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1.
Mol Cell ; 79(3): 472-487.e10, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32531202

RESUMO

It is widely assumed that decreasing transcription factor DNA-binding affinity reduces transcription initiation by diminishing occupancy of sequence-specific regulatory elements. However, in vivo transcription factors find their binding sites while confronted with a large excess of low-affinity degenerate motifs. Here, using the melanoma lineage survival oncogene MITF as a model, we show that low-affinity binding sites act as a competitive reservoir in vivo from which transcription factors are released by mitogen-activated protein kinase (MAPK)-stimulated acetylation to promote increased occupancy of their regulatory elements. Consequently, a low-DNA-binding-affinity acetylation-mimetic MITF mutation supports melanocyte development and drives tumorigenesis, whereas a high-affinity non-acetylatable mutant does not. The results reveal a paradoxical acetylation-mediated molecular clutch that tunes transcription factor availability via genome-wide redistribution and couples BRAF to tumorigenesis. Our results further suggest that p300/CREB-binding protein-mediated transcription factor acetylation may represent a common mechanism to control transcription factor availability.


Assuntos
Regulação Neoplásica da Expressão Gênica , Genoma , Melanoma/genética , Fator de Transcrição Associado à Microftalmia/genética , Processamento de Proteína Pós-Traducional , Neoplasias Cutâneas/genética , Acetilação , Sequência de Aminoácidos , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Sequência Conservada , Elementos Facilitadores Genéticos , Feminino , Xenoenxertos , Humanos , Masculino , Melanócitos/metabolismo , Melanócitos/patologia , Melanoma/metabolismo , Melanoma/patologia , Camundongos , Camundongos Nus , Fator de Transcrição Associado à Microftalmia/química , Fator de Transcrição Associado à Microftalmia/metabolismo , Motivos de Nucleotídeos , Regiões Promotoras Genéticas , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Peixe-Zebra
2.
Mol Cell ; 73(6): 1282-1291.e8, 2019 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-30792174

RESUMO

Toxin-antitoxin (TA) systems regulate fundamental cellular processes in bacteria and represent potential therapeutic targets. We report a new RES-Xre TA system in multiple human pathogens, including Mycobacterium tuberculosis. The toxin, MbcT, is bactericidal unless neutralized by its antitoxin MbcA. To investigate the mechanism, we solved the 1.8 Å-resolution crystal structure of the MbcTA complex. We found that MbcT resembles secreted NAD+-dependent bacterial exotoxins, such as diphtheria toxin. Indeed, MbcT catalyzes NAD+ degradation in vitro and in vivo. Unexpectedly, the reaction is stimulated by inorganic phosphate, and our data reveal that MbcT is a NAD+ phosphorylase. In the absence of MbcA, MbcT triggers rapid M. tuberculosis cell death, which reduces mycobacterial survival in macrophages and prolongs the survival of infected mice. Our study expands the molecular activities employed by bacterial TA modules and uncovers a new class of enzymes that could be exploited to treat tuberculosis and other infectious diseases.


Assuntos
Antitoxinas/metabolismo , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Macrófagos/microbiologia , Mycobacterium tuberculosis/enzimologia , Fosforilases/metabolismo , Sistemas Toxina-Antitoxina , Tuberculose/microbiologia , Animais , Antibióticos Antituberculose/farmacologia , Antitoxinas/química , Antitoxinas/genética , Carga Bacteriana , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Células Cultivadas , Modelos Animais de Doenças , Feminino , Interações Hospedeiro-Patógeno , Humanos , Cinética , Macrófagos/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos SCID , Camundongos Transgênicos , Viabilidade Microbiana , Modelos Moleculares , Mycobacterium smegmatis/enzimologia , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/patogenicidade , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/patogenicidade , NAD/metabolismo , Fosforilases/química , Fosforilases/genética , Conformação Proteica , Sistemas Toxina-Antitoxina/genética , Tuberculose/tratamento farmacológico
3.
Nucleic Acids Res ; 48(2): 934-948, 2020 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-31777941

