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1.
Angew Chem Int Ed Engl ; 63(7): e202313900, 2024 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-38158383

RESUMO

N1 -methyladenosine (m1 A) is a prevalent post-transcriptional RNA modification, and the distribution and dynamics of the modification play key epitranscriptomic roles in cell development. At present, the human AlkB Fe(II)/α-ketoglutarate-dependent dioxygenase family member ALKBH3 is the only known mRNA m1 A demethylase, but its catalytic mechanism remains unclear. Here, we present the structures of ALKBH3-oligo crosslinked complexes obtained with the assistance of a synthetic antibody crystallization chaperone. Structural and biochemical results showed that ALKBH3 utilized two ß-hairpins (ß4-loop-ß5 and ß'-loop-ß'') and the α2 helix to facilitate single-stranded substrate binding. Moreover, a bubble-like region around Asp194 and a key residue inside the active pocket (Thr133) enabled specific recognition and demethylation of m1 A- and 3-methylcytidine (m3 C)-modified substrates. Mutation of Thr133 to the corresponding residue in the AlkB Fe(II)/α-ketoglutarate-dependent dioxygenase family members FTO or ALKBH5 converted ALKBH3 substrate selectivity from m1 A to N6 -methyladenosine (m6 A), as did Asp194 deletion. Our findings provide a molecular basis for understanding the mechanisms of substrate recognition and m1 A demethylation by ALKBH3. This study is expected to aid structure-guided design of chemical probes for further functional studies and therapeutic applications.


Assuntos
Dioxigenase FTO Dependente de alfa-Cetoglutarato , RNA , Humanos , RNA/química , Dioxigenase FTO Dependente de alfa-Cetoglutarato/metabolismo , RNA Mensageiro/metabolismo , Desmetilação , Compostos Ferrosos , Homólogo AlkB 3 da Dioxigenase Dependente de alfa-Cetoglutarato/metabolismo
2.
MAbs ; 13(1): 1933690, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34190031

RESUMO

In order to direct T cells to specific features of solid cancer cells, we engineered a bispecific antibody format, named Dual Antigen T cell Engager (DATE), by fusing a single-chain variable fragment targeting CD3 to a tumor-targeting antigen-binding fragment. In this format, multiple novel paratopes against different tumor antigens were able to recruit T-cell cytotoxicity to tumor cells in vitro and in an in vivo pancreatic ductal adenocarcinoma xenograft model. Since unique surface antigens in solid tumors are limited, in order to enhance selectivity, we further engineered "double-DATEs" targeting two tumor antigens simultaneously. The double-DATE contains an additional autonomous variable heavy-chain domain, which binds a second tumor antigen without itself eliciting a cytotoxic response. This novel modality provides a strategy to enhance the selectivity of immune redirection through binary targeting of native tumor antigens. The modularity and use of a common, stable human framework for all components enables a pipeline approach to rapidly develop a broad repertoire of tailored DATEs and double-DATEs with favorable biophysical properties and high potencies and selectivities.


Assuntos
Anticorpos Biespecíficos/farmacologia , Antígenos de Neoplasias/imunologia , Antineoplásicos/farmacologia , Imunoterapia/métodos , Linfócitos T/imunologia , Animais , Anticorpos Monoclonais Humanizados/farmacologia , Complexo CD3/imunologia , Carcinoma Ductal Pancreático/imunologia , Humanos , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Camundongos , Neoplasias Pancreáticas/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto
3.
Acta Crystallogr D Struct Biol ; 76(Pt 9): 876-888, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32876063

