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1.
Cell ; 162(6): 1286-98, 2015 Sep 10.
Artículo en Inglés | MEDLINE | ID: mdl-26359986

RESUMEN

Heat causes protein misfolding and aggregation and, in eukaryotic cells, triggers aggregation of proteins and RNA into stress granules. We have carried out extensive proteomic studies to quantify heat-triggered aggregation and subsequent disaggregation in budding yeast, identifying >170 endogenous proteins aggregating within minutes of heat shock in multiple subcellular compartments. We demonstrate that these aggregated proteins are not misfolded and destined for degradation. Stable-isotope labeling reveals that even severely aggregated endogenous proteins are disaggregated without degradation during recovery from shock, contrasting with the rapid degradation observed for many exogenous thermolabile proteins. Although aggregation likely inactivates many cellular proteins, in the case of a heterotrimeric aminoacyl-tRNA synthetase complex, the aggregated proteins remain active with unaltered fidelity. We propose that most heat-induced aggregation of mature proteins reflects the operation of an adaptive, autoregulatory process of functionally significant aggregate assembly and disassembly that aids cellular adaptation to thermal stress.


Asunto(s)
Respuesta al Choque Térmico , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/fisiología , Cicloheximida/farmacología , Gránulos Citoplasmáticos/metabolismo , Agregado de Proteínas , Biosíntesis de Proteínas/efectos de los fármacos , Inhibidores de la Síntesis de la Proteína/farmacología , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo
2.
J Proteome Res ; 19(5): 1900-1912, 2020 05 01.
Artículo en Inglés | MEDLINE | ID: mdl-32163288

RESUMEN

A Think-Tank Meeting was convened by the National Cancer Institute (NCI) to solicit experts' opinion on the development and application of multiomic single-cell analyses, and especially single-cell proteomics, to improve the development of a new generation of biomarkers for cancer risk, early detection, diagnosis, and prognosis as well as to discuss the discovery of new targets for prevention and therapy. It is anticipated that such markers and targets will be based on cellular, subcellular, molecular, and functional aberrations within the lesion and within individual cells. Single-cell proteomic data will be essential for the establishment of new tools with searchable and scalable features that include spatial and temporal cartographies of premalignant and malignant lesions. Challenges and potential solutions that were discussed included (i) The best way/s to analyze single-cells from fresh and preserved tissue; (ii) Detection and analysis of secreted molecules and from single cells, especially from a tissue slice; (iii) Detection of new, previously undocumented cell type/s in the premalignant and early stage cancer tissue microenvironment; (iv) Multiomic integration of data to support and inform proteomic measurements; (v) Subcellular organelles-identifying abnormal structure, function, distribution, and location within individual premalignant and malignant cells; (vi) How to improve the dynamic range of single-cell proteomic measurements for discovery of differentially expressed proteins and their post-translational modifications (PTM); (vii) The depth of coverage measured concurrently using single-cell techniques; (viii) Quantitation - absolute or semiquantitative? (ix) Single methodology or multiplexed combinations? (x) Application of analytical methods for identification of biologically significant subsets; (xi) Data visualization of N-dimensional data sets; (xii) How to construct intercellular signaling networks in individual cells within premalignant tumor microenvironments (TME); (xiii) Associations between intrinsic cellular processes and extrinsic stimuli; (xiv) How to predict cellular responses to stress-inducing stimuli; (xv) Identification of new markers for prediction of progression from precursor, benign, and localized lesions to invasive cancer, based on spatial and temporal changes within individual cells; (xvi) Identification of new targets for immunoprevention or immunotherapy-identification of neoantigens and surfactome of individual cells within a lesion.


Asunto(s)
Vacunas contra el Cáncer , Neoplasias , Biomarcadores , Biomarcadores de Tumor/genética , Inmunoterapia , National Cancer Institute (U.S.) , Proteómica , Estados Unidos
3.
Nat Chem Biol ; 13(2): 174-180, 2017 02.
Artículo en Inglés | MEDLINE | ID: mdl-27918561

RESUMEN

Proteomic detection of non-annotated microproteins indicates the translation of hundreds of small open reading frames (smORFs) in human cells, but whether these microproteins are functional or not is unknown. Here, we report the discovery and characterization of a 7-kDa human microprotein we named non-annotated P-body dissociating polypeptide (NoBody). NoBody interacts with mRNA decapping proteins, which remove the 5' cap from mRNAs to promote 5'-to-3' decay. Decapping proteins participate in mRNA turnover and nonsense-mediated decay (NMD). NoBody localizes to mRNA-decay-associated RNA-protein granules called P-bodies. Modulation of NoBody levels reveals that its abundance is anticorrelated with cellular P-body numbers and alters the steady-state levels of a cellular NMD substrate. These results implicate NoBody as a novel component of the mRNA decapping complex and demonstrate potential functionality of a newly discovered microprotein.