RESUMO

Interrupted dimeric coiled coil segments are found in a broad range of proteins and generally confer selective functional properties such as binding to specific ligands. However, there is only one documented case of a basic-helix-loop-helix leucine zipper transcription factor-microphthalmia-associated transcription factor (MITF)-in which an insertion of a three-residue stammer serves as a determinant of conditional partner selectivity. To unravel the molecular principles of this selectivity, we have analyzed the high-resolution structures of stammer-containing MITF and an engineered stammer-less MITF variant, which comprises an uninterrupted symmetric coiled coil. Despite this fundamental difference, both MITF structures reveal identical flanking in-phase coiled coil arrangements, gained by helical over-winding and local asymmetry in wild-type MITF across the stammer region. These conserved structural properties allow the maintenance of a proper functional readout in terms of nuclear localization and binding to specific DNA-response motifs regardless of the presence of the stammer. By contrast, MITF heterodimer formation with other bHLH-Zip transcription factors is only permissive when both factors contain either the same type of inserted stammer or no insert. Our data illustrate a unique principle of conditional partner selectivity within the wide arsenal of transcription factors with specific partner-dependent functional readouts.


Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/química , Núcleo Celular/química , Fator de Transcrição Associado à Microftalmia/química , Conformação Proteica , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Ligantes , Camundongos , Fator de Transcrição Associado à Microftalmia/genética , Ligação Proteica , Domínios Proteicos/genética , Multimerização Proteica
4.
Genes Dev ; 26(23): 2647-58, 2012 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-23207919

RESUMO

Microphthalmia-associated transcription factor (MITF) is a master regulator of melanocyte development and an important oncogene in melanoma. MITF heterodimeric assembly with related basic helix-loop-helix leucine zipper transcription factors is highly restricted, and its binding profile to cognate DNA sequences is distinct. Here, we determined the crystal structure of MITF in its apo conformation and in the presence of two related DNA response elements, the E-box and M-box. In addition, we investigated mouse and human Mitf mutations to dissect the functional significance of structural features. Owing to an unusual three-residue shift in the leucine zipper register, the MITF homodimer shows a marked kink in one of the two zipper helices to allow an out-of-register assembly. Removal of this insertion relieves restricted heterodimerization by MITF and permits assembly with the transcription factor MAX. Binding of MITF to the M-box motif is mediated by an unusual nonpolar interaction by Ile212, a residue that is mutated in mice and humans with Waardenburg syndrome. As several related transcription factors have low affinity for the M-box sequence, our analysis unravels how these proteins discriminate between similar target sequences. Our data provide a rational basis for targeting MITF in the treatment of important hereditary diseases and cancer.


Assuntos
Zíper de Leucina , Fator de Transcrição Associado à Microftalmia/química , Fator de Transcrição Associado à Microftalmia/metabolismo , Modelos Moleculares , Sequência de Aminoácidos , Animais , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Dimerização , Elementos Facilitadores Genéticos/genética , Humanos , Zíper de Leucina/genética , Camundongos , Fator de Transcrição Associado à Microftalmia/genética , Dados de Sequência Molecular , Mutação , Ligação Proteica , Estrutura Terciária de Proteína , Alinhamento de Sequência , Síndrome de Waardenburg/genética
5.
J Cell Sci ; 130(9): 1675-1687, 2017 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-28325759

RESUMO

Tail-anchored (TA) proteins contain a single transmembrane domain (TMD) at the C-terminus that anchors them to the membranes of organelles where they mediate critical cellular processes. Accordingly, mutations in genes encoding TA proteins have been identified in a number of severe inherited disorders. Despite the importance of correctly targeting a TA protein to its appropriate membrane, the mechanisms and signals involved are not fully understood. In this study, we identify additional peroxisomal TA proteins, discover more proteins that are present on multiple organelles, and reveal that a combination of TMD hydrophobicity and tail charge determines targeting to distinct organelle locations in mammals. Specifically, an increase in tail charge can override a hydrophobic TMD signal and re-direct a protein from the ER to peroxisomes or mitochondria and vice versa. We show that subtle changes in those parameters can shift TA proteins between organelles, explaining why peroxisomes and mitochondria have many of the same TA proteins. This enabled us to associate characteristic physicochemical parameters in TA proteins with particular organelle groups. Using this classification allowed successful prediction of the location of uncharacterized TA proteins for the first time.


