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1.
iScience ; 23(3): 100968, 2020 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-32199293

RESUMO

R-loops, three-stranded DNA-DNA:RNA hybrid structures, are best known for their deleterious effects on genome stability. The regulatory factors of this fundamental genetic structure remain unclear. Here, we reveal an epigenetic factor that controls R-loop stability. METTL8, a member of the methyltransferase-like protein family that methylates 3-methylcytidine (m3C), is a key factor in the R-loop regulating methyltransferase complex. Biochemical studies show that METTL8 forms a large SUMOylated nuclear RNA-binding protein complex (∼0.8 mega daltons) that contains well-reported R-loop related factors. Genetic ablation of METTL8 results in an overall reduction of R-loops in cells. Interaction assays indicated METTL8 binds to RNAs and is responsible for R-loop stability on selected gene regions. Our results demonstrate that the SUMOylated METTL8 promotes tumorigenesis by affecting genetic organization primarily in, or in close proximity to, the nucleolus and impacts the formation of regulatory R-loops through its methyltransferase activity on m3C.

2.
Sci Rep ; 9(1): 9998, 2019 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-31292492

RESUMO

Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that has been linked with the development of systemic lupus erythematosus (SLE). Thus far, molecular mimicry has been implicated as the principal mechanism that explains this association. In this study, we characterise a potential alternative process whereby HCMV contributes to SLE. In a cohort of SLE patients, we show a significant association between HCMV infection and SLE through a human antibody response that targets UL44. UL44 is an obligate nuclear-resident, non-structural viral protein vital for HCMV DNA replication. The intracellular nature of this viral protein complicates its targeting by the humoral response - the mechanism remains unresolved. To characterise this response, we present a thorough molecular analysis of the first human monoclonal antibody specific for UL44 derived from a HCMV seropositive donor. This human antibody immunoprecipitates UL44 from HCMV-infected cells together with known nuclear-resident SLE autoantigens - namely, nucleolin, dsDNA and ku70. We also show that UL44 is redistributed to the cell surface during virus-induced apoptosis as part of a complex with these autoantigens. This phenomenon represents a potential mechanism for the bystander presentation of SLE autoantigens to the humoral arm of our immune system under circumstances that favour a break in peripheral tolerance.

3.
Sci Rep ; 9(1): 7591, 2019 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-31110185

RESUMO

Atherosclerosis is the main killer in the western world. Today's clinical markers include the total level of cholesterol and high-/low-density lipoproteins, which often fails to accurately predict the disease. The relationship between the lipid exchange capacity and lipoprotein structure should explain the extent by which they release or accept lipid cargo and should relate to the risk for developing atherosclerosis. Here, small-angle neutron scattering and tailored deuteration have been used to follow the molecular lipid exchange between human lipoprotein particles and cellular membrane mimics made of natural, "neutron invisible" phosphatidylcholines. We show that lipid exchange occurs via two different processes that include lipid transfer via collision and upon direct particle tethering to the membrane, and that high-density lipoprotein excels at exchanging the human-like unsaturated phosphatidylcholine. By mapping the specific lipid content and level of glycation/oxidation, the mode of action of specific lipoproteins can now be deciphered. This information can prove important for the development of improved diagnostic tools and in the treatment of atherosclerosis.

4.
Proc Natl Acad Sci U S A ; 116(18): 8685-8692, 2019 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-30975751

RESUMO

Biomineralization, the process by which mineralized tissues grow and harden via biogenic mineral deposition, is a relatively lengthy process in many mineral-producing organisms, resulting in challenges to study the growth and biomineralization of complex hard mineralized tissues. Arthropods are ideal model organisms to study biomineralization because they regularly molt their exoskeletons and grow new ones in a relatively fast timescale, providing opportunities to track mineralization of entire tissues. Here, we monitored the biomineralization of the mantis shrimp dactyl club-a model bioapatite-based mineralized structure with exceptional mechanical properties-immediately after ecdysis until the formation of the fully functional club and unveil an unusual development mechanism. A flexible membrane initially folded within the club cavity expands to form the new club's envelope. Mineralization proceeds inwards by mineral deposition from this membrane, which contains proteins regulating mineralization. Building a transcriptome of the club tissue and probing it with proteomic data, we identified and sequenced Club Mineralization Protein 1 (CMP-1), an abundant mildly phosphorylated protein from the flexible membrane suggested to be involved in calcium phosphate mineralization of the club, as indicated by in vitro studies using recombinant CMP-1. This work provides a comprehensive picture of the development of a complex hard tissue, from the secretion of its organic macromolecular template to the formation of the fully functional club.