RESUMO

The genome-packaging motor of tailed bacteriophages and herpesviruses is a multisubunit protein complex formed by several copies of a large (TerL) and a small (TerS) terminase subunit. The motor assembles transiently at the portal protein vertex of an empty precursor capsid to power the energy-dependent packaging of viral DNA. Both the ATPase and nuclease activities associated with genome packaging reside in TerL. Structural studies of TerL from bacteriophage P22 have been hindered by the conformational flexibility of this enzyme and its susceptibility to proteolysis. Here, an unbiased, synthetic phage-display Fab library was screened and a panel of high-affinity Fabs against P22 TerL were identified. This led to the discovery of a recombinant antibody fragment, Fab4, that binds a 33-amino-acid α-helical hairpin at the N-terminus of TerL with an equilibrium dissociation constant Kd of 71.5 nM. A 1.51 Šresolution crystal structure of Fab4 bound to the TerL epitope (TLE) together with a 1.15 Šresolution crystal structure of the unliganded Fab4, which is the highest resolution ever achieved for a Fab, elucidate the principles governing the recognition of this novel helical epitope. TLE adopts two different conformations in the asymmetric unit and buries as much as 1250 Å2 of solvent-accessible surface in Fab4. TLE recognition is primarily mediated by conformational changes in the third complementarity-determining region of the Fab4 heavy chain (CDR H3) that take place upon epitope binding. It is demonstrated that TLE can be introduced genetically at the N-terminus of a target protein, where it retains high-affinity binding to Fab4.


Assuntos
Bacteriófago P22/enzimologia , Endodesoxirribonucleases , Fragmentos Fab das Imunoglobulinas , Proteínas Virais , Endodesoxirribonucleases/química , Sequências Hélice-Volta-Hélice , Fragmentos Fab das Imunoglobulinas/química , Modelos Moleculares , Biblioteca de Peptídeos , Ligação Proteica , Proteínas Virais/química
4.
Proc Natl Acad Sci U S A ; 115(40): 10010-10015, 2018 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-30224453

RESUMO

The Gcn5 histone acetyltransferase (HAT) subunit of the SAGA transcriptional coactivator complex catalyzes acetylation of histone H3 and H2B N-terminal tails, posttranslational modifications associated with gene activation. Binding of the SAGA subunit partner Ada2 to Gcn5 activates Gcn5's intrinsically weak HAT activity on histone proteins, but the mechanism for this activation by the Ada2 SANT domain has remained elusive. We have employed Fab antibody fragments as crystallization chaperones to determine crystal structures of a yeast Ada2/Gcn5 complex. Our structural and biochemical results indicate that the Ada2 SANT domain does not activate Gcn5's activity by directly affecting histone peptide binding as previously proposed. Instead, the Ada2 SANT domain enhances Gcn5 binding of the enzymatic cosubstrate acetyl-CoA. This finding suggests a mechanism for regulating chromatin modification enzyme activity: controlling binding of the modification cosubstrate instead of the histone substrate.


Assuntos
Acetilcoenzima A/química , Histona Acetiltransferases/química , Histonas/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Fatores de Transcrição/química , Acetilcoenzima A/metabolismo , Cristalografia por Raios X , Ativação Enzimática , Histona Acetiltransferases/metabolismo , Histonas/metabolismo , Ligação Proteica , Domínios Proteicos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo
5.
Methods Mol Biol ; 1575: 93-119, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28255876

RESUMO

Phage display is commonly used to identify and isolate binders from large combinatorial libraries. Here we present phage selection protocols enabling generation of synthetic antibodies capable of recognizing multiprotein complexes and conformational states. The procedure describes stages of the experiment design, optimization, and screening, as well as provides the framework for building downstream assays with an end goal of isolating bioactive antibodies for future therapeutic use. The methods described are also applicable to screening directly on cells and can be ported to other in vitro directed evolution systems utilizing non-immunoglobulin scaffolds.


Assuntos
Anticorpos/metabolismo , Complexos Multiproteicos/química , Anticorpos/genética , Especificidade de Anticorpos , Humanos , Conformação Molecular , Complexos Multiproteicos/imunologia , Biblioteca de Peptídeos
6.
PLoS One ; 10(10): e0139695, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26437229

