of C. crescentus suggested that it has a more hydrophilic interior and possibly a larger diameter.
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Proteínas de la Membrana Bacteriana Externa/metabolismo , Cationes/metabolismo , Caulobacter crescentus/metabolismo , Canales Iónicos/metabolismo , Secuencia de Aminoácidos , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/genética , Caulobacter crescentus/genética , Técnicas de Silenciamiento del Gen , Canales Iónicos/química , Canales Iónicos/genética , MutaciónRESUMEN
P13 is one of the major outer membrane proteins of Borrelia burgdorferi. Previous studies described P13 as a porin. In the present study some structure and function aspects of P13 were studied. P13 showed according to lipid bilayer studies a channel-forming activity of 0.6 nanosiemens in 1 m KCl. Single channel and selectivity measurements demonstrated that P13 had no preference for either cations or anions and showed no voltage-gating up to ±100 mV. Blue native polyacrylamide gel electrophoresis was used to isolate and characterize the P13 protein complex in its native state. The complex had a high molecular mass of about 300 kDa and was only composed of P13 monomers. The channel size was investigated using non-electrolytes revealing an apparent diameter of about 1.4 nm with a 400-Da molecular mass cut-off. Multichannel titrations with different substrates reinforced the idea that P13 forms a general diffusion channel. The identity of P13 within the complex was confirmed by second dimension SDS-PAGE, Western blotting, mass spectrometry, and the use of a p13 deletion mutant strain. The results suggested that P13 is the protein responsible for the 0.6-nanosiemens pore-forming activity in the outer membrane of B. burgdorferi.
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Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Borrelia burgdorferi , Porinas/química , Porinas/metabolismo , Relación Dosis-Respuesta a Droga , Activación del Canal Iónico/efectos de los fármacos , Membrana Dobles de Lípidos/metabolismo , Porosidad , Sales (Química)/farmacología , Especificidad por SustratoRESUMEN
BACKGROUND: Known genetic variants can account for 10% to 20% of all cases with autism spectrum disorders (ASD). Overlapping cellular pathomechanisms common to neurons of the central nervous system (CNS) and in tissues of peripheral organs, such as immune dysregulation, oxidative stress and dysfunctions in mitochondrial and protein synthesis metabolism, were suggested to support the wide spectrum of ASD on unifying disease phenotype. Here, we studied in patient-derived lymphoblastoid cell lines (LCLs) how an ASD-specific mutation in ribosomal protein RPL10 (RPL10[H213Q]) generates a distinct protein signature. We compared the RPL10[H213Q] expression pattern to expression patterns derived from unrelated ASD patients without RPL10[H213Q] mutation. In addition, a yeast rpl10 deficiency model served in a proof-of-principle study to test for alterations in protein patterns in response to oxidative stress. METHODS: Protein extracts of LCLs from patients, relatives and controls, as well as diploid yeast cells hemizygous for rpl10, were subjected to two-dimensional gel electrophoresis and differentially regulated spots were identified by mass spectrometry. Subsequently, Gene Ontology database (GO)-term enrichment and network analysis was performed to map the identified proteins into cellular pathways. RESULTS: The protein signature generated by RPL10[H213Q] is a functionally related subset of the ASD-specific protein signature, sharing redox-sensitive elements in energy-, protein- and redox-metabolism. In yeast, rpl10 deficiency generates a specific protein signature, harboring components of pathways identified in both the RPL10[H213Q] subjects' and the ASD patients' set. Importantly, the rpl10 deficiency signature is a subset of the signature resulting from response of wild-type yeast to oxidative stress. CONCLUSIONS: Redox-sensitive protein signatures mapping into cellular pathways with pathophysiology in ASD have been identified in both LCLs carrying the ASD-specific mutation RPL10[H213Q] and LCLs from ASD patients without this mutation. At pathway levels, this redox-sensitive protein signature has also been identified in a yeast rpl10 deficiency and an oxidative stress model. These observations point to a common molecular pathomechanism in ASD, characterized in our study by dysregulation of redox balance. Importantly, this can be triggered by the known ASD-RPL10[H213Q] mutation or by yet unknown mutations of the ASD cohort that act upstream of RPL10 in differential expression of redox-sensitive proteins.
