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1.
Int J Mol Sci ; 20(7)2019 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-30965558

RESUMO

The growing integration of quality-by-design (QbD) concepts in biomanufacturing calls for a detailed and quantitative knowledge of the profile of impurities and their impact on the product safety and efficacy. Particularly valuable is the determination of the residual level of host cell proteins (HCPs) secreted, together with the product of interest, by the recombinant cells utilized for production. Though often referred to as a single impurity, HCPs comprise a variety of species with diverse abundance, size, function, and composition. The clearance of these impurities is a complex issue due to their cell line to cell line, product-to-product, and batch-to-batch variations. Improvements in HCP monitoring through proteomic-based methods have led to identification of a subset of "problematic" HCPs that are particularly challenging to remove, both at the product capture and product polishing steps, and compromise product stability and safety even at trace concentrations. This paper describes the development of synthetic peptide ligands capable of capturing a broad spectrum of Chinese hamster ovary (CHO) HCPs with a combination of peptide species that allow for advanced mixed-mode binding. Solid phase peptide libraries were screened for identification and characterization of peptides that capture CHO HCPs while showing minimal binding of human IgG, utilized here as a model product. Tetrameric and hexameric ligands featuring either multipolar or hydrophobic/positive amino acid compositions were found to be the most effective. Tetrameric multipolar ligands exhibited the highest targeted binding ratio (ratio of HCP clearance over IgG loss), more than double that of commercial mixed-mode and anion exchange resins utilized by industry for IgG polishing. All peptide resins tested showed preferential binding to HCPs compared to IgG, indicating potential uses in flow-through mode or weak-partitioning-mode chromatography.


Assuntos
Peptídeos/isolamento & purificação , Animais , Células CHO , Cromatografia de Afinidade , Cromatografia Líquida , Cricetinae , Cricetulus , Humanos , Peptídeos/química , Proteômica/métodos
2.
Plant Physiol ; 178(1): 372-389, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30006378

RESUMO

Geminiviruses are single-stranded DNA viruses that infect a wide variety of plants and cause severe crop losses worldwide. The geminivirus replication initiator protein (Rep) binds to the viral replication origin and catalyzes DNA cleavage and ligation to initiate rolling circle replication. In this study, we found that the Tomato golden mosaic virus (TGMV) Rep is phosphorylated at serine-97 by sucrose nonfermenting 1-related protein kinase 1 (SnRK1), a master regulator of plant energy homeostasis and metabolism. Phosphorylation of Rep or the phosphomimic S97D mutation impaired Rep binding to viral DNA. A TGMV DNA-A replicon containing the Rep S97D mutation replicated less efficiently in tobacco (Nicotiana tabacum) protoplasts than in wild-type or Rep phosphorylation-deficient replicons. The TGMV Rep-S97D mutant also was less infectious than the wild-type virus in Nicotiana benthamiana and was unable to infect tomato (Solanum lycopersicum). Nearly all geminivirus Rep proteins have a serine residue at the position equivalent to TGMV Rep serine-97. SnRK1 phosphorylated the equivalent serines in the Rep proteins of Tomato mottle virus and Tomato yellow leaf curl virus and reduced DNA binding, suggesting that SnRK1 plays a key role in combating geminivirus infection. These results established that SnRK1 phosphorylates Rep and interferes with geminivirus replication and infection, underscoring the emerging role for SnRK1 in the host defense response against plant pathogens.