5.
ACS Omega ; 3(9): 11050-11061, 2018 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-30320257

RESUMO

Caloric restriction (CR) is an intervention that can increase maximal lifespan in organisms, but its application to humans remains challenging. A more feasible approach to achieve lifespan extension is to develop CR mimetics that target biochemical pathways affected by CR. Recent studies in the engineering and structural characterization of polyketide synthases (PKSs) have facilitated their use as biocatalysts to produce novel polyketides. Here, we show that by establishing a combinatorial biosynthetic route in Escherichia coli and exploring the substrate promiscuity of a mutant PKS from alfalfa, 413 potential anti-ageing polyketides were biosynthesized. In this approach, novel acyl-coenzyme A (CoA) precursors generated by promiscuous acid-CoA ligases were utilized by PKS to generate polyketides which were then fed to Caenorhabditis elegans to study their potential efficacy in lifespan extension. It was found that CR mimetics like resveratrol can counter the age-associated decline in mitochondrial function and increase the lifespan of C. elegans. Using the mitochondrial respiration profile of C. elegans supplemented for 8 days with 50 µM resveratrol as a blueprint, we can screen our novel polyketides for potential CR mimetics with improved potency. This study highlights the utility of synthetic enzymology in the development of novel anti-ageing therapeutics.

6.
Sci Rep ; 8(1): 11296, 2018 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-30050044

RESUMO

Meibomian gland (MG) dysfunction is the leading cause of evaporative dry eye and it leads to inflammation of the ocular surface. Eicosanoids may be involved in inflammation of dry eye. This study aimed to profile tear eicosanoid levels in healthy individuals and those with MG dysfunction, and to examine if these levels are associated with clinical factors and expressibility of MG. Forty participants with MG dysfunction and 30 healthy controls were recruited in this study. Clinical signs of MG dysfunction were assessed, and tear lactoferrin concentration was evaluated. Tear eicosanoids were extracted from Schirmer's strips and analyzed using mass spectrometry. We were able to quantify 38 tear eicosanoids and levels were increased in older individuals. In participants with MG dysfunction, higher 5-HETE, LTB4, 18-HEPE, 12-HEPE and 14-HDoHE were associated with poorer MG expressibility. The eicosanoids PGF2α, 18-HEPE, 20-HDoHE and 17-HDoHE were elevated with increased corneal staining; higher 5-HETE, LTB4 were associated with lower tear lactoferrin levels. The receiver-operating-characteristics analysis shows higher levels of 5-HETE, LTB4 and 18-HEPE were able to predict poor expressibility of MGs. In conclusion, tear eicosanoid levels are age-dependent and specific eicosanoids may be indicators of clinical obstruction of MG or the severity of ocular surface damage.


Assuntos
Síndromes do Olho Seco/patologia , Eicosanoides/análise , Voluntários Saudáveis , Glândulas Tarsais/fisiopatologia , Lágrimas/química , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Adulto Jovem
7.
J Invest Dermatol ; 138(5): 1137-1145, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29246799