RESUMO

We developed and optimized a high-throughput project workflow to generate renewable recombinant antibodies to human proteins involved in epigenetic signalling. Three different strategies to produce phage display compatible protein antigens in bacterial systems were compared, and we found that in vivo biotinylation through the use of an Avi tag was the most productive method. Phage display selections were performed on 265 in vivo biotinylated antigen domains. High-affinity Fabs (<20nM) were obtained for 196. We constructed and optimized a new expression vector to produce in vivo biotinylated Fabs in E. coli. This increased average yields up to 10-fold, with an average yield of 4 mg/L. For 118 antigens, we identified Fabs that could immunoprecipitate their full-length endogenous targets from mammalian cell lysates. One Fab for each antigen was converted to a recombinant IgG and produced in mammalian cells, with an average yield of 15 mg/L. In summary, we have optimized each step of the pipeline to produce recombinant antibodies, significantly increasing both efficiency and yield, and also showed that these Fabs and IgGs can be generally useful for chromatin immunoprecipitation (ChIP) protocols.


Assuntos
Formação de Anticorpos/fisiologia , Antígenos/imunologia , Fragmentos Fab das Imunoglobulinas/imunologia , Proteínas Recombinantes/imunologia , Clonagem Molecular , Humanos , Biblioteca de Peptídeos
7.
Science ; 350(6256): 56-64, 2015 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-26316600

RESUMO

The nuclear pore complex (NPC) constitutes the sole gateway for bidirectional nucleocytoplasmic transport. We present the reconstitution and interdisciplinary analyses of the ~425-kilodalton inner ring complex (IRC), which forms the central transport channel and diffusion barrier of the NPC, revealing its interaction network and equimolar stoichiometry. The Nsp1•Nup49•Nup57 channel nucleoporin heterotrimer (CNT) attaches to the IRC solely through the adaptor nucleoporin Nic96. The CNT•Nic96 structure reveals that Nic96 functions as an assembly sensor that recognizes the three-dimensional architecture of the CNT, thereby mediating the incorporation of a defined CNT state into the NPC. We propose that the IRC adopts a relatively rigid scaffold that recruits the CNT to primarily form the diffusion barrier of the NPC, rather than enabling channel dilation.


Assuntos
Chaetomium/ultraestrutura , Proteínas Fúngicas/ultraestrutura , Complexo de Proteínas Formadoras de Poros Nucleares/ultraestrutura , Poro Nuclear/ultraestrutura , Proteínas Nucleares/ultraestrutura , Sequência de Aminoácidos , Chaetomium/metabolismo , Proteínas Fúngicas/química , Dados de Sequência Molecular , Poro Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/química , Proteínas Nucleares/química , Ligação Proteica , Multimerização Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína
8.
Mol Cell Proteomics ; 14(10): 2833-47, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26290498

RESUMO

Antibodies are key reagents in biology and medicine, but commercial sources are rarely recombinant and thus do not provide a permanent and renewable resource. Here, we describe an industrialized platform to generate antigens and validated recombinant antibodies for 346 transcription factors (TFs) and 211 epigenetic antigens. We describe an optimized automated phage display and antigen expression pipeline that in aggregate produced about 3000 sequenced Fragment antigen-binding domain that had high affinity (typically EC50<20 nm), high stability (Tm∼80 °C), good expression in E. coli (∼5 mg/L), and ability to bind antigen in complex cell lysates. We evaluated a subset of Fabs generated to homologous SCAN domains for binding specificities. These Fragment antigen-binding domains were monospecific to their target SCAN antigen except in rare cases where they cross-reacted with a few highly related antigens. Remarkably, immunofluorescence experiments in six cell lines for 270 of the TF antigens, each having multiple antibodies, show that ∼70% stain predominantly in the cytosol and ∼20% stain in the nucleus which reinforces the dominant role that translocation plays in TF biology. These cloned antibody reagents are being made available to the academic community through our web site recombinant-antibodies.org to allow a more system-wide analysis of TF and chromatin biology. We believe these platforms, infrastructure, and automated approaches will facilitate the next generation of renewable antibody reagents to the human proteome in the coming decade.