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ICPL_ESIQuant is a proteomics software tool for quantitatively analyzing large mass spectrometric datasets acquired from ICPL based proteomics experiments. It is able to process mass spectrometric data from various vendors and implements results from the Mascot search engine to generate protein and peptide result tables. This protocol briefly introduces ICPL_ESIQuant and presents a detailed step by step tutorial, how to use the software with MS datasets obtained from ICPL duplex, triplex and quadruplex experiments. Requiring MS raw data in .mzXML file format and Mascot search results in .dat format as input, ICPL_ESIQuant reliably quantifies ICPL labeled proteins and provides additional information about all detected, sequenced and identified features in the sample. The software supports both the shotgun and the directed proteomics strategy, enabling the user to apply mass inclusion lists for identifying peptides not fragmented in the first MS cycle. The software together with a test dataset is freely available under http://sourceforge.net/projects/icplquant/. This article is part of a Special Issue entitled: Computational Proteomics in the Post-Identification Era. Guest Editors: Martin Eisenacher and Christian Stephan.
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Proteómica , Programas Informáticos , ComputadoresRESUMEN
Ricinosomes are specialized ER-derived organelles that store the inactive pro-forms of KDEL-tailed cysteine endopeptidases (KDEL-CysEP) associated with programmed cell death (PCD). The Arabidopsis genome encodes three KDEL-CysEP (AtCEP1, AtCEP2, and AtCEP3) that are differentially expressed in vegetative and generative tissues undergoing PCD. These Arabidopsis proteases have not been characterized at a biochemical level, nor have they been localized intracellularly. In this study, we characterized AtCEP2. A 3xHA-mCherry-AtCEP2 gene fusion including pro-peptide and KDEL targeting sequences expressed under control of the endogenous promoter enabled us to isolate AtCEP2 "ex vivo". The purified protein was shown to be activated in a pH-dependent manner. After activation, however, protease activity was pH-independent. Analysis of substrate specificity showed that AtCEP2 accepts proline near the cleavage site, which is a rare feature specific for KDEL-CysEPs. mCherry-AtCEP2 was detected in the epidermal layers of leaves, hypocotyls and roots; in the root, it was predominantly found in the elongation zone and root cap. Co-localization with an ER membrane marker showed that mCherry-AtCEP2 was stored in two different types of ER-derived organelles: 10 µm long spindle shaped organelles as well as round vesicles with a diameter of approximately 1 µm. The long organelles appear to be ER bodies, which are found specifically in Brassicacae. The round vesicles strongly resemble the ricinosomes first described in castor bean. This study provides a first evidence for the existence of ricinosomes in Arabidopsis, and may open up new avenues of research in the field of PCD and developmental tissue remodeling.