Asunto(s)
Proteínas Portadoras/metabolismo , Endorribonucleasas/química , Endorribonucleasas/metabolismo , ARN Mensajero/metabolismo , Animales , Células COS , Células Cultivadas , Chlorocebus aethiops , Humanos , Caperuzas de ARN/metabolismo , ARN Mensajero/química , ARN Mensajero/genética
4.
J Biol Chem ; 289(16): 10950-10957, 2014 Apr 18.
Artículo en Inglés | MEDLINE | ID: mdl-24610814

RESUMEN

The recent discovery of numerous human short open reading frame (sORF)-encoded polypeptides (SEPs) has raised important questions about the functional roles of these molecules in cells. Here, we show that a 69-amino acid SEP, MRI-2, physically interacts with the Ku heterodimer to stimulate DNA double-strand break ligation via nonhomologous end joining. The characterization of MRI-2 suggests that this SEP may participate in DNA repair and underscores the potential of SEPs to serve important biological functions in mammalian cells.


Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades/fisiología , ADN Helicasas/metabolismo , Sistemas de Lectura Abierta/fisiología , Línea Celular , ADN Helicasas/genética , Humanos , Autoantígeno Ku
5.
Nat Chem Biol ; 9(1): 59-64, 2013 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-23160002

RESUMEN

The complete extent to which the human genome is translated into polypeptides is of fundamental importance. We report a peptidomic strategy to detect short open reading frame (sORF)-encoded polypeptides (SEPs) in human cells. We identify 90 SEPs, 86 of which are previously uncharacterized, which is the largest number of human SEPs ever reported. SEP abundances range from 10-1,000 molecules per cell, identical to abundances of known proteins. SEPs arise from sORFs in noncoding RNAs as well as multicistronic mRNAs, and many SEPs initiate with non-AUG start codons, indicating that noncanonical translation may be more widespread in mammals than previously thought. In addition, coding sORFs are present in a small fraction (8 out of 1,866) of long intergenic noncoding RNAs. Together, these results provide strong evidence that the human proteome is more complex than previously appreciated.


Asunto(s)
Sistemas de Lectura Abierta , Péptidos/química , Proteoma , Codón , Humanos , ARN Mensajero/genética
6.
J Proteome Res ; 13(3): 1757-65, 2014 Mar 07.
Artículo en Inglés | MEDLINE | ID: mdl-24490786

RESUMEN

The existence of nonannotated protein-coding human short open reading frames (sORFs) has been revealed through the direct detection of their sORF-encoded polypeptide (SEP) products. The discovery of novel SEPs increases the size of the genome and the proteome and provides insights into the molecular biology of mammalian cells, such as the prevalent usage of non-AUG start codons. Through modifications of the existing SEP-discovery workflow, we discover an additional 195 SEPs in K562 cells and extend this methodology to identify novel human SEPs in additional cell lines and human tissue for a final tally of 237 new SEPs. These results continue to expand the human genome and proteome and demonstrate that SEPs are a ubiquitous class of nonannotated polypeptides that require further investigation.


Asunto(s)
Neoplasias de la Mama/química , Genoma Humano , Sistemas de Lectura Abierta , Péptidos/análisis , Proteoma/análisis , Neoplasias de la Mama/genética , Línea Celular , Cromatografía Liquida , Codón Iniciador/química , Codón Iniciador/genética , Femenino , Humanos , Células K562 , Péptidos/química , Biosíntesis de Proteínas , Proteoma/química , Espectrometría de Masas en Tándem
7.
Proc Natl Acad Sci U S A ; 108(2): 680-5, 2011 Jan 11.
Artículo en Inglés | MEDLINE | ID: mdl-21187411