RESUMO

Skin provides the first defense against pathogenic micro-organisms and is also colonized by a diverse microbiota. Phylogenetic analysis of whole skin microbiome at different skin sites in health and disease has generated important insights on possible microbial involvement in modulating skin health. However, functional roles of the skin microbial community remain unclear. The most common sebaceous skin commensal yeasts are the basidiomycetes, Malassezia. Here, we characterized the dominant secreted Malassezia globosa protease in culture and subsequently named it Malassezia globosa Secreted Aspartyl Protease 1 (MgSAP1). We defined recombinant MgSAP1's substrate cleavage profile using an unbiased, mass-spectrometry-based technique. We show that this enzyme is physiologically relevant as mgsap1 expression was detected on at least one facial skin site of 17 healthy human volunteers. In addition, we demonstrated that this protease rapidly hydrolyzes Staphylococcus aureus protein A, an important S. aureus virulence factor involved in immune evasion and biofilm formation. We further observed that MgSAP1 has anti-biofilm properties against S. aureus. Taken together, our study defines a role for the skin fungus Malassezia in inter-kingdom interactions and suggests that this fungus and the enzymes it produces may be beneficial for skin health.


Assuntos
Biofilmes , Malassezia/enzimologia , Peptídeo Hidrolases/fisiologia , Pele/microbiologia , Staphylococcus aureus/fisiologia , Ácido Aspártico Proteases/fisiologia , Humanos
8.
J Chromatogr B Analyt Technol Biomed Life Sci ; 1061-1062: 438-444, 2017 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-28820982

RESUMO

Pre-analytical treatment of blood plasma is a time consuming and often rate limiting step in the workflow of LC/MS analysis. We present in this pilot study a new approach for quantitative LC/MS based on weak affinity chromatography (WAC) of crude plasma. The steroid hormone cortisol was selected as a clinically relevant biomarker, as it currently requires extensive pre-analytical preparation. A WAC unit with saturating, immobilized albumin as a prototypic weak binder was used in combination with an ion-funnel MS/MS detector to perform zonal affinity chromatography of cortisol directly from a plasma sample, followed by quantitative multiple reaction monitoring (MRM). This procedure also allowed us to determine the amount of bioavailable cortisol in the clinical plasma sample which is of significant therapeutic interest. This WAC-MS approach showed an excellent correlation (R2=0.86 (P<0.0001 (highly significant); n=60) with a state-of-the-art, clinical competitive immunoassay procedure for plasma cortisol analysis. With integration of WAC into LC/MS workflow, it may be possible to both accelerate and improve assay performance by eliminating the sample extraction step. Preliminary data with other steroid hormones indicate that WAC-MS can be applied to various biomolecules using a plasma transport protein such as albumin.


Assuntos
Cromatografia de Afinidade/métodos , Cromatografia Líquida/métodos , Hidrocortisona/sangue , Espectrometria de Massas em Tandem/métodos , Disponibilidade Biológica , Humanos , Hidrocortisona/metabolismo , Modelos Lineares , Projetos Piloto , Sensibilidade e Especificidade
9.
Oncotarget ; 6(15): 13539-49, 2015 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-25915536

RESUMO

Transitional bladder carcinoma (BCa) is prevalent in developed countries, particularly among men. Given that these tumors frequently recur or progress, the early detection and subsequent monitoring of BCa at different stages is critical. Current BCa diagnostic biomarkers are not sufficiently sensitive for substituting or complementing invasive cystoscopy. Here, we sought to identify a robust set of urine biomarkers for BCa detection. Using a high-resolution, mass spectrometry-based, quantitative proteomics approach, we measured, compared and validated protein variations in 451 voided urine samples from healthy subjects, non-bladder cancer patients and patients with non-invasive and invasive BCa. We identified five robust biomarkers: Coronin-1A, Apolipoprotein A4, Semenogelin-2, Gamma synuclein and DJ-1/PARK7. In diagnosing Ta/T1 BCa, these biomarkers achieved an AUC of 0.92 and 0.98, respectively, using ELISA and western blot data (sensitivity, 79.2% and 93.9%; specificity, 100% and 96.7%, respectively). In diagnosing T2/T3 BCa, an AUC of 0.94 and 1.0 was attained (sensitivity, 86.4% and 100%; specificity, 100%) using the same methods. Thus, our multiplex biomarker panel offers unprecedented accuracy for the diagnosis of BCa patients and provides the prospect for a non-invasive way to detect bladder cancer.


