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1.
Cell ; 153(6): 1394-405, 2013 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-23746849

RESUMO

Drosophila cryptochrome (dCRY) is a FAD-dependent circadian photoreceptor, whereas mammalian cryptochromes (CRY1/2) are integral clock components that repress mCLOCK/mBMAL1-dependent transcription. We report crystal structures of full-length dCRY, a dCRY loop deletion construct, and the photolyase homology region of mouse CRY1 (mCRY1). Our dCRY structures depict Phe534 of the regulatory tail in the same location as the photolesion in DNA-repairing photolyases and reveal that the sulfur loop and tail residue Cys523 plays key roles in the dCRY photoreaction. Our mCRY1 structure visualizes previously characterized mutations, an NLS, and MAPK and AMPK phosphorylation sites. We show that the FAD and antenna chromophore-binding regions, a predicted coiled-coil helix, the C-terminal lid, and charged surfaces are involved in FAD-independent mPER2 and FBXL3 binding and mCLOCK/mBMAL1 transcriptional repression. The structure of a mammalian cryptochrome1 protein may catalyze the development of CRY chemical probes and the design of therapeutic metabolic modulators.


Assuntos
Relógios Circadianos , Criptocromos/química , Proteínas de Drosophila/química , Drosophila/metabolismo , Proteínas do Olho/química , Sequência de Aminoácidos , Animais , Ritmo Circadiano , Criptocromos/genética , Criptocromos/metabolismo , Análise Mutacional de DNA , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Transporte de Elétrons , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Proteínas F-Box/metabolismo , Regulação da Expressão Gênica , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Circadianas Period/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Alinhamento de Sequência , Transcrição Gênica
2.
Nat Chem Biol ; 6(2): 117-24, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20081827

RESUMO

Deregulation of the phosphoinositide-3-OH kinase (PI(3)K) pathway has been implicated in numerous pathologies including cancer, diabetes, thrombosis, rheumatoid arthritis and asthma. Recently, small-molecule and ATP-competitive PI(3)K inhibitors with a wide range of selectivities have entered clinical development. In order to understand the mechanisms underlying the isoform selectivity of these inhibitors, we developed a new expression strategy that enabled us to determine to our knowledge the first crystal structure of the catalytic subunit of the class IA PI(3)K p110 delta. Structures of this enzyme in complex with a broad panel of isoform- and pan-selective class I PI(3)K inhibitors reveal that selectivity toward p110 delta can be achieved by exploiting its conformational flexibility and the sequence diversity of active site residues that do not contact ATP. We have used these observations to rationalize and synthesize highly selective inhibitors for p110 delta with greatly improved potencies.


Assuntos
Domínio Catalítico , Fosfatidilinositol 3-Quinases/química , Inibidores de Proteínas Quinases/química , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Animais , Linhagem Celular , Simulação por Computador , Cristalografia por Raios X , Humanos , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Fosfatidilinositol 3-Quinases/metabolismo , Domínios e Motivos de Interação entre Proteínas , Spodoptera , Relação Estrutura-Atividade , Especificidade por Substrato
3.
Elife ; 102021 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-34519269

RESUMO

The mTORC1 kinase complex regulates cell growth, proliferation, and survival. Because mis-regulation of DEPTOR, an endogenous mTORC1 inhibitor, is associated with some cancers, we reconstituted mTORC1 with DEPTOR to understand its function. We find that DEPTOR is a unique partial mTORC1 inhibitor that may have evolved to preserve feedback inhibition of PI3K. Counterintuitively, mTORC1 activated by RHEB or oncogenic mutation is much more potently inhibited by DEPTOR. Although DEPTOR partially inhibits mTORC1, mTORC1 prevents this inhibition by phosphorylating DEPTOR, a mutual antagonism that requires no exogenous factors. Structural analyses of the mTORC1/DEPTOR complex showed DEPTOR's PDZ domain interacting with the mTOR FAT region, and the unstructured linker preceding the PDZ binding to the mTOR FRB domain. The linker and PDZ form the minimal inhibitory unit, but the N-terminal tandem DEP domains also significantly contribute to inhibition.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Sítios de Ligação , Microscopia Crioeletrônica , Escherichia coli , Regulação da Expressão Gênica , Humanos , Processamento de Imagem Assistida por Computador , Peptídeos e Proteínas de Sinalização Intracelular/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Modelos Moleculares , Domínios PDZ , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes , Serina-Treonina Quinases TOR/genética
4.
Biochem Soc Trans ; 37(Pt 4): 615-26, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19614567

RESUMO

PI3Ks (phosphoinositide 3-kinases) have important roles in a variety of cellular activities, including survival, proliferation, growth, shape, migration and intracellular sorting. Consistent with their function in cell survival and growth, the gene for the class Ialpha PI3K catalytic subunit is a common site of gain-of-function mutations in cancers. Ongoing structural studies of these enzymes and the complexes they make with their regulatory subunits have helped to clarify the mechanistic basis of this role in tumour development. The broad spectrum of biological activities associated with various isotypes of class I PI3Ks has led to an intense search for isotype-specific inhibitors as tools in mammalian cell biology and for therapeutic application. Structural studies of the class I PI3Ks suggest that flexibility may be a component of the catalytic cycle of the enzymes.