Assuntos
Anticorpos , Fragmentos Fab das Imunoglobulinas , Fatores de Transcrição , Anticorpos/genética , Anticorpos/imunologia , Antígenos/genética , Antígenos/imunologia , Escherichia coli/genética , Ensaios de Triagem em Larga Escala , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/imunologia , Dobramento de Proteína , RNA Interferente Pequeno/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Fatores de Transcrição/genética , Fatores de Transcrição/imunologia
9.
Nat Methods ; 12(8): 725-31, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26121405

RESUMO

Antibodies are used in multiple cell biology applications, but there are no standardized methods to assess antibody quality-an absence that risks data integrity and reproducibility. We describe a mass spectrometry-based standard operating procedure for scoring immunoprecipitation antibody quality. We quantified the abundance of all the proteins in immunoprecipitates of 1,124 new recombinant antibodies for 152 chromatin-related human proteins by comparing normalized spectral abundance factors from the target antigen with those of all other proteins. We validated the performance of the standard operating procedure in blinded studies in five independent laboratories. Antibodies for which the target antigen or a member of its known protein complex was the most abundant protein were classified as 'IP gold standard'. This method generates quantitative outputs that can be stored and archived in public databases, and it represents a step toward a platform for community benchmarking of antibody quality.


Assuntos
Anticorpos Monoclonais/química , Especificidade de Anticorpos , Cromatina/química , Imunoprecipitação/métodos , Proteômica/métodos , Clonagem Molecular , Biologia Computacional/métodos , Escherichia coli/metabolismo , Células HEK293 , Humanos , Fragmentos de Imunoglobulinas/química , Imunoglobulina G/química , Espectrometria de Massas/métodos , Biblioteca de Peptídeos , Proteínas/química , Proteoma , Reprodutibilidade dos Testes
10.
Science ; 347(6226): 1148-52, 2015 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-25745173

RESUMO

The nuclear pore complex (NPC) constitutes the sole gateway for bidirectional nucleocytoplasmic transport. Despite half a century of structural characterization, the architecture of the NPC remains unknown. Here we present the crystal structure of a reconstituted ~400-kilodalton coat nucleoporin complex (CNC) from Saccharomyces cerevisiae at a 7.4 angstrom resolution. The crystal structure revealed a curved Y-shaped architecture and the molecular details of the coat nucleoporin interactions forming the central "triskelion" of the Y. A structural comparison of the yeast CNC with an electron microscopy reconstruction of its human counterpart suggested the evolutionary conservation of the elucidated architecture. Moreover, 32 copies of the CNC crystal structure docked readily into a cryoelectron tomographic reconstruction of the fully assembled human NPC, thereby accounting for ~16 megadalton of its mass.


Assuntos
Complexo de Proteínas Formadoras de Poros Nucleares/química , Poro Nuclear/ultraestrutura , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/ultraestrutura , Cristalografia por Raios X , Humanos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína
11.
Science ; 347(6226): 1152-5, 2015 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-25745174

RESUMO

Tail-anchored (TA) proteins are a physiologically important class of membrane proteins targeted to the endoplasmic reticulum by the conserved guided-entry of TA proteins (GET) pathway. During transit, their hydrophobic transmembrane domains (TMDs) are chaperoned by the cytosolic targeting factor Get3, but the molecular nature of the functional Get3-TA protein targeting complex remains unknown. We reconstituted the physiologic assembly pathway for a functional targeting complex and showed that it comprises a TA protein bound to a Get3 homodimer. Crystal structures of Get3 bound to different TA proteins showed an α-helical TMD occupying a hydrophobic groove that spans the Get3 homodimer. Our data elucidate the mechanism of TA protein recognition and shielding by Get3 and suggest general principles of hydrophobic domain chaperoning by cellular targeting factors.