Assuntos
Biomarcadores Tumorais/urina , Carcinoma de Células de Transição/diagnóstico , Neoplasias da Bexiga Urinária/diagnóstico , Carcinoma de Células de Transição/patologia , Carcinoma de Células de Transição/urina , Estudos de Casos e Controles , Estudos de Coortes , Humanos , Recidiva Local de Neoplasia/diagnóstico , Recidiva Local de Neoplasia/patologia , Recidiva Local de Neoplasia/urina , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Neoplasias da Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/urina
10.
Sci Rep ; 5: 8235, 2015 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-25648885

RESUMO

The Type VI Secretion System cluster 1 (T6SS1) is essential for the pathogenesis of Burkholderia pseudomallei, the causative agent of melioidosis, a disease endemic in the tropics. Inside host cells, B. pseudomallei escapes into the cytosol and through T6SS1, induces multinucleated giant cell (MNGC) formation that is thought to be important for bacterial cell to cell spread. The hemolysin-coregulated protein (Hcp) is both a T6SS substrate, as well as postulated to form part of the T6SS secretion tube. Our structural study reveals that Hcp1 forms hexameric rings similar to the other Hcp homologs but has an extended loop (Asp40-Arg56) that deviates significantly in position compared to other Hcp structures and may act as a key contact point between adjacent hexameric rings. When two residues within the loop were mutated, the mutant proteins were unable to stack as dodecamers, suggesting defective tube assembly. Moreover, infection with a bacterial mutant containing in situ substitution of these hcp1 residues abolishes Hcp1 secretion inside infected cells and MNGC formation. We further show that Hcp has the ability to preferentially bind to the surface of antigen-presenting cells, which may contribute to its immunogenicity in inducing high titers of antibodies seen in melioidosis patients.


Assuntos
Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Burkholderia pseudomallei/fisiologia , Domínios e Motivos de Interação entre Proteínas , Sistemas de Secreção Tipo VI , Sequência de Aminoácidos , Animais , Anticorpos Antibacterianos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/imunologia , Linhagem Celular , Humanos , Macrófagos/imunologia , Macrófagos/metabolismo , Melioidose/imunologia , Melioidose/microbiologia , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Proteínas Recombinantes/metabolismo
11.
Angew Chem Int Ed Engl ; 53(49): 13390-4, 2014 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-25348595

RESUMO

Bioorthogonal cleavable linkers are attractive building blocks for compounds that can be manipulated to study biological and cellular processes. Sodium dithionite sensitive azobenzene-containing (Abc) peptides were applied for the temporary stabilization of recombinant MHC complexes, which can then be employed to generate libraries of MHC tetramers after exchange with a novel epitope. This technology represents an important tool for high-throughput studies of disease-specific T cell responses.


Assuntos
Compostos Azo/química , Antígenos HLA-A/química , Peptídeos/química , Sequência de Aminoácidos , Compostos Azo/imunologia , Ditionita/química , Epitopos/química , Epitopos/imunologia , Antígenos HLA-A/imunologia , Humanos , Ligantes , Modelos Moleculares , Peptídeos/imunologia , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia
12.
Nat Biotechnol ; 31(10): 908-15, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24013196

RESUMO

Efforts to engineer new materials inspired by biological structures are hampered by the lack of genomic data from many model organisms studied in biomimetic research. Here we show that biomimetic engineering can be accelerated by integrating high-throughput RNA-seq with proteomics and advanced materials characterization. This approach can be applied to a broad range of systems, as we illustrate by investigating diverse high-performance biological materials involved in embryo protection, adhesion and predation. In one example, we rapidly engineer recombinant squid sucker ring teeth proteins into a range of structural and functional materials, including nanopatterned surfaces and photo-cross-linked films that exceed the mechanical properties of most natural and synthetic polymers. Integrating RNA-seq with proteomics and materials science facilitates the molecular characterization of natural materials and the effective translation of their molecular designs into a wide range of bio-inspired materials.