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Apoptosis/fisiología , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Cisteína Endopeptidasas/metabolismo , Retículo Endoplásmico/enzimología , Precursores Enzimáticos/metabolismo , Oligopéptidos/metabolismo , Arabidopsis/citología , Arabidopsis/genética , Arabidopsis/fisiología , Proteínas de Arabidopsis/genética , Pared Celular/metabolismo , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/genética , Activación Enzimática , Precursores Enzimáticos/química , Precursores Enzimáticos/genética , Concentración de Iones de Hidrógeno , Hipocótilo/citología , Hipocótilo/enzimología , Hipocótilo/genética , Hipocótilo/fisiología , Oligopéptidos/genética , Especificidad de Órganos , Epidermis de la Planta/citología , Epidermis de la Planta/enzimología , Epidermis de la Planta/genética , Epidermis de la Planta/fisiología , Hojas de la Planta/citología , Hojas de la Planta/enzimología , Hojas de la Planta/genética , Hojas de la Planta/fisiología , Raíces de Plantas/citología , Raíces de Plantas/enzimología , Raíces de Plantas/genética , Raíces de Plantas/fisiología , Plantas Modificadas Genéticamente , Señales de Clasificación de Proteína/genética , Transporte de Proteínas , Proteínas Recombinantes de Fusión , Eliminación de Secuencia , Especificidad por SustratoRESUMEN
A great variety of technologies using stable isotope labeling in combination with mass spectrometry have been described being tools to identify and relatively quantify proteins within complex mixtures. Here, we present a method, termed isotope-coded protein label (ICPL), which is capable of high-throughput quantitative proteome profiling on a global scale. Since ICPL is based on tagging stable isotope derivatives at the free amino groups of intact proteins, the method is applicable to any protein sample, including extracts from tissues or body fluids. All separation methods currently employed in proteome studies can be used to reduce complexity on the protein level. After enzymatic cleavage of the protein fractions, the ratios of peptides from different proteome states can be calculated by simple MS-based mass spectrometric analyses. Only peptides representing different expression levels in the different proteomic states are further analyzed by tandem-mass spectrometry to identify respective proteins. For quantification of proteins from multiplexed ICPL experiments, ICPLQuant was developed, a software package especially designed to cover the whole ICPL workflow. The ICPL method results in accurate and reproducible quantification of proteins and high sequence coverage, indispensable for a comprehensive detection of posttranslational modifications and discrimination of protein isoforms.
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Proteoma/química , Programas Informáticos , Animales , Isótopos de Carbono , Precipitación Química , Cisteína/química , Interpretación Estadística de Datos , Deuterio , Electroforesis en Gel Bidimensional , Humanos , Marcaje Isotópico , Espectrometría de Masas/normas , Metilación , Fragmentos de Péptidos/química , Proteolisis , Proteoma/aislamiento & purificación , Estándares de Referencia , Serina Endopeptidasas/química , SuccinimidasRESUMEN
BACKGROUND: The thermoacidophilic and chemolithotrophic archaeon Acidianus ambivalens is routinely grown with sulfur and CO(2)-enriched air. We had described a membrane-bound, tetrathionate (TT) forming thiosulfate:quinone oxidoreductase. Here we describe the first TT hydrolase (TTH) from Archaea. RESULTS: A. ambivalens cells grown aerobically with TT as sole sulfur source showed doubling times of 9 h and final cell densities of up to 8 × 10(8)/ml. TTH activity (≈0.28 U/mg protein) was found in cell-free extracts of TT-grown but not of sulfur-grown cells. Differential fractionation of freshly harvested cells involving a pH shock showed that about 92% of the TTH activity was located in the pseudo-periplasmic fraction associated with the surface layer, while 7.3% and 0.3% were present in the soluble and membrane fractions, respectively. The enzyme was enriched 54-fold from the cytoplasmic fraction and 2.1-fold from the pseudo-periplasmic fraction. The molecular mass of the single subunit was 54 kDa. The optimal activity was at or above 95°C at pH 1. Neither PQQ nor divalent cations had a significant effect on activity. The gene (tth1) was identified following N-terminal sequencing of the protein. Northern hybridization showed that tth1 was transcribed in TT-grown cells in contrast to a second paralogous tth2 gene. The deduced amino acid sequences showed similarity to the TTH from Acidithiobacillus and other proteins from the PQQ dehydrogenase superfamily. It displayed a ß-propeller structure when being modeled, however, important residues from the PQQ-binding site were absent. CONCLUSION: The soluble, extracellular, and acidophilic TTH identified in TT-grown A. ambivalens cells is essential for TT metabolism during growth but not for the downstream processing of the TQO reaction products in S°-grown cells. The liberation of TTH by pH shock from otherwise intact cells strongly supports the pseudo-periplasm hypothesis of the S-layer of Archaea.