Assuntos
Adenosina Trifosfatases/química , Fatores de Troca do Nucleotídeo Guanina/química , Proteínas de Membrana/química , Proteínas de Saccharomyces cerevisiae/química , Adenosina Trifosfatases/metabolismo , Cristalografia por Raios X , Citosol/enzimologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Proteínas de Membrana/metabolismo , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Multimerização Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Transporte Proteico , Proteínas de Saccharomyces cerevisiae/metabolismo
12.
J Vis Exp ; (95): 51492, 2015 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-25651360

RESUMO

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. However, it is apparent that traditional monoclonal technologies are not alone up to this task. This has led to the development of alternate methods to satisfy the demand for high quality and renewable affinity reagents to all accessible elements of the proteome. Toward this end, high throughput methods for conducting selections from phage-displayed synthetic antibody libraries have been devised for applications involving diverse antigens and optimized for rapid throughput and success. Herein, a protocol is described in detail that illustrates with video demonstration the parallel selection of Fab-phage clones from high diversity libraries against hundreds of targets using either a manual 96 channel liquid handler or automated robotics system. Using this protocol, a single user can generate hundreds of antigens, select antibodies to them in parallel and validate antibody binding within 6-8 weeks. Highlighted are: i) a viable antigen format, ii) pre-selection antigen characterization, iii) critical steps that influence the selection of specific and high affinity clones, and iv) ways of monitoring selection effectiveness and early stage antibody clone characterization. With this approach, we have obtained synthetic antibody fragments (Fabs) to many target classes including single-pass membrane receptors, secreted protein hormones, and multi-domain intracellular proteins. These fragments are readily converted to full-length antibodies and have been validated to exhibit high affinity and specificity. Further, they have been demonstrated to be functional in a variety of standard immunoassays including Western blotting, ELISA, cellular immunofluorescence, immunoprecipitation and related assays. This methodology will accelerate antibody discovery and ultimately bring us closer to realizing the goal of generating renewable, high quality antibodies to the proteome.


Assuntos
Anticorpos/imunologia , Antígenos/imunologia , Ensaios de Triagem em Larga Escala/métodos , Biblioteca de Peptídeos , Reações Antígeno-Anticorpo , Ensaio de Imunoadsorção Enzimática/métodos , Epitopos , Humanos , Imunoensaio/métodos
13.
J Biol Chem ; 289(50): 34851-61, 2014 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-25352592

RESUMO

The γ-secretase complex, composed of presenilin, nicastrin (NCT), anterior pharynx-defective 1 (APH-1), and presenilin enhancer 2 (PEN-2), is assembled in a highly regulated manner and catalyzes the intramembranous proteolysis of many type I membrane proteins, including Notch and amyloid precursor protein. The Notch family of receptors plays important roles in cell fate specification during development and in adult tissues, and aberrant hyperactive Notch signaling causes some forms of cancer. γ-Secretase-mediated processing of Notch at the cell surface results in the generation of the Notch intracellular domain, which associates with several transcriptional coactivators involved in nuclear signaling events. On the other hand, γ-secretase-mediated processing of amyloid precursor protein leads to the production of amyloid ß (Aß) peptides that play an important role in the pathogenesis of Alzheimer disease. We used a phage display approach to identify synthetic antibodies that specifically target NCT and expressed them in the single-chain variable fragment (scFv) format in mammalian cells. We show that expression of a NCT-specific scFv clone, G9, in HEK293 cells decreased the production of the Notch intracellular domain but not the production of amyloid ß peptides that occurs in endosomal and recycling compartments. Biochemical studies revealed that scFvG9 impairs the maturation of NCT by associating with immature forms of NCT and, consequently, prevents its association with the other components of the γ-secretase complex, leading to degradation of these molecules. The reduced cell surface levels of mature γ-secretase complexes, in turn, compromise the intramembranous processing of Notch.


Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Glicoproteínas de Membrana/imunologia , Glicoproteínas de Membrana/metabolismo , Anticorpos de Cadeia Única/imunologia , Secretases da Proteína Precursora do Amiloide/química , Secretases da Proteína Precursora do Amiloide/imunologia , Especificidade de Anticorpos , Células HEK293 , Humanos , Espaço Intracelular/metabolismo , Glicoproteínas de Membrana/química , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Proteólise , Receptores Notch/metabolismo , Anticorpos de Cadeia Única/genética
14.
J Virol ; 88(20): 11713-25, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25122782