Assuntos
Materiais Biomiméticos/química , Biomimética/métodos , Proteômica/métodos , Análise de Sequência de RNA , Adesividade , Sequência de Aminoácidos , Estruturas Animais/ultraestrutura , Animais , Organismos Aquáticos/metabolismo , Dados de Sequência Molecular , Óvulo/metabolismo , Engenharia de Proteínas , Proteínas Recombinantes/química , Seda/química , Espectroscopia de Infravermelho com Transformada de Fourier
13.
J Proteome Res ; 12(6): 2933-45, 2013 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-23659346

RESUMO

Troglitazone, a first-generation thiazolidinedione of antihyperglycaemic properties, was withdrawn from the market due to unacceptable idiosyncratic hepatotoxicity. Despite intensive research, the underlying mechanism of troglitazone-induced liver toxicity remains unknown. Here we report the use of the Sod2(+/-) mouse model of silent mitochondrial oxidative-stress-based and quantitative mass spectrometry-based proteomics to track the mitochondrial proteome changes induced by physiologically relevant troglitazone doses. By quantitative untargeted proteomics, we first globally profiled the Sod2(+/-) hepatic mitochondria proteome and found perturbations including GSH metabolism that enhanced the toxicity of the normally nontoxic troglitazone. Short- and long-term troglitazone administration in Sod2(+/-) mouse led to a mitochondrial proteome shift from an early compensatory response to an eventual phase of intolerable oxidative stress, due to decreased mitochondrial glutathione (mGSH) import protein, decreased dicarboxylate ion carrier (DIC), and the specific activation of ASK1-JNK and FOXO3a with prolonged troglitazone exposure. Furthermore, mapping of the detected proteins onto mouse specific protein-centered networks revealed lipid-associated proteins as contributors to overt mitochondrial and liver injury when under prolonged exposure to the lipid-normalizing troglitazone. By integrative toxicoproteomics, we demonstrated a powerful systems approach in identifying the collapse of specific fragile nodes and activation of crucial proteome reconfiguration regulators when targeted by an exogenous toxicant.


Assuntos
Cromanos/toxicidade , Glutationa/antagonistas & inibidores , Hipoglicemiantes/toxicidade , Mitocôndrias/efeitos dos fármacos , Proteínas Mitocondriais/genética , Proteômica , Tiazolidinedionas/toxicidade , Animais , Transportadores de Ácidos Dicarboxílicos/antagonistas & inibidores , Transportadores de Ácidos Dicarboxílicos/genética , Transportadores de Ácidos Dicarboxílicos/metabolismo , Feminino , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/agonistas , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Glutationa/metabolismo , Humanos , Transporte de Íons/efeitos dos fármacos , MAP Quinase Quinase 4/genética , MAP Quinase Quinase 4/metabolismo , MAP Quinase Quinase Quinase 5/genética , MAP Quinase Quinase Quinase 5/metabolismo , Masculino , Camundongos , Camundongos Knockout , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Transdução de Sinais , Superóxido Dismutase/deficiência , Superóxido Dismutase/genética , Troglitazona
14.
J Cell Biol ; 196(6): 801-10, 2012 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-22412018

RESUMO

We have developed a new technique for proximity-dependent labeling of proteins in eukaryotic cells. Named BioID for proximity-dependent biotin identification, this approach is based on fusion of a promiscuous Escherichia coli biotin protein ligase to a targeting protein. BioID features proximity-dependent biotinylation of proteins that are near-neighbors of the fusion protein. Biotinylated proteins may be isolated by affinity capture and identified by mass spectrometry. We apply BioID to lamin-A (LaA), a well-characterized intermediate filament protein that is a constituent of the nuclear lamina, an important structural element of the nuclear envelope (NE). We identify multiple proteins that associate with and/or are proximate to LaA in vivo. The most abundant of these include known interactors of LaA that are localized to the NE, as well as a new NE-associated protein named SLAP75. Our results suggest BioID is a useful and generally applicable method to screen for both interacting and neighboring proteins in their native cellular environment.