RESUMEN

Evolving lineages face a constant intracellular threat: most new coding sequence mutations destabilize the folding of the encoded protein. Misfolded proteins form insoluble aggregates and are hypothesized to be intrinsically cytotoxic. Here, we experimentally isolate a fitness cost caused by toxicity of misfolded proteins. We exclude other costs of protein misfolding, such as loss of functional protein or attenuation of growth-limiting protein synthesis resources, by comparing growth rates of budding yeast expressing folded or misfolded variants of a gratuitous protein, YFP, at equal levels. We quantify a fitness cost that increases with misfolded protein abundance, up to as much as a 3.2% growth rate reduction when misfolded YFP represents less than 0.1% of total cellular protein. Comparable experiments on variants of the yeast gene orotidine-5'-phosphate decarboxylase (URA3) produce similar results. Quantitative proteomic measurements reveal that, within the cell, misfolded YFP induces coordinated synthesis of interacting cytosolic chaperone proteins in the absence of a wider stress response, providing evidence for an evolved modular response to misfolded proteins in the cytosol. These results underscore the distinct and evolutionarily relevant molecular threat of protein misfolding, independent of protein function. Assuming that most misfolded proteins impose similar costs, yeast cells express almost all proteins at steady-state levels sufficient to expose their encoding genes to selection against misfolding, lending credibility to the recent suggestion that such selection imposes a global constraint on molecular evolution.


Asunto(s)
Citosol/química , Proteínas Fúngicas/química , Proteínas Bacterianas/química , Citosol/metabolismo , Evolución Molecular , Calor , Proteínas Luminiscentes/química , Chaperonas Moleculares/química , Desnaturalización Proteica , Pliegue de Proteína , Proteínas/química , Proteómica/métodos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Transcripción Genética
8.
J Biol Chem ; 284(44): 30016-23, 2009 Oct 30.
Artículo en Inglés | MEDLINE | ID: mdl-19740738

RESUMEN

Insulin stimulates the translocation of the glucose transporter GLUT4 from intracellular locations to the plasma membrane in adipose and muscle cells. Prior studies have shown that Akt phosphorylation of the Rab GTPase-activating protein, AS160 (160-kDa Akt substrate; also known as TBC1D4), triggers GLUT4 translocation, most likely by suppressing its Rab GTPase-activating protein activity. However, the regulation of a very similar protein, TBC1D1 (TBC domain family, member 1), which is mainly found in muscle, in insulin-stimulated GLUT4 translocation has been unclear. In the present study, we have identified likely Akt sites of insulin-stimulated phosphorylation of TBC1D1 in C2C12 myotubes. We show that a mutant of TBC1D1, in which several Akt sites have been converted to alanine, is considerably more inhibitory to insulin-stimulated GLUT4 translocation than wild-type TBC1D1. This result thus indicates that similar to AS160, Akt phosphorylation of TBC1D1 enables GLUT4 translocation. We also show that in addition to Akt activation, activation of the AMP-dependent protein kinase partially relieves the inhibition of GLUT4 translocation by TBC1D1. Finally, we show that the R125W variant of TBC1D1, which has been genetically associated with obesity, is equally inhibitory to insulin-stimulated GLUT4 translocation, as is wild-type TBC1D1, and that healthy and type 2 diabetic individuals express approximately the same level of TBC1D1 in biopsies of vastus lateralis muscle. In conclusion, phosphorylation of TBC1D1 is required for GLUT4 translocation. Thus, the regulation of TBC1D1 resembles that of its paralog, AS160.


Asunto(s)
Transportador de Glucosa de Tipo 4/metabolismo , Insulina/farmacología , Proteínas Nucleares/metabolismo , Células 3T3-L1 , Animales , Diabetes Mellitus Tipo 2/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Ratones , Músculo Esquelético/química , Proteínas Nucleares/análisis , Fosforilación , Transporte de Proteínas/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo
9.
Chem Biol ; 15(8): 808-17, 2008 Aug 25.
Artículo en Inglés | MEDLINE | ID: mdl-18721752

RESUMEN

The first three members of the ErbB family of receptor tyrosine kinases activate a wide variety of signaling pathways and are frequently misregulated in cancer. Much less is known about ErbB4. Here we use tandem mass spectrometry to identify 19 sites of tyrosine phosphorylation on ErbB4, and protein microarrays to quantify biophysical interactions between these sites and virtually every SH2 and PTB domain encoded in the human genome. Our unbiased approach highlighted several previously unrecognized interactions and led to the finding that ErbB4 can recruit and activate STAT1. At a systems level, we found that ErbB4 is much more selective than the other ErbB receptors. This suggests that ErbB4 may enable ErbB2 and ErbB3 to signal independently of EGFR under normal conditions, and provides a possible explanation for the protective properties of ErbB4 in cancer.