3.
Plant Cell ; 29(9): 2285-2303, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28842532

RESUMO

BAK1 is a coreceptor and positive regulator of multiple ligand binding leucine-rich repeat receptor kinases (LRR-RKs) and is involved in brassinosteroid (BR)-dependent growth and development, innate immunity, and cell death control. The BAK1-interacting LRR-RKs BIR2 and BIR3 were previously identified by proteomics analyses of in vivo BAK1 complexes. Here, we show that BAK1-related pathways such as innate immunity and cell death control are affected by BIR3 in Arabidopsis thaliana BIR3 also has a strong negative impact on BR signaling. BIR3 directly interacts with the BR receptor BRI1 and other ligand binding receptors and negatively regulates BR signaling by competitive inhibition of BRI1. BIR3 is released from BAK1 and BRI1 after ligand exposure and directly affects the formation of BAK1 complexes with BRI1 or FLAGELLIN SENSING2. Double mutants of bak1 and bir3 show spontaneous cell death and constitutive activation of defense responses. BAK1 and its closest homolog BKK1 interact with and are stabilized by BIR3, suggesting that bak1 bir3 double mutants mimic the spontaneous cell death phenotype observed in bak1 bkk1 mutants via destabilization of BIR3 target proteins. Our results provide evidence for a negative regulatory mechanism for BAK1 receptor complexes in which BIR3 interacts with BAK1 and inhibits ligand binding receptors to prevent BAK1 receptor complex formation.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas/metabolismo , Arabidopsis/efeitos dos fármacos , Brassinosteroides/metabolismo , Morte Celular/efeitos dos fármacos , Flagelina/farmacologia , Ligantes , Mutação/genética , Padrões Moleculares Associados a Patógenos/metabolismo , Fenótipo , Ligação Proteica/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Transdução de Sinais
4.
J Proteome Res ; 16(4): 1579-1592, 2017 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-28282151

RESUMO

Cilia are essential to many diverse cellular processes. Although many major axonemal components have been identified and studied, how they interact to form a functional axoneme is not completely understood. To further our understanding of the protein composition of human airway cilia, we performed a semiquantitative analysis of ciliary axonemes using label-free LC/MSE, which identified over 400 proteins with high confidence. Tubulins were the most abundant proteins identified, with evidence of 20 different isoforms obtained. Twelve different isoforms of axonemal dynein heavy chain were also identified. Absolute quantification of the nontubulin components demonstrated a greater than 75-fold range of protein abundance (RSPH9;1850 fmol vs CCDC103;24 fmol), adding another level of complexity to axonemal structure. Of the identified proteins, ∼70% are known axonemal proteins. In addition, many previously uncharacterized proteins were identified. Unexpectedly, several of these, including ERICH3, C1orf87, and CCDC181, were present at high relative abundance in the cilia. RT-PCR analysis and immunoblotting confirmed cilia-specific expression for eight uncharacterized proteins, and fluorescence microscopy demonstrated unique axonemal localizations. These studies have provided the first quantitative analysis of the ciliary proteome and have identified and characterized several previously unknown proteins as major constituents of human airway cilia.


Assuntos
Axonema/genética , Cílios/genética , Proteínas/genética , Proteoma/genética , Dineínas/genética , Dineínas/isolamento & purificação , Regulação da Expressão Gênica , Humanos , Proteínas/isolamento & purificação , Proteômica , Tubulina (Proteína)/genética , Tubulina (Proteína)/isolamento & purificação
5.
J Biol Chem ; 292(10): 3988-4002, 2017 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-28154194

RESUMO

Plant calcium (Ca2+)-dependent protein kinases (CPKs) represent the primary Ca2+-dependent protein kinase activities in plant systems. CPKs are composed of a dual specificity (Ser/Thr and Tyr) kinase domain tethered to a calmodulin-like domain (CLD) via an autoinhibitory junction (J). Although regulation of CPKs by Ca2+ has been extensively studied, the contribution of autophosphorylation in controlling CPK activity is less well understood. Furthermore, whether calmodulin (CaM) contributes to CPK regulation, as is the case for Ca2+/CaM-dependent protein kinases outside the plant lineage, remains an open question. We therefore screened a subset of plant CPKs for CaM binding and found that CPK28 is a high affinity Ca2+/CaM-binding protein. Using synthetic peptides and native gel electrophoresis, we coarsely mapped the CaM-binding domain to a site within the CPK28 J domain that overlaps with the known site of intramolecular interaction between the J domain and the CLD. Peptide kinase activity of fully dephosphorylated CPK28 was Ca2+-responsive and was inhibited by Ca2+/CaM. Using in situ autophosphorylated protein, we expand on the known set of CPK28 autophosphorylation sites, and we demonstrate that, unexpectedly, autophosphorylated CPK28 had enhanced kinase activity at physiological concentrations of Ca2+ compared with the dephosphorylated protein, suggesting that autophosphorylation functions to prime CPK28 for Ca2+ activation and might also allow CPK28 to remain active when Ca2+ levels are low. Furthermore, CPK28 autophosphorylation substantially reduced sensitivity of the kinase to Ca2+/CaM inhibition. Overall, our analyses uncover new complexities in the control of CPK28 and provide mechanistic support for Ca2+ signaling specificity through Ca2+ sensor priming.