Assuntos
Compartimento Celular , Mamíferos/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Animais , Retículo Endoplasmático/metabolismo , Células Hep G2 , Humanos , Interações Hidrofóbicas e Hidrofílicas , Membranas Intracelulares/metabolismo , Mitocôndrias/metabolismo , Modelos Biológicos , Peroxissomos/metabolismo , Transporte Proteico , Saccharomyces cerevisiae/metabolismo , Frações Subcelulares/metabolismo
6.
Hum Mol Genet ; 22(21): 4357-67, 2013 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-23787126

RESUMO

The basic-helix-loop-helix-leucine zipper (bHLHZip) protein MITF (microphthalmia-associated transcription factor) is a master regulator of melanocyte development. Mutations in the MITF have been found in patients with the dominantly inherited hypopigmentation and deafness syndromes Waardenburg syndrome type 2A (WS2A) and Tietz syndrome (TS). Additionally, both somatic and germline mutations have been found in MITF in melanoma patients. Here, we characterize the DNA-binding and transcription activation properties of 24 MITF mutations found in WS2A, TS and melanoma patients. We show that most of the WS2A and TS mutations fail to bind DNA and activate expression from melanocyte-specific promoters. Some of the mutations, especially R203K and S298P, exhibit normal activity and may represent neutral variants. Mutations found in melanomas showed normal DNA-binding and minor variations in transcription activation properties; some showed increased potential to form colonies. Our results provide molecular insights into how mutations in a single gene can lead to such different phenotypes.


Assuntos
Albinismo Oculocutâneo/genética , Surdez/genética , Melanoma/genética , Fator de Transcrição Associado à Microftalmia/genética , Fator de Transcrição Associado à Microftalmia/metabolismo , Síndrome de Waardenburg/genética , Adolescente , Adulto , Albinismo Oculocutâneo/metabolismo , Albinismo Oculocutâneo/patologia , Sítios de Ligação , Criança , Pré-Escolar , Surdez/metabolismo , Surdez/patologia , Feminino , Variação Genética , Células HEK293 , Humanos , Masculino , Melanoma/metabolismo , Melanoma/patologia , Mutação de Sentido Incorreto , Regiões Promotoras Genéticas , Ativação Transcricional , Transfecção , Síndrome de Waardenburg/metabolismo , Síndrome de Waardenburg/patologia , Adulto Jovem
7.
J Struct Biol ; 188(3): 249-58, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25450593

RESUMO

Although PIN (PilT N-terminal)-domain proteins are known to have ribonuclease activity, their specific mechanism of action remains unknown. VapCs form a family of ribonucleases that possess a PIN-domain assembly and are known as toxins. The activities of VapCs are impaired by VapB antitoxins. Here we present the crystal structure of the VapBC-15 toxin-antitoxin complex from Mycobacterium tuberculosis determined to 2.1Å resolution. The VapB-15 and VapC-15 components assemble into one heterotetramer (VapB2C2) and two heterotrimers (VapBC2) in each asymmetric unit of the crystal. The active site of VapC-15 toxin consists of a cluster of acidic amino acid residues and two divalent metal ions, forming a well organised ribonuclease active site. The distribution of the catalytic-site residues of the VapC-15 toxin is similar to that of T4 RNase H and of Methanococcus jannaschii FEN-1, providing strong evidence that these three proteins share a similar mechanism of activity. The presence of both VapB2C2 and VapBC2 emphasizes the fact that the same antitoxin can bind the toxin in 1:1 and 1:2 ratios. The crystal structure determination of the VapBC-15 complex reveals for the first time a PIN-domain ribonuclease protein that shows two metal ions at the active site and a variable mode of toxin-antitoxin assembly. The structure further shows that VapB-15 antitoxin binds to the same groove meant for the binding of putative substrate (RNA), resulting in the inhibition of VapC-15's toxicity.


Assuntos
Antitoxinas/metabolismo , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Glicoproteínas de Membrana/metabolismo , Mycobacterium tuberculosis/metabolismo , Ribonucleases/metabolismo , Modelos Moleculares , Estrutura Terciária de Proteína , Difração de Raios X
8.
Cell Signal ; 121: 111270, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-38909932