RESUMO

Paramyxoviruses are enveloped negative-strand RNA viruses that are significant human and animal pathogens. Most paramyxoviruses infect host cells via the concerted action of a tetrameric attachment protein (variously called HN, H, or G) that binds either sialic acid or protein receptors on target cells and a trimeric fusion protein (F) that merges the viral envelope with the plasma membrane at neutral pH. F initially folds to a metastable prefusion conformation that becomes activated via a cleavage event during cellular trafficking. Upon receptor binding, the attachment protein, which consists of a globular head anchored to the membrane via a helical tetrameric stalk, triggers a major conformation change in F which results in fusion of virus and host cell membranes. We recently proposed a model for F activation in which the attachment protein head domains move following receptor binding to expose HN stalk residues critical for triggering F. To test the model in the context of wild-type viral glycoproteins, we used a restricted-diversity combinatorial Fab library and phage display to rapidly generate synthetic antibodies (sAbs) against multiple domains of the paramyxovirus parainfluenza 5 (PIV5) pre- and postfusion F and HN. As predicted by the model, sAbs that bind to the critical F-triggering region of the HN stalk do not disrupt receptor binding or neuraminidase (NA) activity but are potent inhibitors of fusion. An inhibitory prefusion F-specific sAb recognized a quaternary antigenic site and may inhibit fusion by preventing F refolding or by blocking the F-HN interaction. Importance: The paramyxovirus family of negative-strand RNA viruses cause significant disease in humans and animals. The viruses bind to cells via their receptor binding protein and then enter cells by fusion of their envelope with the host cell plasma membrane, a process mediated by a metastable viral fusion (F) protein. To understand the steps in viral membrane fusion, a library of synthetic antibodies to F protein and the receptor binding protein was generated in bacteriophage. These antibodies bound to different regions of the F protein and the receptor binding protein, and the location of antibody binding affected different processes in viral entry into cells.


Assuntos
Anticorpos/imunologia , Glicoproteínas/fisiologia , Paramyxoviridae/fisiologia , Proteínas Virais/fisiologia , Animais , Especificidade de Anticorpos , Antígenos Virais/imunologia , Linhagem Celular , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Glicoproteínas/imunologia , Humanos , Microscopia Eletrônica , Proteínas Virais/imunologia
15.
Cell Rep ; 8(1): 297-310, 2014 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-24981860

RESUMO

Chromatin regulation is driven by multicomponent protein complexes, which form functional modules. Deciphering the components of these modules and their interactions is central to understanding the molecular pathways these proteins are regulating, their functions, and their relation to both normal development and disease. We describe the use of affinity purifications of tagged human proteins coupled with mass spectrometry to generate a protein-protein interaction map encompassing known and predicted chromatin-related proteins. On the basis of 1,394 successful purifications of 293 proteins, we report a high-confidence (85% precision) network involving 11,464 protein-protein interactions among 1,738 different human proteins, grouped into 164 often overlapping protein complexes with a particular focus on the family of JmjC-containing lysine demethylases, their partners, and their roles in chromatin remodeling. We show that RCCD1 is a partner of histone H3K36 demethylase KDM8 and demonstrate that both are important for cell-cycle-regulated transcriptional repression in centromeric regions and accurate mitotic division.


Assuntos
Proteínas de Transporte/metabolismo , Cromatina/metabolismo , Segregação de Cromossomos , Histona Desmetilases/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Transporte/genética , Células HEK293 , Humanos , Proteínas de Membrana/genética , Ligação Proteica
16.
Nat Struct Mol Biol ; 21(3): 244-52, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24487958

RESUMO

The transduction of transmembrane electric fields into protein motion has an essential role in the generation and propagation of cellular signals. Voltage-sensing domains (VSDs) carry out these functions through reorientations of positive charges in the S4 helix. Here, we determined crystal structures of the Ciona intestinalis VSD (Ci-VSD) in putatively active and resting conformations. S4 undergoes an ~5-Å displacement along its main axis, accompanied by an ~60° rotation. This movement is stabilized by an exchange in countercharge partners in helices S1 and S3 that generates an estimated net charge transfer of ~1 eo. Gating charges move relative to a ''hydrophobic gasket' that electrically divides intra- and extracellular compartments. EPR spectroscopy confirms the limited nature of S4 movement in a membrane environment. These results provide an explicit mechanism for voltage sensing and set the basis for electromechanical coupling in voltage-dependent enzymes and ion channels.