Assuntos
Arabidopsis/metabolismo , Cálcio/farmacologia , Calmodulina/farmacologia , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Proteínas Quinases/química , Sequência de Aminoácidos , Arabidopsis/efeitos dos fármacos , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/metabolismo , Cinética , Fosforilação/efeitos dos fármacos , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Quinases/metabolismo , Homologia de Sequência de Aminoácidos
6.
Plant Physiol ; 171(1): 71-81, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-26960732

RESUMO

Symbiosis receptor kinase (SYMRK) is indispensable for activation of root nodule symbiosis (RNS) at both epidermal and cortical levels and is functionally conserved in legumes. Previously, we reported SYMRK to be phosphorylated on "gatekeeper" Tyr both in vitro as well as in planta. Since gatekeeper phosphorylation was not necessary for activity, the significance remained elusive. Herein, we show that substituting gatekeeper with nonphosphorylatable residues like Phe or Ala significantly affected autophosphorylation on selected targets on activation segment/αEF and ß3-αC loop of SYMRK. In addition, the same gatekeeper mutants failed to restore proper symbiotic features in a symrk null mutant where rhizobial invasion of the epidermis and nodule organogenesis was unaffected but rhizobia remain restricted to the epidermis in infection threads migrating parallel to the longitudinal axis of the root, resulting in extensive infection patches at the nodule apex. Thus, gatekeeper phosphorylation is critical for synchronizing epidermal/cortical responses in RNS.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Plantas/metabolismo , Raízes de Plantas/metabolismo , Proteínas Quinases/metabolismo , Nódulos Radiculares de Plantas/metabolismo , Simbiose , Tirosina/metabolismo , Sequência de Aminoácidos , Proteínas de Transporte/genética , Fabaceae/metabolismo , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Mutagênese , Mutação , Fenótipo , Fosfoaminoácidos/análise , Fosforilação , Epiderme Vegetal , Proteínas de Plantas/genética , Nodulação , Raízes de Plantas/microbiologia , Proteínas Quinases/genética , Rhizobium/fisiologia , Nódulos Radiculares de Plantas/enzimologia , Nódulos Radiculares de Plantas/genética
7.
Methods ; 89: 64-73, 2015 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-26091612

RESUMO

In this article, we describe our methods and protocols using collision-induced dissociative chemical crosslinking-tandem mass spectrometry (CID-CXL-MS/MS) analysis and the practical considerations when implementing these reagents and methodology for protein crosslinking studies. The synthesis of our novel chemical crosslinkers is described as well as their use for effectively labeling protein and protein complexes. Several sample preparation methods for liquid chromatography-tandem mass spectrometry are provided including the enrichment of interpeptide crosslinks. For identification of CID-CXL-MS/MS crosslinks, details regarding MS acquisition parameters and the utilization of various mass spectrometers are addressed along with post-data acquisition analysis to identify interpeptide crosslinks. Once the CID-CXL-MS/MS approach is optimized for a protein target or a set of targets, it can be used as a tool for biological research for studying protein structure when integrated with data obtained using other techniques, such as NMR, X-ray crystallography, and cryo-electron microscopy, or extended to the study of protein-protein interactions in physiological environments.


Assuntos
Reagentes para Ligações Cruzadas/química , Reagentes para Ligações Cruzadas/metabolismo , Soroalbumina Bovina/análise , Soroalbumina Bovina/metabolismo , Espectrometria de Massas em Tandem/métodos , Animais , Bovinos , Cromatografia Líquida/métodos , Ligação Proteica/fisiologia
8.
Plant J ; 82(6): 1042-60, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25912465