Asunto(s)
Receptores ErbB/química , Receptores ErbB/metabolismo , Fosfotirosina/metabolismo , Secuencia de Aminoácidos , Línea Celular , Humanos , Datos de Secuencia Molecular , Fosforilación , Análisis por Matrices de Proteínas , Unión Proteica , Receptor ErbB-2/metabolismo , Receptor ErbB-3/metabolismo , Receptor ErbB-4 , Factor de Transcripción STAT1/metabolismo , Transducción de Señal , Especificidad por Sustrato
10.
Biochim Biophys Acta ; 1764(12): 1870-80, 2006 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-17118724

RESUMEN

Mass spectrometry has been an analytical tool of choice for glycosylation analysis of individual proteins. Over the last 5 years several previously and newly developed mass spectrometry methods have been extended to global glycoprotein studies. In this review we discuss the importance of these global studies and the advances that have been made in enrichment analyses and fragmentation methods. We also briefly describe relevant sample preparation methods that have been used for the analysis of a single glycoprotein that could be extrapolated to global studies. Finally this review covers aspects of improvements and advances on the instrument front which are important to future global glycoproteomic studies.


Asunto(s)
Glicosilación , Espectrometría de Masas/métodos , Acetilglucosamina/química , Cromatografía de Afinidad/métodos , Transporte de Electrón , Electrones , Cromatografía de Gases y Espectrometría de Masas/métodos , Lectinas/aislamiento & purificación , Procesamiento Proteico-Postraduccional
11.
J Am Soc Mass Spectrom ; 17(4): 576-585, 2006 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-16503151

RESUMEN

To explore the mechanism of electron capture dissociation (ECD) of linear peptides, a set of 16-mer peptides were synthesized with deuterium labeled on the alpha-carbon position of four glycines. The ECD spectra of these peptides showed that such peptides exhibit a preference for the radical to migrate to the alpha-carbon position on glycine via hydrogen (or deuterium) abstraction before the final cleavage and generation of the detected product ions. The data show c-type fragment ions, ions corresponding to the radical cation of the c-type fragments, c*, and they also show c*-1 peaks in the deuterated peptides only. The presence of the c*-1 peaks is best explained by radical-mediated scrambling of the deuterium atoms in the long-lived, metastable, radical intermediate complex formed by initial electron capture, followed by dissociation of the complex. These data suggest the presence of at least two mechanisms, one slow, one fast. The abundance of H* and -CO losses from the precursor ion changed upon deuterium labeling indicating the presence of a kinetic isotope effect, which suggests that the values reported here represent an underestimation of radical migration and H/D scrambling in the observed fragments.


Asunto(s)
Espectrometría de Masas/métodos , Péptidos/química , Secuencia de Aminoácidos , Deuterio , Radicales Libres/química , Glicina/química , Hidrógeno/química , Estructura Molecular , Péptidos/síntesis química
12.
J Am Soc Mass Spectrom ; 16(12): 1985-99, 2005 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-16271296

RESUMEN

The use of a new electrospray qQq Fourier transform ion cyclotron mass spectrometer (qQq-FTICR MS) instrument for biologic applications is described. This qQq-FTICR mass spectrometer was designed for the study of post-translationally modified proteins and for top-down analysis of biologically relevant protein samples. The utility of the instrument for the analysis of phosphorylation, a common and important post-translational modification, was investigated. Phosphorylation was chosen as an example because it is ubiquitous and challenging to analyze. In addition, the use of the instrument for top-down sequencing of proteins was explored since this instrument offers particular advantages to this approach. Top-down sequencing was performed on different proteins, including commercially available proteins and biologically derived samples such as the human E2 ubiquitin conjugating enzyme, UbCH10. A good sequence tag was obtained for the human UbCH10, allowing the unambiguous identification of the protein. The instrument was built with a commercially produced front end: a focusing rf-only quadrupole (Q0), followed by a resolving quadrupole (Q1), and a LINAC quadrupole collision cell (Q2), in combination with an FTICR mass analyzer. It has utility in the analysis of samples found in substoichiometric concentrations, as ions can be isolated in the mass resolving Q1 and accumulated in Q2 before analysis in the ICR cell. The speed and efficacy of the Q2 cooling and fragmentation was demonstrated on an LCMS-compatible time scale, and detection limits for phosphopeptides in the 10 amol/muL range (pM) were demonstrated. The instrument was designed to make several fragmentation methods available, including nozzle-skimmer fragmentation, Q2 collisionally activated dissociation (Q2 CAD), multipole storage assisted dissociation (MSAD), electron capture dissociation (ECD), infrared multiphoton induced dissociation (IRMPD), and sustained off resonance irradiation (SORI) CAD, thus allowing a variety of MS(n) experiments. A particularly useful aspect of the system was the use of Q1 to isolate ions from complex mixtures with narrow windows of isolation less than 1 m/z. These features enable top-down protein analysis experiments as well structural characterization of minor components of complex mixtures.