RESUMO

BACKGROUND: In breast cancer, over one third of all patients harbor a somatic mutation in the PIK3CA gene, encoding the p110α catalytic subunit of the phosphatidylinositol 3-kinase (PI3K) in their tumor cells. Circulating tumor cells (CTCs) are cells shed from the primary tumor into the blood stream. Recently, the long-term stable breast cancer CTC-ITB-01 cell line with tumorigenic and metastatic capacity was established from liquid biopsy derived cells. The oncogenic hotspot PIK3CA mutation H1047R (kinase domain) was detected in the primary tumor, CTCs and metastasis of the same patient. Other PIK3CA mutations located within the C2 domain (E418K and E453K) were detected in the CTCs and the vaginal metastasis but not in the primary tumor. The goal of our study was to functionally characterize the impact of the rare E418K and E453K mutations within the C2 domain that were not detected in the primary tumor. METHODS: PIK3CA mutations E418K, E453K, H1047R were generated by site-directed mutagenesis and stably overexpressed in breast cancer cells by lentiviral transduction. Subsequent signaling pathway activation was examined by western blot analysis. The impact of PIK3CA mutations on biological processes was studied by live cell imaging using the Incucyte Zoom system. Structural modeling was conducted in Pymol. The membrane localization of the mutants was evaluated by separating the cytosolic and membrane fraction using ultracentrifugation. Drug susceptibility of CTC-ITB-01 cells was analyzed by live cell imaging. RESULTS: Western blot analysis of human MDA-MB-231, MCF-7 and T47D breast cancer cells stably overexpressing either the PIK3CA wildtype (WT) or one of the E418K, E453K or H1047R mutants revealed a significant increase in AKT phosphorylation in both C2 mutants (E418K and E453K) and the kinase domain mutant H1047R. Functional analysis showed a significantly increased proliferation of MDA-MB-231 cells overexpressing the E453K and H1047R mutants. Migration was increased in all cells overexpressing WT and each of the mutants. Interestingly, invasion and chemotaxis were only enhanced in the MDA-MB-231 cells overexpressing the C2 domain mutants, i.e. E418K and E453K. In addition, membrane localization of the two C2 domain mutants was increased. Structural modeling of the E453K mutation suggests a disruption of the interaction between the negative regulatory domain of the p85α subunit and the p110α catalytic subunit as a potential mechanism leading to the observed activation of PI3K/AKT/mTOR signaling. Dual targeting of AKT/mTOR pathway by MK2206 and RAD001 leads to very strong synergistic effects (IC50 MK2206: 148 nM, IC50 RAD001: 15 nM) with respect to proliferation in the CTC-ITB-01 line through apoptosis induction. CONCLUSIONS: Our results demonstrate that PIK3CA C2 domain mutations activate PI3K downstream AKT signaling and can increase proliferation, migration and invasion after stable lentiviral transduction. Although both investigated mutations - E418K and E453K - are located within the C2 domain, a different molecular mechanism can be proposed. The PIK3CA mutated CTC-ITB-01 shows a high susceptibility against dual inhibition of AKT/mTOR. Further studies are required to fully elucidate the oncogenic potential of rare PIK3CA mutations.


Assuntos
Neoplasias da Mama , Classe I de Fosfatidilinositol 3-Quinases , Mutação , Células Neoplásicas Circulantes , Humanos , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Feminino , Células Neoplásicas Circulantes/metabolismo , Células Neoplásicas Circulantes/patologia , Classe I de Fosfatidilinositol 3-Quinases/genética , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Linhagem Celular Tumoral , Metástase Neoplásica , Domínios Proteicos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proliferação de Células , Movimento Celular/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositol 3-Quinases/genética
9.
Nat Commun ; 14(1): 2245, 2023 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-37076474

RESUMO

Bacterial pathogens often make use of post-translational modifications to manipulate host cells. Legionella pneumophila, the causative agent of Legionnaires disease, secretes the enzyme AnkX that uses cytidine diphosphate-choline to post-translationally modify the human small G-Protein Rab1 with a phosphocholine moiety at Ser76. Later in the infection, the Legionella enzyme Lem3 acts as a dephosphocholinase, hydrolytically removing the phosphocholine. While the molecular mechanism for Rab1 phosphocholination by AnkX has recently been resolved, structural insights into the activity of Lem3 remained elusive. Here, we stabilise the transient Lem3:Rab1b complex by substrate mediated covalent capture. Through crystal structures of Lem3 in the apo form and in complex with Rab1b, we reveal Lem3's catalytic mechanism, showing that it acts on Rab1 by locally unfolding it. Since Lem3 shares high structural similarity with metal-dependent protein phosphatases, our Lem3:Rab1b complex structure also sheds light on how these phosphatases recognise protein substrates.