RESUMO

Leucine-rich repeat receptor-like kinases (LRR RLKs) form a large family of plant signaling proteins consisting of an extracellular domain connected by a single-pass transmembrane sequence to a cytoplasmic kinase domain. Autophosphorylation on specific Ser and/or Thr residues in the cytoplasmic domain is often critical for the activation of several LRR RLK family members with proven functional roles in plant growth regulation, morphogenesis, disease resistance, and stress responses. While identification and functional characterization of in vivo phosphorylation sites is ultimately required for a full understanding of LRR RLK biology and function, bacterial expression of recombinant LRR RLK cytoplasmic catalytic domains for identification of in vitro autophosphorylation sites provides a useful resource for further targeted identification and functional analysis of in vivo sites. In this study we employed high-throughput cloning and a variety of mass spectrometry approaches to generate an autophosphorylation site database representative of more than 30% of the approximately 223 LRR RLKs in Arabidopsis thaliana. We used His-tagged constructs of complete cytoplasmic domains to identify a total of 592 phosphorylation events across 73 LRR RLKs, with 497 sites uniquely assigned to specific Ser (268 sites) or Thr (229 sites) residues in 68 LRR RLKs. Multiple autophosphorylation sites per LRR RLK were the norm, with an average of seven sites per cytoplasmic domain, while some proteins showed more than 20 unique autophosphorylation sites. The database was used to analyze trends in the localization of phosphorylation sites across cytoplasmic kinase subdomains and to derive a statistically significant sequence motif for phospho-Ser autophosphorylation.


Assuntos
Proteínas de Arabidopsis/metabolismo , Bases de Dados Factuais , Proteínas Quinases/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Citoplasma/metabolismo , Escherichia coli/genética , Dados de Sequência Molecular , Fosforilação , Proteínas Quinases/genética , Estrutura Terciária de Proteína , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo
9.
Artigo em Inglês | MEDLINE | ID: mdl-25777480

RESUMO

Phosphorylation is an important post-translational modification that is involved in regulating many signaling pathways. Of particular interest are the growth factor mediated Ras and phosphoinositide 3-kinase (PI3K) signaling pathways which, if misregulated, can contribute to the progression of cancer. Phosphoproteomic methods have been developed to study regulation of signaling pathways; however, due to the low stoichiometry of phosphorylation, understanding these pathways is still a challenge. In this study, we have developed a multi-dimensional method incorporating electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) with tandem IMAC/TiO2 enrichment for subsequent phosphopeptide identification by LC/MS/MS. We applied this method to PDGF-stimulated NIH 3T3 cells to provide over 11,000 unique phosphopeptide identifications. Upon motif analysis, IMAC was found to enrich for basophilic kinase substrates while the subsequent TiO2 step enriched for acidophilic kinase substrates, suggesting that both enrichment methods are necessary to capture the full complement of kinase substrates. Biological functions that were over-represented at each PDGF stimulation time point, together with the phosphorylation dynamics of several phosphopeptides containing known kinase phosphorylation sites, illustrate the feasibility of this approach in quantitative phosphoproteomic studies.


Assuntos
Cromatografia Líquida/métodos , Fosfopeptídeos/análise , Proteômica/métodos , Transdução de Sinais/fisiologia , Espectrometria de Massas em Tandem/métodos , Sequência de Aminoácidos , Dados de Sequência Molecular , Fosfopeptídeos/química
10.
Anal Chem ; 86(14): 6731-40, 2014 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-24983800

RESUMO

Advances in resolution and sensitivity of analytical techniques have provided novel applications, including the analyses of fossil material. However, the recovery of original proteinaceous components from very old fossil samples (defined as >1 million years (1 Ma) from previously named limits in the literature) is far from trivial. Here, we discuss the challenges to recovery of proteinaceous components from fossils, and the need for new sample preparation techniques, analytical methods, and bioinformatics to optimize and fully utilize the great potential of information locked in the fossil record. We present evidence for survival of original components across geological time, and discuss the potential benefits of recovery, analyses, and interpretation of fossil materials older than 1 Ma, both within and outside of the fields of evolutionary biology.