Assuntos
Biotina/metabolismo , Carbono-Nitrogênio Ligases/metabolismo , Proteínas de Escherichia coli/metabolismo , Laminina/metabolismo , Mapeamento de Interação de Proteínas/métodos , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/metabolismo , Animais , Sítios de Ligação , Biotinilação , Carbono-Nitrogênio Ligases/genética , Proteínas de Escherichia coli/genética , Células HEK293 , Humanos , Laminina/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Repressoras/genética
15.
Neuron ; 73(2): 304-16, 2012 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-22284185

RESUMO

Adenosine-to-inosine RNA editing is crucial for generating molecular diversity, and serves to regulate protein function through recoding of genomic information. Here, we discover editing within Ca(v)1.3 Ca²âº channels, renown for low-voltage Ca²âº-influx and neuronal pacemaking. Significantly, editing occurs within the channel's IQ domain, a calmodulin-binding site mediating inhibitory Ca²âº-feedback (CDI) on channels. The editing turns out to require RNA adenosine deaminase ADAR2, whose variable activity could underlie a spatially diverse pattern of Ca(v)1.3 editing seen across the brain. Edited Ca(v)1.3 protein is detected both in brain tissue and within the surface membrane of primary neurons. Functionally, edited Ca(v)1.3 channels exhibit strong reduction of CDI; in particular, neurons within the suprachiasmatic nucleus show diminished CDI, with higher frequencies of repetitive action-potential and calcium-spike activity, in wild-type versus ADAR2 knockout mice. Our study reveals a mechanism for fine-tuning Ca(v)1.3 channel properties in CNS, which likely impacts a broad spectrum of neurobiological functions.


Assuntos
Encéfalo/metabolismo , Canais de Cálcio Tipo L/genética , Cálcio/metabolismo , Edição de RNA , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Animais , Canais de Cálcio Tipo L/metabolismo , Sinalização do Cálcio/fisiologia , Camundongos , Camundongos Knockout , Neurônios/metabolismo , Proteínas de Ligação a RNA , Ratos , Ratos Sprague-Dawley , Núcleo Supraquiasmático/metabolismo
16.
Curr Opin Chem Biol ; 15(4): 570-5, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21763176

RESUMO

Drug target deconvolution is a process where the action of a drug, a small molecule, is characterised by identifying the proteins binding the drug and initiating the biological effect. The biological relevant target has to be extracted, or deconvoluted, from a list of proteins identified in such an approach. Beside the medically desired action of the drug, the identification of other proteins binding the drug can help to identify side effects and toxicity at a very early stage of drug development. The current approach to identify the proteins binding to the drug is an affinity-enrichment based approach, where the drug molecule is immobilised to a matrix through a linker and the proteins binding to the drug are identified by proteomics.


Assuntos
Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/prevenção & controle , Preparações Farmacêuticas/química , Proteínas/química , Proteômica/métodos , Sítios de Ligação , Cromatografia de Afinidade , Desenho de Fármacos , Ligação Proteica , Proteínas/metabolismo
17.
Biochim Biophys Acta ; 1808(9): 2224-32, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21640070

RESUMO

The serotonin (5-HT(1A)) receptor, a G-protein-coupled receptor (GPCR), plays important roles in serotonergic signaling in the central nervous system. The third intracellular loop (ICL3) of the 5-HT(1A) receptor has been shown to be important for the regulation of this receptor through interactions with proteins such as G-proteins and calmodulin. In this study, the ICL3 of 5-HT(1A) receptor was expressed in E. coli and purified. Gel filtration and mass spectrometry were used to confirm the molecular weight of the purified ICL3. Secondary structure analysis using circular dichroism (CD) demonstrated the presence of α-helical structures. Backbone assignment of ICL3 was achieved using three-dimensional experiments. A chemical shift index and Talos+ analysis showed that residues E326 to R339 form α-helical structure. Residues G256 to S269 of ICL3 were shown to be a novel region that has a molecular interaction with calmodulin in titration assays. Peptide derived from the ICL3 containing residues from G256 to S269 also showed molecular interaction with calmodulin.