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The photosynthetic alphaproteobacterium Rhodobacter sphaeroides has to cope with photooxidative stress that is caused by the bacteriochlorophyll a-mediated formation of singlet oxygen ((1)O(2)). Exposure to (1)O(2) induces the alternative sigma factors RpoE and RpoH(II) which then promote transcription of photooxidative stress-related genes, including small RNAs (sRNAs). The ubiquitous RNA chaperone Hfq is well established to interact with and facilitate the base-pairing of sRNAs and target mRNAs to influence mRNA stability and/or translation. Here we report on the pleiotropic phenotype of a Δhfq mutant of R. sphaeroides, which is less pigmented, produces minicells and is more sensitive to (1)O(2). The higher (1)O(2) sensitivity of the Δhfq mutant is paralleled by a reduced RpoE activity and a disordered induction of RpoH(II)-dependent genes. We used co-immunoprecipitation of FLAG-tagged Hfq combined with RNA-seq to identify association of at least 25 sRNAs and of mRNAs encoding cell division proteins and ribosomal proteins with Hfq. Remarkably, > 70% of the Hfq-bound sRNAs are (1)O(2)-affected. Proteomics analysis of the Hfq-deficient strain revealed an impact of Hfq on amino acid transport and metabolic functions. Our data demonstrate for the first time an involvement of Hfq in regulation of photosynthesis genes and in the photooxidative stress response.
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Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Proteína de Factor 1 del Huésped/metabolismo , Estrés Oxidativo , Unión Proteica , Rhodobacter sphaeroides/metabolismo , Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica/efectos de la radiación , Proteína de Factor 1 del Huésped/genética , Estrés Oxidativo/efectos de la radiación , Fotosíntesis , Rhodobacter sphaeroides/genética , Rhodobacter sphaeroides/efectos de la radiaciónRESUMEN
Stable isotope labeling in combination with mass spectrometry has emerged as a powerful tool to identify and quantify thousands of proteins within complex protein mixtures. Isotope-coded protein label (ICPL) is capable of high-throughput quantitative proteome profiling on a global scale. Since ICPL is based on stable isotope tagging at the free amino groups of intact proteins, it is applicable to any protein sample, including extracts from tissues or body fluids. After labeling of up to four different proteome states, the samples can be combined and the complexity reduced by any separation method currently employed in protein chemistry. After enzymatic cleavage of the protein fractions the ratios of peptides in the different proteome states can be calculated by simple MS-based mass spectrometric analyses. Only peptides that exhibit regulations in the different proteome states are further investigated for identification by tandem-mass spectrometry. The quantification of multiplexed ICPL experiments is greatly facilitated by the recently published ICPLQuant software, which was especially designed to cover the whole ICPL workflow. The method shows highly accurate and reproducible quantification of proteins, yields high sequence coverage, and is indispensable for the comprehensive detection of posttranslational modifications and protein isoforms.
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Marcaje Isotópico/métodos , Proteoma/análisis , Proteómica/métodos , Espectrometría de Masas/métodos , Procesamiento Proteico-Postraduccional , Proteínas/análisisRESUMEN
2-DE proved to be a key technology in protein science since the two orthogonal separation dimensions are capable of protein isoform separation. Recently, Agilent introduced the OFFGEL 3100 fractionator for in solution IEF (off-gel) of proteins with the help of a 12- or 24-well frame. With this instrument also conventional focusing in IPG strips after passive in-tray rehydration can be performed. In this study, two novel IEF applications using the OFFGEL electrophoresis were developed. First, a sample cup was built and a cup-loading method for the OFFGEL device was implemented. Applying proteins via cup resulted in higher reproducibility and less protein loss compared with conventional in-tray rehydration loading. Especially, the recovery of basic and high-molecular-mass proteins seems to be favored by cup loading. These effects are more pronounced with low microgram sample amounts. Second, a 48-well OFFGEL frame was developed, which doubles the resolution of the commercially available 24-well frame. It is capable of separating proteins with small pI differences and shows potential for isoform/PTM separation.