Assuntos
Legionella pneumophila , Legionella , Doença dos Legionários , Humanos , Legionella/metabolismo , Fosforilcolina/metabolismo , Legionella pneumophila/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas de Bactérias/metabolismo , Proteínas rab1 de Ligação ao GTP/metabolismo
10.
Nat Commun ; 14(1): 6051, 2023 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-37770430

RESUMO

The ability of transcription factors to discriminate between different classes of binding sites associated with specific biological functions underpins effective gene regulation in development and homeostasis. How this is achieved is poorly understood. The microphthalmia-associated transcription factor MITF is a lineage-survival oncogene that plays a crucial role in melanocyte development and melanoma. MITF suppresses invasion, reprograms metabolism and promotes both proliferation and differentiation. How MITF distinguishes between differentiation and proliferation-associated targets is unknown. Here we show that compared to many transcription factors MITF exhibits a very long residence time which is reduced by p300/CBP-mediated MITF acetylation at K206. While K206 acetylation also decreases genome-wide MITF DNA-binding affinity, it preferentially directs DNA binding away from differentiation-associated CATGTG motifs toward CACGTG elements. The results reveal an acetylation-mediated switch that suppresses differentiation and provides a mechanistic explanation of why a human K206Q MITF mutation is associated with Waardenburg syndrome.


Assuntos
Melanoma , Fator de Transcrição Associado à Microftalmia , Humanos , Linhagem Celular Tumoral , Fator de Transcrição Associado à Microftalmia/genética , Fator de Transcrição Associado à Microftalmia/metabolismo , Acetilação , Melanoma/genética , Melanoma/metabolismo , Melanócitos/metabolismo
11.
Commun Biol ; 6(1): 1124, 2023 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-37932372

RESUMO

The intracellular bacterial pathogen Coxiella burnetii evades the host response by secreting effector proteins that aid in establishing a replication-friendly niche. Bacterial filamentation induced by cyclic AMP (Fic) enzymes can act as effectors by covalently modifying target proteins with the posttranslational AMPylation by transferring adenosine monophosphate (AMP) from adenosine triphosphate (ATP) to a hydroxyl-containing side chain. Here we identify the gene product of C. burnetii CBU_0822, termed C. burnetii Fic 2 (CbFic2), to AMPylate host cell histone H3 at serine 10 and serine 28. We show that CbFic2 acts as a bifunctional enzyme, both capable of AMPylation as well as deAMPylation, and is regulated by the binding of DNA via a C-terminal helix-turn-helix domain. We propose that CbFic2 performs AMPylation in its monomeric state, switching to a deAMPylating dimer upon DNA binding. This study unveils reversible histone modification by a specific enzyme of a pathogenic bacterium.


Assuntos
Coxiella burnetii , AMP Cíclico , Histonas , DNA , Serina
12.
J Struct Biol ; 175(2): 135-46, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21571073

RESUMO

Control of transcription allows the regulation of cell activity in response to external stimuli and research in the field has greatly benefited from efforts in structural biology. In this review, based on specific examples from the European SPINE2-COMPLEXES initiative, we illustrate the impact of structural proteomics on our understanding of the molecular basis of gene expression. While most atomic structures were obtained by X-ray crystallography, the impact of solution NMR and cryo-electron microscopy is far from being negligible. Here, we summarize some highlights and illustrate the importance of specific technologies on the structural biology of protein-protein or protein/DNA transcription complexes: structure/function analysis of components the eukaryotic basal and activated transcription machinery with focus on the TFIID and TFIIH multi-subunit complexes as well as transcription regulators such as members of the nuclear hormone receptor families. We also discuss molecular aspects of promoter recognition and epigenetic control of gene expression.


Assuntos
Complexos Multiproteicos/química , Conformação Proteica , Transcrição Gênica , Regulação Alostérica , Animais , Microscopia Crioeletrônica , Cristalografia por Raios X , Epigênese Genética , Humanos , Espectroscopia de Ressonância Magnética , Complexos Multiproteicos/isolamento & purificação , Complexos Multiproteicos/metabolismo , Regiões Promotoras Genéticas , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo
13.
Nat Commun ; 12(1): 2426, 2021 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-33893288