Assuntos
Dinossauros , Fósseis , Proteínas/análise , Proteômica/métodos , Animais , Osso e Ossos , Colágeno/análise , Colágeno/química , Hidroxiprolina/análise , Queratinas/análise , Queratinas/química , Espectrometria de Massas/métodos
11.
J Virol ; 88(18): 10598-612, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24990996

RESUMO

UNLABELLED: Geminivirus AL2/C2 proteins play key roles in establishing infection and causing disease in their plant hosts. They are involved in viral gene expression, counter host defenses by suppressing transcriptional gene silencing, and interfere with the host signaling involved in pathogen resistance. We report here that begomovirus and curtovirus AL2/C2 proteins interact strongly with host geminivirus Rep-interacting kinases (GRIKs), which are upstream activating kinases of the protein kinase SnRK1, a global regulator of energy and nutrient levels in plants. We used an in vitro kinase system to show that GRIK-activated SnRK1 phosphorylates recombinant AL2/C2 proteins from several begomoviruses and to map the SnRK1 phosphorylation site to serine-109 in the AL2 proteins of two New World begomoviruses: Cabbage Leaf Curl Virus (CaLCuV) and Tomato mottle virus. A CaLCuV AL2 S109D phosphomimic mutation did not alter viral DNA levels in protoplast replication assays. In contrast, the phosphomimic mutant was delayed for symptom development and viral DNA accumulation during infection of Arabidopsis thaliana, demonstrating that SnRK1 contributes to host defenses against CaLCuV. Our observation that serine-109 is not conserved in all AL2/C2 proteins that are SnRK1 substrates in vitro suggested that phosphorylation of viral proteins by plant kinases contributes to the evolution of geminivirus-host interactions. IMPORTANCE: Geminiviruses are single-stranded DNA viruses that cause serious diseases in many crops. Dicot-infecting geminiviruses carry genes that encode multifunctional AL2/C2 proteins that are essential for infection. However, it is not clear how AL2/C2 proteins are regulated. Here, we show that the host protein kinase SnRK1, a central regulator of energy balance and nutrient metabolism in plants, phosphorylates serine-109 in AL2 proteins of three subgroups of New World begomoviruses, resulting in a delay in viral DNA accumulation and symptom appearance. Our results support SnRK1's antiviral role and reveal a novel mechanism underlying this function. Phylogenetic analysis suggested that AL2 S109 evolved as begomoviruses migrated from the Old World to the New World and may have provided a selective advantage as begomoviruses adapted to a different environment and different plant hosts. This study provides new insights into the interaction of viral pathogens with their plant hosts at the level of viral protein modification by the host.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Arabidopsis/virologia , Begomovirus/metabolismo , Doenças das Plantas/virologia , Fatores de Transcrição/metabolismo , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Begomovirus/química , Begomovirus/classificação , Begomovirus/genética , Interações Hospedeiro-Patógeno , Dados de Sequência Molecular , Filogenia , Ligação Proteica , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Fatores de Transcrição/genética , Proteínas Virais/química , Proteínas Virais/genética
12.
Mol Syst Biol ; 10: 718, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24489118

RESUMO

The extracellular signal-regulated kinase (ERK) signaling pathway controls cell proliferation and differentiation in metazoans. Two hallmarks of its dynamics are adaptation of ERK phosphorylation, which has been linked to negative feedback, and nucleocytoplasmic shuttling, which allows active ERK to phosphorylate protein substrates in the nucleus and cytosol. To integrate these complex features, we acquired quantitative biochemical and live-cell microscopy data to reconcile phosphorylation, localization, and activity states of ERK. While maximal growth factor stimulation elicits transient ERK phosphorylation and nuclear translocation responses, ERK activities available to phosphorylate substrates in the cytosol and nuclei show relatively little or no adaptation. Free ERK activity in the nucleus temporally lags the peak in nuclear translocation, indicating a slow process. Additional experiments, guided by kinetic modeling, show that this process is consistent with ERK's modification of and release from nuclear substrate anchors. Thus, adaptation of whole-cell ERK phosphorylation is a by-product of transient protection from phosphatases. Consistent with this interpretation, predictions concerning the dose-dependence of the pathway response and its interruption by inhibition of MEK were experimentally confirmed.