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Proteínas de Escherichia coli/aislamiento & purificación , Focalización Isoeléctrica/instrumentación , Focalización Isoeléctrica/métodos , Proteómica/métodos , Electroforesis en Gel Bidimensional , Diseño de Equipo , Proteínas de Escherichia coli/química , Procesamiento de Imagen Asistido por Computador , Punto Isoeléctrico , Análisis de Componente Principal , Isoformas de Proteínas , Fuerza Protón-Motriz , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Most mitochondrial proteins are encoded in the nucleus as precursor proteins and carry N-terminal presequences for import into the organelle. The vast majority of presequences are proteolytically removed by the mitochondrial processing peptidase (MPP) localized in the matrix. A subset of precursors with a characteristic amino acid motif is additionally processed by the mitochondrial intermediate peptidase (MIP) octapeptidyl aminopeptidase 1 (Oct1), which removes an octapeptide from the N-terminus of the precursor intermediate. However, the function of this second cleavage step is elusive. In this paper, we report the identification of a novel Oct1 substrate protein with an unusual cleavage motif. Inspection of the Oct1 substrates revealed that the N-termini of the intermediates typically carry a destabilizing amino acid residue according to the N-end rule of protein degradation, whereas mature proteins carry stabilizing N-terminal residues. We compared the stability of intermediate and mature forms of Oct1 substrate proteins in organello and in vivo and found that Oct1 cleavage increases the half-life of its substrate proteins, most likely by removing destabilizing amino acids at the intermediate's N-terminus. Thus Oct1 converts unstable precursor intermediates generated by MPP into stable mature proteins.
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Metaloendopeptidasas/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Transportador 1 de Catión Orgánico/metabolismo , Precursores de Proteínas/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Semivida , Mitocondrias/enzimología , Datos de Secuencia Molecular , Orgánulos/metabolismo , Procesamiento Proteico-Postraduccional , Estabilidad Proteica , Saccharomyces cerevisiae/enzimología , Peptidasa de Procesamiento MitocondrialRESUMEN
We investigated the overlap shared between the immunoglobulin (Ig) proteome of the cerebrospinal fluid (CSF) and the B cell Ig-transcriptome of CSF and the central nervous system (CNS) tissue of three patients with multiple sclerosis. We determined the IgG-proteomes of CSF by mass spectrometry, and compared them to the IgG-transcriptomes from CSF and brain lesions, which were analyzed by cDNA cloning. Characteristic peptides that were identified in the CSF-proteome could also be detected in the transcriptomes of both, brain lesions and CSF, providing evidence for a strong overlap of the IgG repertoires in brain lesions and in the CSF.
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Subgrupos de Linfocitos B/inmunología , Subgrupos de Linfocitos B/metabolismo , Inmunoglobulinas/biosíntesis , Inmunoglobulinas/líquido cefalorraquídeo , Esclerosis Múltiple/líquido cefalorraquídeo , Esclerosis Múltiple/inmunología , Subgrupos de Linfocitos B/patología , Células Clonales/inmunología , Células Clonales/metabolismo , Células Clonales/patología , Humanos , Esclerosis Múltiple/genéticaRESUMEN
Roseobacter clade aerobic anoxygenic phototrophic bacteria (AAnP) are abundant in photic zone environments of marine ecosystems. These bacteria form a photosynthetic apparatus at oxygen saturation, a situation expected to generate high levels of singlet oxygen (¹O2) when light is present. Rhodobacter sphaeroides, an anaerobic anoxygenic phototroph, represses photosynthesis genes at high oxygen tension. Here we report that Roseobacter denitrificans showed higher sensitivity to ¹O2 compared with Rhb. sphaeroides. While photosynthetic membranes of Rsb. denitrificans generated more ¹O2 during light exposure, key regulator genes rpoE and rpoH(II) were more strongly induced in response to ¹O2 stress compared with Rhb. sphaeroides. The regulon controlled by RpoE was different in Rsb. denitrificans and Rhb. sphaeroides. Patterns of synthesized soluble proteins strongly changed upon high light exposure in Rsb. denitrificans but not in Rhb. sphaeroides, and most changes were not further promoted by artificial ¹O2 generation. The strong increase of small RNA RDs2461 levels by photooxidative stress implies a role for sRNAs in post-transcriptional regulation of the response to ¹O2 in AAnPs. Our data reveal similarities but also significant differences in the response of Rsb. denitrificans and Rhb. sphaeroides to ¹O2, most likely a consequence of their different lifestyles.