RESUMO

To adapt to fluctuating protein folding loads in the endoplasmic reticulum (ER), the Hsp70 chaperone BiP is reversibly modified with adenosine monophosphate (AMP) by the ER-resident Fic-enzyme FICD/HYPE. The structural basis for BiP binding and AMPylation by FICD has remained elusive due to the transient nature of the enzyme-substrate-complex. Here, we use thiol-reactive derivatives of the cosubstrate adenosine triphosphate (ATP) to covalently stabilize the transient FICD:BiP complex and determine its crystal structure. The complex reveals that the TPR-motifs of FICD bind specifically to the conserved hydrophobic linker of BiP and thus mediate specificity for the domain-docked conformation of BiP. Furthermore, we show that both AMPylation and deAMPylation of BiP are not directly regulated by the presence of unfolded proteins. Together, combining chemical biology, crystallography and biochemistry, our study provides structural insights into a key regulatory mechanism that safeguards ER homeostasis.


Assuntos
Proteínas de Choque Térmico/metabolismo , Proteínas de Membrana/metabolismo , Nucleotidiltransferases/metabolismo , Processamento de Proteína Pós-Traducional , Repetições de Tetratricopeptídeos , Monofosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Retículo Endoplasmático/metabolismo , Chaperona BiP do Retículo Endoplasmático , Células HEK293 , Proteínas de Choque Térmico/química , Homeostase , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Simulação de Dinâmica Molecular , Nucleotidiltransferases/química , Nucleotidiltransferases/genética , Ligação Proteica , Conformação Proteica , Especificidade por Substrato
14.
Nat Chem ; 12(8): 732-739, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32632184

RESUMO

Various pathogenic bacteria use post-translational modifications to manipulate the central components of host cell functions. Many of the enzymes released by these bacteria belong to the large Fic family, which modify targets with nucleotide monophosphates. The lack of a generic method for identifying the cellular targets of Fic family enzymes hinders investigation of their role and the effect of the post-translational modification. Here, we establish an approach that uses reactive co-substrate-linked enzymes for proteome profiling. We combine synthetic thiol-reactive nucleotide derivatives with recombinantly produced Fic enzymes containing strategically placed cysteines in their active sites to yield reactive binary probes for covalent substrate capture. The binary complexes capture their targets from cell lysates and permit subsequent identification. Furthermore, we determined the structures of low-affinity ternary enzyme-nucleotide-substrate complexes by applying a covalent-linking strategy. This approach thus allows target identification of the Fic enzymes from both bacteria and eukarya.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Membrana/metabolismo , Nucleotidiltransferases/metabolismo , Monofosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Bartonella/metabolismo , Biocatálise , Cristalografia por Raios X , Células HeLa , Humanos , Proteínas de Membrana/química , Nucleotidiltransferases/química , Pasteurellaceae/metabolismo , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Alinhamento de Sequência , Especificidade por Substrato , Proteína cdc42 de Ligação ao GTP/genética , Proteína cdc42 de Ligação ao GTP/metabolismo
15.
Pigment Cell Melanoma Res ; 32(1): 41-54, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-29938923

RESUMO

Microphthalmia-associated transcription factor (MITF) is a member of the basic helix-loop-helix leucine zipper (bHLH-Zip) family and functions as the master regulator of the melanocytic lineage. MITF-M is the predominant isoform expressed in melanocytes and melanoma cells, and, unlike other MITF isoforms, it is constitutively nuclear. Mutational analysis revealed three karyophilic signals in the bHLH-Zip domain of MITF-M, spanning residues 197-206, 214-217, and 255-265. Structural characterization of the MITF protein showed that basic residues within these signals are exposed for interactions in the absence of DNA. Moreover, our data indicate that neither DNA binding nor dimerization of MITF-M are required for its nuclear localization. Finally, dimerization-deficient MITF-M mutants exhibited a significantly reduced stability in melanoma cells when compared to the wild-type protein. Taken together, we have shown that, in addition to its well-established role in DNA binding and dimer formation, the bHLH-Zip domain of MITF modulates the transcription factor's subcellular localization and stability.