Assuntos
Transporte Ativo do Núcleo Celular , MAP Quinases Reguladas por Sinal Extracelular/química , Retroalimentação Fisiológica , Modelos Teóricos , Animais , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Cinética , Camundongos , Células NIH 3T3 , Fosforilação , Transporte Proteico/genética , Receptores de Fatores de Crescimento/genética , Transdução de Sinais/genética
13.
Plant Cell ; 25(9): 3377-88, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24064770

RESUMO

Brassinosteroid (BR) hormones are primarily perceived at the cell surface by the leucine-rich repeat receptor-like kinase brassinosteroid insensitive1 (BRI1). In Arabidopsis thaliana, BRI1 has two close homologs, BRI1-LIKE1 (BRL1) and BRL3, respectively, which are expressed in the vascular tissues and regulate shoot vascular development. Here, we identify novel components of the BRL3 receptor complex in planta by immunoprecipitation and mass spectrometry analysis. Whereas BRI1 associated kinase1 (BAK1) and several other known BRI1 interactors coimmunoprecipitated with BRL3, no evidence was found of a direct interaction between BRI1 and BRL3. In addition, we confirmed that BAK1 interacts with the BRL1 receptor by coimmunoprecipitation and fluorescence microscopy analysis. Importantly, genetic analysis of brl1 brl3 bak1-3 triple mutants revealed that BAK1, BRL1, and BRL3 signaling modulate root growth and development by contributing to the cellular activities of provascular and quiescent center cells. This provides functional relevance to the observed protein-protein interactions of the BRL3 signalosome. Overall, our study demonstrates that cell-specific BR receptor complexes can be assembled to perform different cellular activities during plant root growth, while highlighting that immunoprecipitation of leucine-rich repeat receptor kinases in plants is a powerful approach for unveiling signaling mechanisms with cellular resolution in plant development.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Proteínas Serina-Treonina Quinases/genética , Receptores de Superfície Celular/genética , Transdução de Sinais , Arabidopsis/citologia , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Brassinosteroides/metabolismo , Ciclo Celular , Cromatografia Líquida , Genes Reporter , Complexos Multiproteicos , Mutação , Fenótipo , Floema/citologia , Floema/genética , Floema/crescimento & desenvolvimento , Floema/metabolismo , Raízes de Plantas/citologia , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas , Mapeamento de Interação de Proteínas , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Superfície Celular/metabolismo , Proteínas Recombinantes de Fusão , Espectrometria de Massas em Tandem
14.
FEBS Lett ; 587(18): 2972-9, 2013 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-23962520

RESUMO

Plant receptor-like kinases (RLKs) share their evolutionary origin with animal interleukin-1 receptor-associated kinase (IRAK)/Pelle family of soluble kinases and are distinguished by having tyrosine as 'gatekeeper'. This position is adjacent to the hinge region and is hidden in a hydrophobic pocket of the catalytic cleft of protein kinases and is therefore least probable to be a target for any modification. This communication illustrates the accessibility of the gatekeeper site (Y670) towards both autophosphorylation and dephosphorylation in the recombinant cytoplasmic domain of symbiosis receptor kinase from Arachis hypogaea (AhSYMRK). Autophosphorylation on gatekeeper tyrosine was detected prior to extraction but never under in vitro conditions. We hypothesize gatekeeper phosphorylation to be associated with synthesis/maturation of AhSYMRK and this phenomenon may be prevalent among RLKs.


Assuntos
Arachis/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Tirosina/metabolismo , Motivos de Aminoácidos , Arachis/genética , Domínio Catalítico , Linhagem Celular , Mutação , Fosforilação , Proteínas de Plantas/genética , Ligação Proteica , Receptores Proteína Tirosina Quinases/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Tirosina/genética
15.
Plant Physiol ; 163(1): 30-42, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23843605