Asunto(s)
Mapeo Peptídico/métodos , Procesamiento Proteico-Postraduccional , Proteínas/análisis , Proteínas/química , Análisis de Secuencia de Proteína/instrumentación , Espectrometría de Masa por Ionización de Electrospray/instrumentación , Espectroscopía Infrarroja por Transformada de Fourier/instrumentación , Diseño de Equipo , Análisis de Falla de Equipo , Fosfopéptidos/análisis , Fosfopéptidos/química , Fosforilación , Análisis de Secuencia de Proteína/métodos , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectroscopía Infrarroja por Transformada de Fourier/métodos
13.
J Am Soc Mass Spectrom ; 15(7): 1087-98, 2004 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-15234367

RESUMEN

First results are reported on the application of ECD in analysis of 2+ and 3+ ions of stereoisomers of Trp-cage (NLYIQWLKDGGPSSGRPPPS), the smallest and fastest-folding protein, which exhibits a tightly folded tertiary structure in solution. The chiral recognition based on the ratios of the abundances of z(18) and z(19) fragments in ECD of 2+ ions was excellent even for a single amino acid (Tyr) D-substitution (R(chiral) = 8.6). The chiral effect decreased with an increase of temperature at the electrospray ion source, as well as at a higher degree of ionization, 3+ ions (R(chiral) = 1.5). A general approach is suggested for charge localization in n+ ions by analysis of ECD mass spectra of (n + 1)+ ions. Application of this approach to 3+ Trp-cage ions revealed the protonation probability order in 2+ ions: Arg(16) >> Gln(5) > approximately N-terminus. The ECD results for native form of the 2+ ions favor the preservation of the solution-phase tertiary structure, and chiral recognition through the interaction between the charges and the neutral bond network. Conversely, ECD of 3+ ions supports the dominance of ionic hydrogen bonding which determines a different gas-phase structure than found in solution. Vibrational activation of 2+ ions indicated greater stability of the native form, but the fragmentation patterns did not provide stereoisomer differentiation, thus underlying the special position of ECD among other MS/MS fragmentation techniques. Further ECD studies should yield more structural information as well as quantitative single-amino acid D/L content measurements in proteins.


Asunto(s)
Aminoácidos/análisis , Aminoácidos/química , Cristalografía/métodos , Cromatografía de Gases y Espectrometría de Masas/métodos , Espectrometría de Masas/métodos , Electrones , Pliegue de Proteína , Estructura Terciaria de Proteína , Reproducibilidad de los Resultados , Sensibilidad y Especificidad
14.
J Am Soc Mass Spectrom ; 15(1): 128-32, 2004 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-14698563

RESUMEN

A new design for a high pressure MALDI-FTMS instrument is described and initial data are shown. The instrument incorporates a large, 10 cm x 10 cm, sample translation stage to accommodate and position the MALDI target. The new instrument allows coupling to a wide variety of surface techniques such as gel electrophoresis or surface plasmon resonance. Coupling to thin layer chromatography is shown. Furthermore, a new nozzle design allows high pressure collisional cooling sufficient to stabilize gangliosides while minimizing the gas load on the system.