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Luz , Estrés Oxidativo , Fotosíntesis , Roseobacter/metabolismo , Regulación Bacteriana de la Expresión Génica , Genes Reguladores , Oxígeno/metabolismo , Fotosíntesis/genética , Rhodobacter sphaeroides/genética , Rhodobacter sphaeroides/metabolismo , Rhodobacter sphaeroides/efectos de la radiación , Roseobacter/genética , Roseobacter/efectos de la radiación , Oxígeno Singlete/metabolismoRESUMEN
Studies of skin aging are usually performed at the genomic level by investigating differentially regulated genes identified through subtractive hybridization or microarray analyses. In contrast, relatively few studies have investigated changes in protein expression of aged skin using proteomic profiling by two-dimensional (2-D) gel electrophoresis and mass spectrometry, although this approach at the protein level is suggested to reflect more accurately the aging phenotype. We undertook such a proteomic analysis of intrinsic human skin aging by quantifying proteins extracted and fluorescently labeled from sun-protected human foreskin samples pooled from 'young' and 'old' men. In addition, we analyzed these candidate gene products by 1-D and 2-D western blotting to obtain corroborative protein expression data, and by both real-time PCR (RT-PCR) and microarray analyses to confirm expression at the mRNA level. We discovered 30 putative proteins for skin aging, including previously unrecognized, post-translationally regulated candidates such as phosphatidyl-ethanolamine binding protein (PEBP) and carbonic anhydrase 1 (CA1).
Asunto(s)
Prepucio/citología , Prepucio/fisiología , Perfilación de la Expresión Génica , Proteómica , Envejecimiento de la Piel/fisiología , Adolescente , Adulto , Anciano , Western Blotting , Anhidrasas Carbónicas/genética , Anhidrasas Carbónicas/metabolismo , Niño , Preescolar , Electroforesis en Gel Bidimensional , Humanos , Lactante , Masculino , Espectrometría de Masas , Persona de Mediana Edad , Proteínas de Unión a Fosfatidiletanolamina/genética , Proteínas de Unión a Fosfatidiletanolamina/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Adulto JovenRESUMEN
Despite worldwide efforts biomarker discovery by plasma proteomics was not successful so far. Several reasons for this failure are obvious. Mainly, proteome diversity is remarkable between different individuals and is caused by genetic, environmental and life style parameters. To recognize disease related proteins that could serve as potential biomarkers is only feasible by investigating a non realizable large number of patients. Furthermore, plasma proteomics comprises enormous technical hurdles for quantitative analysis. High reproducibility of blood sampling in clinical routine is hard to achieve. Quantitative proteome analysis has to struggle with the complexity of millions of protein species comprising typical plasma proteins, cellular leakage proteins and antibodies and concentration differences of more than 1011 between high and low abundant proteins. Therefore, no successful quantitative and comprehensive plasma proteome analysis is reported so far. A novel proteomics strategy is proposed for biomarker discovery in plasma. Instead of comparing the plasma proteome of different individuals it is recommended to analyze the proteomes of different time points of a single individual during the development of a disease. This strategy is realized by the use of plasma of the Bavarian Red Cross Blood Bank, were three million samples are stored under standardized conditions. To achieve reliable data the isotope coded protein labelling proteomics technology was used.