RESUMO

Brassinosteroids (BRs) are plant hormones that are perceived at the cell surface by a membrane-bound receptor kinase, BRASSINOSTEROID INSENSITIVE1 (BRI1). BRI1 interacts with BRI1-ASSOCIATED RECEPTOR KINASE1 (BAK1) to initiate a signal transduction pathway in which autophosphorylation and transphosphorylation of BRI1 and BAK1, as well as phosphorylation of multiple downstream substrates, play critical roles. Detailed mechanisms of BR signaling have been examined in Arabidopsis (Arabidopsis thaliana), but the role of BRI1 and BAK1 phosphorylation in crop plants is unknown. As a foundation for understanding the mechanism of BR signaling in tomato (Solanum lycopersicum), we used liquid chromatography-tandem mass spectrometry to identify multiple in vitro phosphorylation sites of the tomato BRI1 and BAK1 cytoplasmic domains. Kinase assays showed that both tomato BRI1 and BAK1 are active in autophosphorylation as well as transphosphorylation of each other and specific peptide substrates with a defined sequence motif. Site-directed mutagenesis revealed that the highly conserved kinase domain activation loop residue threonine-1054 was essential for tomato BRI1 autophosphorylation and peptide substrate phosphorylation in vitro. Furthermore, analysis of transgenic lines expressing full-length tomato BRI1-Flag constructs in the weak tomato bri1 allele, curl3(-abs1), demonstrated that threonine-1054 is also essential for normal BRI1 signaling and tomato growth in planta. Finally, we cloned the tomato ortholog of TGF-ß Receptor Interacting Protein (TRIP1), which was previously shown to be a BRI1-interacting protein and kinase domain substrate in Arabidopsis, and found that tomato TRIP1 is a substrate of both tomato BRI1 and BAK1 kinases in vitro.


Assuntos
Lycopersicon esculentum/enzimologia , Proteínas de Plantas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Sítios de Ligação , Cinética , Fosforilação , Proteínas de Plantas/química , Proteínas Serina-Treonina Quinases/química , Transdução de Sinais
16.
Biochem Biophys Res Commun ; 431(2): 253-7, 2013 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-23313475

RESUMO

To fully understand the modes of action of multi-protein complexes, it is essential to determine their overall global architecture and the specific relationships between domains and subunits. The transcription factor AbrB is a functional homotetramer consisting of two domains per monomer. Obtaining the high-resolution structure of tetrameric AbrB has been extremely challenging due to the independent character of these domains. To facilitate the structure determination process, we solved the NMR structures of both domains independently and utilized gas-phase cleavable chemical crosslinking and LC/MS(n) analysis to correctly position the domains within the full tetrameric AbrB protein structure.


Assuntos
Proteínas de Bactérias/química , Reagentes para Ligações Cruzadas/química , Proteínas de Ligação a DNA/química , Fatores de Transcrição/química , Sequência de Aminoácidos , Cromatografia Líquida , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Espectrometria de Massas em Tandem
17.
J Proteome Res ; 11(12): 5663-77, 2012 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-23106611

RESUMO

Vaccination is an effective strategy to reduce the loss of chickens in the poultry industry caused by Marek's Disease (MD), an avian lymphoproliferative disease. The vaccines currently used are from attenuated serotype 1 Marek's disease virus (MDV) or naturally nononcogenic MDV strains. To prepare for future immunity breaks, functional genomic and proteomic studies have been used to better understand the underlying mechanisms of MDV pathogenicity and the effects induced by the vaccine viruses. In this study, a combined approach of quantitative GeLC-MSE and qualitative ERLIC/IMAC/LC-MS/MS analysis were used to identify abundance changes of proteins and the variations of phosphorylation status resulting from the perturbations due to infection with an attenuated oncogenic virus strain (Md11/75C) and several nononcogenic virus strains (CVI988, FC126 and 301B) in vitro. Using this combined approach, several signal transduction pathways mapped by the identified proteins were found to be altered at both the level of protein abundance and phosphorylation. On the basis of this study, a kinase-dependent pathway to regulate phosphorylation of 4E-BP1 to modulate assembly of the protein translation initiation complex was revealed. The differences of 4E-BP1 phosphorylation patterns as well as the measured abundance changes among several other proteins that regulate host transcriptional and translational activities across the virus strains used in this study provide new insight for future functional and biochemical characterization of specific proteins involved in MDV pathogenesis.


Assuntos
Fibroblastos/virologia , Herpesvirus Galináceo 2/patogenicidade , Fosfoproteínas/análise , Proteoma/análise , Proteômica/métodos , Sequência de Aminoácidos , Animais , Células Cultivadas , Embrião de Galinha , Cromatografia de Afinidade/métodos , Fator de Iniciação 4E em Eucariotos/metabolismo , Fibroblastos/metabolismo , Herpesvirus Galináceo 2/classificação , Doença de Marek , Espectrometria de Massas/métodos , Dados de Sequência Molecular , Complexos Multiproteicos/análise , Complexos Multiproteicos/metabolismo , Iniciação Traducional da Cadeia Peptídica , Fosfopeptídeos/análise , Fosfopeptídeos/metabolismo , Fosfoproteínas/metabolismo , Fosforilação , Proteoma/metabolismo , Transcrição Genética , Ensaio de Placa Viral
18.
Methods Mol Biol ; 876: 1-15, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22576082