Asunto(s)
Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/instrumentación , Espectroscopía Infrarroja por Transformada de Fourier/instrumentación , Cromatografía en Capa Delgada , Gangliósidos/química
15.
J Mass Spectrom ; 37(11): 1141-4, 2002 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-12447890

RESUMEN

Ionization energies (IE) of [M + zH](z+) (z+) electrospray-produced polypeptides were determined by electron ionization in a Penning cell of 4.7 and 9.4 T Fourier transform mass spectrometers. For z = 1+ and substance P, the found IE value of 11.0 +/- 0.4 eV is in agreement with that obtained earlier for ions generated with matrix-assisted laser desorption/ionization. For higher z, the following values were found: 11.7 +/- 0.3 eV for 2+ of [Arg-8]-vasopressin, 11.1 +/- 0.6 eV for 2+ of substance P, 12.2 +/- 0.7 eV for 2+ of renin substrate, 13.3 +/- 0.4 eV for 3+ of B-chain of insulin and 14.6 +/- 0.6 eV for 4+ and 15.1 +/- 0.4 eV for 5+ of melittin. It was found that 90% of existing IE data on polypeptides in the 1.0-3.5 kDa mass range are described with

Asunto(s)
Análisis de Fourier , Espectrometría de Masas/métodos , Péptidos/química , Angiotensinógeno/química , Insulina/química , Iones/química , Meliteno/química , Protones , Sustancia P/química , Vasopresinas/química
16.
Phytochemistry ; 59(5): 501-11, 2002 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-11853745

RESUMEN

Nineteen species of Passiflora (Passifloraceae) were examined for the presence of cyanogenic glycosides. Passibiflorin, a bisglycoside containing the 6-deoxy-beta-D-gulopyranosyl residue, was isolated from P. apetala, P. biflora, P. cuneata, P. indecora, P. murucuja and P. perfoliata. In some cases this glycoside co-occurs with simple beta-D-glucopyranosides: tetraphyllin A, deidaclin, tetraphyllin B, volkenin, epivolkenin and taraktophyllin. P. citrina contains passicapsin, a rare glycoside with the 2,6-dideoxy-beta-D-xylo-hexopyranosyl moiety, while P. herbertiana contains tetraphyllin A, deidaclin, epivolkenin and taraktophyllin, P. discophora tetraphyllin B and volkenin, and P. x violacea tetraphyllin B sulfate. The remaining species were noncyanogenic. The glycosides were identified by 1H and 13C NMR spectroscopy following isolation by reversed-phase preparative HPLC. From P. guatemalensis, a new glucoside named passiguatemalin was isolated and identified as a 1-(beta-D-glucopyranosyloxy)-2,3-dihydroxycyclopentane-1-carbonitrile. An isomeric glycoside was prepared by catalytic hydrogenation of gynocardin. alpha-Hydroxyamides corresponding to the cyanogenic glycosides were isolated from several Passiflora species. These alpha-hydroxyamides, presumably formed during processing of the plant material, behave as cyanogenic compounds when treated with commercial Helix pomatia crude enzyme preparation. Thus, the enzyme preparation appears to contain an amide dehydratase, which converts alpha-hydroxyamides to cyanohydrins that liberate cyanide; this finding is of interest in connection with analysis of plant tissues and extracts using Helix pomatia enzymes.


Asunto(s)
Amidas/metabolismo , Glicósidos/metabolismo , Nitrilos/metabolismo , Passiflora/metabolismo , Cromatografía Líquida de Alta Presión , Glicósidos/química , Espectroscopía de Resonancia Magnética , Nitrilos/química
17.
J Chromatogr A ; 962(1-2): 95-103, 2002 Jul 12.
Artículo en Inglés | MEDLINE | ID: mdl-12198976

RESUMEN

Two mass spectrometry methods, high-performance liquid chromatography combined on-line with electrospray ionization mass spectrometry (HPLC-ESI-MS) and electron-capture (EC) dissociation tandem mass spectrometry (MS-MS), were applied for structural analysis of bovine and human osteocalcins. Osteocalcin contains gamma-carboxyglutamic acid (Gla) residues, which bind metal ions, among its amino acids. Ethylenediaminetetraacetic acid (EDTA) was added to all samples in order to chelate bound metal ions. After elimination of interfering metal ions MS spectra became uncomplicated to interpret. EDTA is incompatible with ESI and it was removed from samples using either on-line HPLC or micropurification method. The number of Gla residues varies in osteocalcin. These subforms, which contain different amounts of Gla residues, were separated using the HPLC-ESI-MS method. In order to determine locations of Gla residues in human osteocalcin, which contained two Gla residues, dissociation MS-MS method was successfully applied.