Assuntos
Calmodulina/química , Escherichia coli/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Receptor 5-HT1A de Serotonina/química , Sequência de Aminoácidos , Cromatografia em Gel/métodos , Cromatografia por Troca Iônica/métodos , Dicroísmo Circular , Clonagem Molecular , Biologia Computacional/métodos , Humanos , Dados de Sequência Molecular , Peso Molecular , Peptídeos/química , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Serotonina/química
18.
Biochem Biophys Res Commun ; 403(1): 126-32, 2010 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-21055387

RESUMO

The human Ether-à-go-go Related Gene (hERG) potassium channel mediates the rapid delayed rectifier current (IKr) in the cardiac action potential. Mutations in the 135 amino acid residue N-terminal domain (NTD) cause channel dysfunction or mis-translocation. To study the structure of NTD, it was overexpressed and purified from Escherichia coli cells using affinity purification and gel filtration chromatography. The purified protein behaved as a monomer under purification conditions. Far- and near-UV, circular dichroism (CD) and solution nuclear magnetic resonance (NMR) studies showed that the purified protein was well-folded. The solution structure of NTD was obtained and the N-terminal residues 13-23 forming an amphipathic helix which may be important for the protein-protein or protein-membrane interactions. NMR titration experiment also demonstrated that residues from 88 to 94 in NTD are important for the molecular interaction with the peptide derived from the S4-S5 linker.


Assuntos
Canais de Potássio Éter-A-Go-Go/química , Humanos , Ressonância Magnética Nuclear Biomolecular , Peptídeos/química , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína
19.
Biomol NMR Assign ; 4(2): 211-3, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20607461

RESUMO

The human ether à go-go related gene (hERG) voltage-gated potassium controls the rapid delayed rectifier potassium current (I(ks)) in heart. The N-terminal 135 amino acids (NTD) form a Per-Arnt-Sim (PAS) domain which involves in signal transduction and protein-protein interactions. NTD was shown to be necessary for the regulation of the channel activity through its interaction with the channel pore region of hERG. Mutations in NTD were related to serious heart diseases. We report the (1)H, (13)C and (15)N chemical shift assignments for NTD using 2D and 3D heteronuclear NMR experiments. More than 95% backbone resonance assignments were obtained.


Assuntos
Canais de Potássio Éter-A-Go-Go/química , Ressonância Magnética Nuclear Biomolecular , Sequência de Aminoácidos , Isótopos de Carbono , Canal de Potássio ERG1 , Humanos , Hidrogênio , Ativação do Canal Iônico , Dados de Sequência Molecular , Isótopos de Nitrogênio , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína
20.
Nat Biotechnol ; 25(9): 1035-44, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17721511

RESUMO

We describe a chemical proteomics approach to profile the interaction of small molecules with hundreds of endogenously expressed protein kinases and purine-binding proteins. This subproteome is captured by immobilized nonselective kinase inhibitors (kinobeads), and the bound proteins are quantified in parallel by mass spectrometry using isobaric tags for relative and absolute quantification (iTRAQ). By measuring the competition with the affinity matrix, we assess the binding of drugs to their targets in cell lysates and in cells. By mapping drug-induced changes in the phosphorylation state of the captured proteome, we also analyze signaling pathways downstream of target kinases. Quantitative profiling of the drugs imatinib (Gleevec), dasatinib (Sprycel) and bosutinib in K562 cells confirms known targets including ABL and SRC family kinases and identifies the receptor tyrosine kinase DDR1 and the oxidoreductase NQO2 as novel targets of imatinib. The data suggest that our approach is a valuable tool for drug discovery.


Assuntos
Inibidores de Proteínas Quinases/farmacologia , Proteômica/métodos , Proteínas Proto-Oncogênicas c-abl/antagonistas & inibidores , Benzamidas , Extratos Celulares , Cromatografia de Afinidade , Receptor com Domínio Discoidina 1 , Enzimas Imobilizadas/antagonistas & inibidores , Células HeLa , Humanos , Mesilato de Imatinib , Concentração Inibidora 50 , Células K562 , Preparações Farmacêuticas , Fosforilação/efeitos dos fármacos , Piperazinas/farmacologia , Pirimidinas/farmacologia , Quinona Redutases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos
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