RESUMO

Ligand binding by the extracellular domain of receptor kinases leads to phosphorylation and activation of the cytoplasmic domain of these important membrane-bound signaling proteins. To thoroughly characterize receptor kinase function, it is essential to identify specific phosphorylation sites by mass spectrometry. In this chapter, we summarize an efficient protein purification and modification protocol to prepare receptor kinases for liquid chromatography/tandem mass spectrometry analysis. Both recombinant receptor kinase cytoplasmic domains expressed in bacteria and full-length receptor kinase proteins expressed in living plant tissue are considered, and multiple methods of mass spectrometry are described that allow optimal identification of phosphorylated peptides of both in vitro- and in vivo-derived samples.


Assuntos
Fosfotransferases/metabolismo , Proteínas de Plantas/metabolismo , Cromatografia de Afinidade , Cromatografia Líquida , Espectrometria de Massas , Fosfopeptídeos/metabolismo , Fosforilação , Transdução de Sinais , Espectrometria de Massas em Tandem
19.
Proteomics ; 12(3): 401-5, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22213719

RESUMO

We describe a novel two-step LC/MS(n) strategy to effectively and confidently identify numerous crosslinked peptides from complex mixtures. This method incorporates the use of our gas-phase cleavable crosslinking reagent, disuccinimidyl-succinamyl-aspartyl-proline (SuDP), and a new data-processing algorithm CXLinkS (Cleavable Crosslink Selection), which enables unequivocal crosslink peptide selection and identification on the basis of mass measurement accuracy, high resolving power, and the unique fragmentation pattern of each crosslinked peptide. We demonstrate our approach with well-characterized monomeric and multimeric protein systems with and without database searching restrictions where inter-peptide crosslink identification is increased 8-fold over our previously published data-dependent LC/MS³ method and discuss its applicability to other CID-cleavable crosslinkers and more complex protein systems.


Assuntos
Misturas Complexas/análise , Hemoglobinas/química , Peptídeos/análise , Proteínas/análise , Algoritmos , Cromatografia Líquida/métodos , Bases de Dados de Proteínas , Humanos , Espectrometria de Massas/métodos , Peptídeos/metabolismo , Proteínas/metabolismo
20.
Bioconjug Chem ; 22(10): 1970-82, 2011 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-21854060

RESUMO

Plant virus nanoparticle (PVN) formulations constructed from Red clover necrotic mosaic virus by drug infusion and targeting peptide conjugation can be employed as drug delivery tools. In this investigation, we studied the cross-linked structures formed by application of sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sSMCC) and succinimidyl-[(N-maleimidopropionamido)-hexaethylene glycol] ester (SMPEG) as heterobifunctional linkers in the bioconjugation process. The plant virus formulations using several targeting peptides cross-linked to the plant virus capsid were characterized by LC/MS(E) analysis, which produced at least 69% sequence coverage using trypsin and chymotrypsin digestion. The results showed evidence for several types of modification located in three domains of the capsid protein. Extensive linker modifications on lysines or cysteines were detected in all the domains, including both intended peptide-capsid cross-links and unintended intracapsid cross-links. Surprisingly, the most extensive peptide modification was observed in the R domain, which is thought to be quite inaccessible to peptides and cross-linking reagents in solution, since it is on the interior of the virus. These results show that heterobifunctional linkers may not be the most efficient method for attachment of peptides to plant virus capsids. As an alternative conjugation strategy, maleimide peptides were used to conjugate with the virus in a one-step reaction. Analysis by LC/MS(E) showed that these one-step maleimide coupling reactions were more specific, such as modifications of C154 and to a lesser extent C267, and provide a means for achieving more effective PVN formulations.


Assuntos
Reagentes para Ligações Cruzadas/química , Sistemas de Liberação de Medicamentos , Peptídeos/química , Tombusviridae/química , Sequência de Aminoácidos , Capsídeo/química , Espectrometria de Massas , Modelos Moleculares , Dados de Sequência Molecular
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