Asunto(s)
Ácido 1-Carboxiglutámico/análisis , Cromatografía Líquida de Alta Presión/métodos , Osteocalcina/química , Espectrometría de Masa por Ionización de Electrospray/métodos , Secuencia de Aminoácidos , Animales , Bovinos , Quelantes/química , Ácido Edético/química , Humanos , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido
18.
Cell Rep ; 7(3): 705-14, 2014 May 08.
Artículo en Inglés | MEDLINE | ID: mdl-24767987

RESUMEN

Fermenting glucose in the presence of enough oxygen to support respiration, known as aerobic glycolysis, is believed to maximize growth rate. We observed increasing aerobic glycolysis during exponential growth, suggesting additional physiological roles for aerobic glycolysis. We investigated such roles in yeast batch cultures by quantifying O2 consumption, CO2 production, amino acids, mRNAs, proteins, posttranslational modifications, and stress sensitivity in the course of nine doublings at constant rate. During this course, the cells support a constant biomass-production rate with decreasing rates of respiration and ATP production but also decrease their stress resistance. As the respiration rate decreases, so do the levels of enzymes catalyzing rate-determining reactions of the tricarboxylic-acid cycle (providing NADH for respiration) and of mitochondrial folate-mediated NADPH production (required for oxidative defense). The findings demonstrate that exponential growth can represent not a single metabolic/physiological state but a continuum of changing states and that aerobic glycolysis can reduce the energy demands associated with respiratory metabolism and stress survival.


Asunto(s)
Metabolismo Energético , Glucólisis , Saccharomyces cerevisiae/crecimiento & desarrollo , Adenosina Trifosfato/metabolismo , Técnicas de Cultivo Celular por Lotes , Biomasa , Ciclo del Ácido Cítrico , Mitocondrias/metabolismo , NADP/metabolismo , Procesamiento Proteico-Postraduccional , ARN Mensajero/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
19.
Science ; 339(6118): 460-4, 2013 Jan 25.
Artículo en Inglés | MEDLINE | ID: mdl-23349292

RESUMEN

Signaling pathways can induce different dynamics of transcription factor (TF) activation. We explored how TFs process signaling inputs to generate diverse dynamic responses. The budding yeast general stress-responsive TF Msn2 acted as a tunable signal processor that could track, filter, or integrate signals in an input-dependent manner. This tunable signal processing appears to originate from dual regulation of both nuclear import and export by phosphorylation, as mutants with one form of regulation sustained only one signal-processing function. Versatile signal processing by Msn2 is crucial for generating distinct dynamic responses to different natural stresses. Our findings reveal how complex signal-processing functions are integrated into a single molecule and provide a guide for the design of TFs with "programmable" signal-processing functions.


Asunto(s)
Núcleo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transducción de Señal , Factores de Transcripción/metabolismo , Transporte Activo de Núcleo Celular , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Citoplasma/metabolismo , Modelos Biológicos , Señales de Exportación Nuclear , Señales de Localización Nuclear , Presión Osmótica , Estrés Oxidativo , Fosforilación , Proteínas/farmacología , Saccharomyces cerevisiae/genética , Estrés Fisiológico
20.
Genetics ; 195(4): 1307-17, 2013 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-24077307

RESUMEN

The Toll signaling pathway has a highly conserved function in innate immunity and is regulated by multiple factors that fine tune its activity. One such factor is ß-arrestin Kurtz (Krz), which we previously implicated in the inhibition of developmental Toll signaling in the Drosophila melanogaster embryo. Another level of controlling Toll activity and immune system homeostasis is by protein sumoylation. In this study, we have uncovered a link between these two modes of regulation and show that Krz affects sumoylation via a conserved protein interaction with a SUMO protease, Ulp1. Loss of function of krz or Ulp1 in Drosophila larvae results in a similar inflammatory phenotype, which is manifested as increased lamellocyte production; melanotic mass formation; nuclear accumulation of Toll pathway transcriptional effectors, Dorsal and Dif; and expression of immunity genes, such as Drosomycin. Moreover, mutations in krz and Ulp1 show dosage-sensitive synergistic genetic interactions, suggesting that these two proteins are involved in the same pathway. Using Dorsal sumoylation as a readout, we found that altering Krz levels can affect the efficiency of SUMO deconjugation mediated by Ulp1. Our results demonstrate that ß-arrestin controls Toll signaling and systemic inflammation at the level of sumoylation.


Asunto(s)
Arrestinas/metabolismo , Cisteína Endopeptidasas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Transducción de Señal , Receptores Toll-Like/metabolismo , Animales , Arrestinas/genética , Línea Celular , Cisteína Endopeptidasas/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Inflamación/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Unión Proteica , Sumoilación , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
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