Assuntos
Sequências Hélice-Alça-Hélice , Fator de Transcrição Associado à Microftalmia/química , Fator de Transcrição Associado à Microftalmia/metabolismo , Sequência de Aminoácidos , Arginina/metabolismo , Linhagem Celular , Núcleo Celular/metabolismo , Humanos , Domínios Proteicos , Multimerização Proteica , Estabilidade Proteica , Transporte Proteico , Frações Subcelulares/metabolismo
16.
Sci Rep ; 9(1): 1055, 2019 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-30705290

RESUMO

The MITF transcription factor is a master regulator of melanocyte development and a critical factor in melanomagenesis. The related transcription factors TFEB and TFE3 regulate lysosomal activity and autophagy processes known to be important in melanoma. Here we show that MITF binds the CLEAR-box element in the promoters of lysosomal and autophagosomal genes in melanocytes and melanoma cells. The crystal structure of MITF bound to the CLEAR-box reveals how the palindromic nature of this motif induces symmetric MITF homodimer binding. In metastatic melanoma tumors and cell lines, MITF positively correlates with the expression of lysosomal and autophagosomal genes, which, interestingly, are different from the lysosomal and autophagosomal genes correlated with TFEB and TFE3. Depletion of MITF in melanoma cells and melanocytes attenuates the response to starvation-induced autophagy, whereas the overexpression of MITF in melanoma cells increases the number of autophagosomes but is not sufficient to induce autophagic flux. Our results suggest that MITF and the related factors TFEB and TFE3 have separate roles in regulating a starvation-induced autophagy response in melanoma. Understanding the normal and pathophysiological roles of MITF and related transcription factors may provide important clinical insights into melanoma therapy.


Assuntos
Melanoma/metabolismo , Fator de Transcrição Associado à Microftalmia/metabolismo , Autofagia/genética , Autofagia/fisiologia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/fisiologia , Células HEK293 , Humanos , Immunoblotting , Lisossomos/metabolismo , Melanócitos/metabolismo , Melanoma/genética , Fator de Transcrição Associado à Microftalmia/genética , Reação em Cadeia da Polimerase em Tempo Real
17.
Sci Rep ; 7(1): 9903, 2017 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-28852099

RESUMO

The RNA-chaperone Hfq catalyses the annealing of bacterial small RNAs (sRNAs) with target mRNAs to regulate gene expression in response to environmental stimuli. Hfq acts on a diverse set of sRNA-mRNA pairs using a variety of different molecular mechanisms. Here, we present an unusual crystal structure showing two Hfq-RNA complexes interacting via their bound RNA molecules. The structure contains two Hfq6:A18 RNA assemblies positioned face-to-face, with the RNA molecules turned towards each other and connected via interdigitating base stacking interactions at the center. Biochemical data further confirm the observed interaction, and indicate that RNA-mediated contacts occur between Hfq-RNA complexes with various (ARN)X motif containing RNA sequences in vitro, including the stress response regulator OxyS and its target, fhlA. A systematic computational survey also shows that phylogenetically conserved (ARN)X motifs are present in a subset of sRNAs, some of which share similar modular architectures. We hypothesise that Hfq can co-opt RNA-RNA base stacking, an unanticipated structural trick, to promote the interaction of (ARN)X motif containing sRNAs with target mRNAs on a "speed-dating" fashion, thereby supporting their regulatory function.


Assuntos
Proteínas de Escherichia coli/química , Fator Proteico 1 do Hospedeiro/química , Conformação de Ácido Nucleico , RNA/química , Motivos de Aminoácidos , Sequência de Bases , Sítios de Ligação , Proteínas de Escherichia coli/metabolismo , Fator Proteico 1 do Hospedeiro/metabolismo , Modelos Moleculares , Estrutura Molecular , Ligação Proteica , RNA/metabolismo , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Soluções/química , Relação Estrutura-Atividade
18.
Biochem J ; 390(Pt 1): 29-39, 2005 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-15836438

RESUMO

The three-dimensional structure of CD4M33, a mimic of the host-cell receptor-antigen CD4 and a powerful inhibitor of CD4-gp120 (viral envelope glycoprotein 120) interaction and HIV-1 entry into cells [Martin, Stricher, Misse, Sironi, Pugniere, Barthe, Prado-Gotor, Freulon, Magne, Roumestand et al. (2003) Nat. Biotechnol. 21, 71-76], was solved by 1H-NMR and its structure was modelled in its complex with gp120. In this complex, CD4M33 binds in a CD4-like mode and inserts its unnatural and prominent Bip23 (biphenylalanine-23) side-chain into the gp120 interior 'Phe43 cavity', thus filling its volume. CD4M33 was specifically labelled with fluorescein and shown by fluorescence anisotropy to bind to different gp120 glycoproteins with dissociation constants in the nanomolar range. Fluorescent CD4M33 was also used in a miniaturized 384-well-plate assay to study direct binding to a large panel of gp120 glycoproteins and in a competition assay to study binding of CD4 or other ligands targeting the CD4 binding site of gp120. Furthermore, by using the fluorescently labelled CD4M33 and the [Phe23]M33 mutant, which possesses a natural Phe23 residue and thus cannot penetrate the gp120 Phe43 cavity, we show that a recently discovered small-molecule-entry inhibitor, BMS-378806, does not target the CD4 binding site nor the Phe43 cavity of gp120. The fluorescently labelled CD4M33 mimic, its mutants and their derivatives represent useful tools with which to discover new molecules which target the CD4 binding site and/or the Phe43 cavity of gp120 glycoproteins in a high-throughput fluorescence-polarization assay and to characterize their mechanism of action.