Assuntos
Ciona intestinalis/química , Estrutura Terciária de Proteína , Sequência de Aminoácidos , Animais , Membrana Celular/metabolismo , Cristalografia por Raios X , Espectroscopia de Ressonância de Spin Eletrônica , Eletrofisiologia , Escherichia coli/metabolismo , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Oócitos/metabolismo , Homologia de Sequência de Aminoácidos , Eletricidade Estática , Xenopus laevis/metabolismo
17.
Nature ; 497(7447): 137-41, 2013 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-23604254

RESUMO

The functions of G-protein-coupled receptors (GPCRs) are primarily mediated and modulated by three families of proteins: the heterotrimeric G proteins, the G-protein-coupled receptor kinases (GRKs) and the arrestins. G proteins mediate activation of second-messenger-generating enzymes and other effectors, GRKs phosphorylate activated receptors, and arrestins subsequently bind phosphorylated receptors and cause receptor desensitization. Arrestins activated by interaction with phosphorylated receptors can also mediate G-protein-independent signalling by serving as adaptors to link receptors to numerous signalling pathways. Despite their central role in regulation and signalling of GPCRs, a structural understanding of ß-arrestin activation and interaction with GPCRs is still lacking. Here we report the crystal structure of ß-arrestin-1 (also called arrestin-2) in complex with a fully phosphorylated 29-amino-acid carboxy-terminal peptide derived from the human V2 vasopressin receptor (V2Rpp). This peptide has previously been shown to functionally and conformationally activate ß-arrestin-1 (ref. 5). To capture this active conformation, we used a conformationally selective synthetic antibody fragment (Fab30) that recognizes the phosphopeptide-activated state of ß-arrestin-1. The structure of the ß-arrestin-1-V2Rpp-Fab30 complex shows marked conformational differences in ß-arrestin-1 compared to its inactive conformation. These include rotation of the amino- and carboxy-terminal domains relative to each other, and a major reorientation of the 'lariat loop' implicated in maintaining the inactive state of ß-arrestin-1. These results reveal, at high resolution, a receptor-interacting interface on ß-arrestin, and they indicate a potentially general molecular mechanism for activation of these multifunctional signalling and regulatory proteins.


Assuntos
Arrestinas/química , Arrestinas/metabolismo , Fosfopeptídeos/química , Fosfopeptídeos/metabolismo , Receptores de Vasopressinas/química , Animais , Arrestinas/imunologia , Cristalografia por Raios X , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos Fab das Imunoglobulinas/metabolismo , Modelos Moleculares , Fosforilação , Ligação Proteica , Conformação Proteica , Estabilidade Proteica , Ratos , Rotação , beta-Arrestina 1 , beta-Arrestinas
18.
Methods ; 60(1): 3-14, 2013 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-23280336

RESUMO

A set of phage display sorting strategies and validation methodologies are presented that are capable of producing high performance synthetic antibodies (sABs) with customized properties. Exquisite control of antigen and conditions during the phage display selection process can yield sABs that: (1) recognize conformational states, (2) target specific regions of the surface of a protein, (3) induce conformational changes, and (4) capture and stabilize multi-protein complexes. These unique capabilities open myriad opportunities to study complex macromolecular processes inaccessible to traditional affinity reagent technology. We present detailed protocols for de novo isolation of binders, as well as examples of downstream biophysical characterization. The methods described are generalizable and can be adapted to other in vitro direct evolution approaches based on yeast or mRNA display.