Assuntos
Antígenos CD4/química , Imunoensaio de Fluorescência por Polarização/métodos , Proteína gp120 do Envelope de HIV/química , HIV-1/química , Mimetismo Molecular , Sistemas de Liberação de Medicamentos , Mutação , Ligação Proteica , Conformação Proteica
19.
Cancer Discov ; 5(5): 506-19, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25716347

RESUMO

UNLABELLED: Regulatory pathways that drive early hematogenous dissemination of tumor cells are insufficiently defined. Here, we used the presence of disseminated tumor cells (DTC) in the bone marrow to define patients with early disseminated breast cancer and identified low retinoic acid-induced 2 (RAI2) expression to be significantly associated with DTC status. Low RAI2 expression was also shown to be an independent poor prognostic factor in 10 different cancer datasets. Depletion of RAI2 protein in luminal breast cancer cell lines resulted in dedifferentiation marked by downregulation of ERα, FOXA1, and GATA3, together with increased invasiveness and activation of AKT signaling. Functional analysis of the previously uncharacterized RAI2 protein revealed molecular interaction with CtBP transcriptional regulators and an overlapping function in controlling the expression of a number of key target genes involved in breast cancer. These results suggest that RAI2 is a new metastasis-associated protein that sustains differentiation of luminal breast epithelial cells. SIGNIFICANCE: We identified downregulation of RAI2 as a novel metastasis-associated genetic alteration especially associated with early occurring bone metastasis in ERα-positive breast tumors. We specified the role of the RAI2 protein to function as a transcriptional regulator that controls the expression of several key regulators of breast epithelial integrity and cancer.


Assuntos
Medula Óssea/patologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Células Neoplásicas Circulantes/patologia , Proteínas/genética , Oxirredutases do Álcool/metabolismo , Sequência de Aminoácidos , Neoplasias da Mama/mortalidade , Linhagem Celular Tumoral , Análise por Conglomerados , Proteínas de Ligação a DNA/metabolismo , Feminino , Expressão Gênica , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Dados de Sequência Molecular , Prognóstico , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas/química , Proteínas/metabolismo , Reprodutibilidade dos Testes , Alinhamento de Sequência , Transcriptoma
20.
Structure ; 22(3): 466-77, 2014 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-24530283

RESUMO

The ability of basic leucine zipper transcription factors for homo- or heterodimerization provides a paradigm for combinatorial control of eukaryotic gene expression. It has been unclear, however, how facultative dimerization results in alternative DNA-binding repertoires on distinct regulatory elements. To unravel the molecular basis of such coupled preferences, we determined two high-resolution structures of the transcription factor MafB as a homodimer and as a heterodimer with c-Fos bound to variants of the Maf-recognition element. The structures revealed several unexpected and dimer-specific coiled-coil-heptad interactions. Based on these findings, we have engineered two MafB mutants with opposite dimerization preferences. One of them showed a strong preference for MafB/c-Fos heterodimerization and enabled selection of heterodimer-favoring over homodimer-specific Maf-recognition element variants. Our data provide a concept for transcription factor design to selectively activate dimer-specific pathways and binding repertoires.


Assuntos
Fator de Transcrição MafB/química , Fator de Transcrição MafB/metabolismo , Sequência de Aminoácidos , Animais , Fatores de Transcrição de Zíper de Leucina Básica/química , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Sítios de Ligação , DNA/metabolismo , Fator de Transcrição MafB/genética , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Conformação Proteica , Engenharia de Proteínas/métodos , Multimerização Proteica , Proteínas Proto-Oncogênicas c-fos/química , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo
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