Assuntos
Anticorpos/química , Especificidade de Anticorpos , Proteínas/metabolismo , Anticorpos/metabolismo , Ensaio de Imunoadsorção Enzimática , Conformação Molecular , Biblioteca de Peptídeos , Proteínas/química
19.
Proc Natl Acad Sci U S A ; 109(22): 8534-9, 2012 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-22586122

RESUMO

The γ-secretase complex, composed of presenilin, anterior-pharynx-defective 1, nicastrin, and presenilin enhancer 2, catalyzes the intramembranous processing of a wide variety of type I membrane proteins, including amyloid precursor protein (APP) and Notch. Earlier studies have revealed that nicastrin, a type I membrane-anchored glycoprotein, plays a role in γ-secretase assembly and trafficking and has been proposed to bind substrates. To gain more insights regarding nicastrin structure and function, we generated a conformation-specific synthetic antibody and used it as a molecular probe to map functional domains within nicastrin ectodomain. The antibody bound to a conformational epitope within a nicastrin segment encompassing residues 245-630 and inhibited the processing of APP and Notch substrates in in vitro γ-secretase activity assays, suggesting that a functional domain pertinent to γ-secretase activity resides within this region. Epitope mapping and database searches revealed the presence of a structured segment, located downstream of the previously identified DAP domain (DYIGS and peptidase; residues 261-502), that is homologous to a tetratricopeptide repeat (TPR) domain commonly involved in peptide recognition. Mutagenesis analyses within the predicted TPR-like domain showed that disruption of the signature helical structure resulted in the loss of γ-secretase activity but not the assembly of the γ-secretase and that Leu571 within the TPR-like domain plays an important role in mediating substrate binding. Taken together, these studies offer provocative insights pertaining to the structural basis for nicastrin function as a "substrate receptor" within the γ-secretase complex.


Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Anticorpos/metabolismo , Glicoproteínas de Membrana/metabolismo , Oligopeptídeos/metabolismo , Sequência de Aminoácidos , Secretases da Proteína Precursora do Amiloide/química , Secretases da Proteína Precursora do Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Sítios de Ligação/genética , Biocatálise , Western Blotting , Células Cultivadas , Dicroísmo Circular , Epitopos/química , Epitopos/genética , Epitopos/metabolismo , Células HEK293 , Humanos , Imuno-Histoquímica/métodos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Knockout , Mutação , Oligopeptídeos/química , Oligopeptídeos/genética , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Sequências Repetitivas de Aminoácidos/genética , Ressonância de Plasmônio de Superfície , Espectrometria de Massas em Tandem
20.
Bioconjug Chem ; 23(1): 42-6, 2012 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-22175275

RESUMO

The use of proteins or nucleic acids as therapeutic agents has been severely hampered by their intrinsic inability to cross the cell membrane. Moreover, common techniques for driving the delivery of macromolecules lack the ability to distinguish between healthy and diseased tissue, precluding their clinical use. Recently, receptor-mediated delivery (RMD) has emerged as a technology with the potential to circumvent the obstacles associated with the delivery of drug targets by utilizing the natural endocytosis of a ligand upon binding to its receptor. Here, we describe the synthesis of variants of substance P (SP), an eleven amino acid neuropeptide ligand of the neurokinin type 1 receptor (NK1R), for the delivery of various types of cargo. The variants of SP were synthesized with an N-terminal maleimide moiety that allows conjugation to surface thiols, resulting in a nonreducible thioether. Cargos lacking an available thiol are conjugated to SP using commercially available cross-linkers. In addition to the delivery of proteins, we expand the use of SP to include nuclear delivery of DNA fragments that are actively expressed in the target cells. We also show that SP can be used to deliver whole bacteriophage particles as well as polystyrene beads up to 1 µm in diameter. The results show the ability of SP to deliver cargo of various sizes and chemical properties that retain their function within the cell. Furthermore, the overexpression of the NK1R in many tumors provides the potential for developing targeted delivery reagents that are specific toward diseased tissue.


Assuntos
DNA/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Substância P/metabolismo , Bacteriófagos/genética , Bacteriófagos/metabolismo , Linhagem Celular Tumoral , Reagentes de Ligações Cruzadas/química , DNA/genética , Proteínas de Fluorescência Verde/genética , Humanos , Microesferas , Poliestirenos , Substância P/síntese química , Substância P/química , Compostos de Sulfidrila/química , Compostos de Sulfidrila/metabolismo
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