Assuntos
Fosfatidilinositol 3-Quinases/química , Fosfatidilinositol 3-Quinases/metabolismo , Animais , Humanos , Fosfatidilinositol 3-Quinases/genética , Fosfatos de Fosfatidilinositol/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Ligação Proteica , Estrutura Secundária de Proteína , Transdução de Sinais/fisiologia
5.
Science ; 366(6462): 203-210, 2019 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-31601764

RESUMO

The Rag guanosine triphosphatases (GTPases) recruit the master kinase mTORC1 to lysosomes to regulate cell growth and proliferation in response to amino acid availability. The nucleotide state of Rag heterodimers is critical for their association with mTORC1. Our cryo-electron microscopy structure of RagA/RagC in complex with mTORC1 shows the details of RagA/RagC binding to the RAPTOR subunit of mTORC1 and explains why only the RagAGTP/RagCGDP nucleotide state binds mTORC1. Previous kinetic studies suggested that GTP binding to one Rag locks the heterodimer to prevent GTP binding to the other. Our crystal structures and dynamics of RagA/RagC show the mechanism for this locking and explain how oncogenic hotspot mutations disrupt this process. In contrast to allosteric activation by RHEB, Rag heterodimer binding does not change mTORC1 conformation and activates mTORC1 by targeting it to lysosomes.


Assuntos
Alvo Mecanístico do Complexo 1 de Rapamicina/química , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas Monoméricas de Ligação ao GTP/química , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Proteína Regulatória Associada a mTOR/metabolismo , Microscopia Crioeletrônica , Cristalografia por Raios X , Dimerização , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Lisossomos/metabolismo , Espectrometria de Massas , Modelos Moleculares , Proteínas Monoméricas de Ligação ao GTP/sangue , Proteínas Monoméricas de Ligação ao GTP/genética , Mutação , Ligação Proteica , Conformação Proteica , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , Proteína Regulatória Associada a mTOR/química , Proteínas de Saccharomyces cerevisiae/sangue , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo
7.
ChemMedChem ; 12(18): 1542-1554, 2017 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-28857471

RESUMO

Activation of the phosphoinositide 3-kinase (PI3K) pathway is a key signaling event in cancer, inflammation, and other proliferative diseases. PI3K inhibitors are already approved for some specific clinical indications, but their systemic on-target toxicity limits their larger use. In particular, whereas toxicity is tolerable in acute treatment of life-threatening diseases, this is less acceptable in chronic conditions. In the past, the strategy to overcome this drawback was to block selected isoforms mainly expressed in leukocytes, but redundancy within the PI3K family members challenges the effectiveness of this approach. On the other hand, decreasing exposure to selected target cells represents a so-far unexplored alternative to circumvent systemic toxicity. In this manuscript, we describe the generation of a library of triazolylquinolones and the development of the first prodrug pan-PI3K inhibitor.


Assuntos
Ácidos Carboxílicos/química , Inibidores Enzimáticos/química , Inibidores de Fosfoinositídeo-3 Quinase , Pró-Fármacos/química , Animais , Sítios de Ligação , Ácidos Carboxílicos/metabolismo , Ácidos Carboxílicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Desenho de Fármacos , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Humanos , Ligação de Hidrogênio , Concentração Inibidora 50 , Camundongos , Microssomos/metabolismo , Simulação de Dinâmica Molecular , Fosfatidilinositol 3-Quinases/metabolismo , Pró-Fármacos/metabolismo , Pró-Fármacos/farmacologia , Ligação Proteica , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/metabolismo , Quinolonas/química , Quinolonas/metabolismo , Quinolonas/farmacologia , Relação Estrutura-Atividade
8.
Nat Commun ; 7: 11016, 2016 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-27072897

RESUMO

The target of rapamycin (Tor) is a Ser/Thr protein kinase that regulates a range of anabolic and catabolic processes. Tor is present in two complexes, TORC1 and TORC2, in which the Tor-Lst8 heterodimer forms a common sub-complex. We have determined the cryo-electron microscopy (EM) structure of Tor bound to Lst8. Two Tor-Lst8 heterodimers assemble further into a dyad-symmetry dimer mediated by Tor-Tor interactions. The first 1,300 residues of Tor form a HEAT repeat-containing α-solenoid with four distinct segments: a highly curved 800-residue N-terminal 'spiral', followed by a 400-residue low-curvature 'bridge' and an extended 'railing' running along the bridge leading to the 'cap' that links to FAT region. This complex topology was verified by domain insertions and offers a new interpretation of the mTORC1 structure. The spiral of one TOR interacts with the bridge of another, which together form a joint platform for the Regulatory Associated Protein of TOR (RAPTOR) regulatory subunit.


Assuntos
Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Multimerização Proteica , Saccharomyces cerevisiae/metabolismo , Serina-Treonina Quinases TOR/química , Serina-Treonina Quinases TOR/metabolismo , Animais , Domínio Catalítico , Microscopia Crioeletrônica , Humanos , Kluyveromyces/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina , Alvo Mecanístico do Complexo 2 de Rapamicina , Camundongos , Modelos Moleculares , Complexos Multiproteicos/ultraestrutura , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas de Saccharomyces cerevisiae/metabolismo , Serina-Treonina Quinases TOR/ultraestrutura
9.
Sci Signal ; 4(195): re2, 2011 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-22009150

RESUMO

Phosphoinositide 3-kinases (PI3Ks) are implicated in a broad spectrum of cellular activities, such as growth, proliferation, differentiation, migration, and metabolism. Activation of class I PI3Ks by mutation or overexpression correlates with the development and maintenance of various human cancers. These PI3Ks are heterodimers, and the activity of the catalytic subunits is tightly controlled by the associated regulatory subunits. Although the same p85 regulatory subunits associate with all class IA PI3Ks, the functional outcome depends on the isotype of the catalytic subunit. New PI3K partners that affect the signaling by the PI3K heterodimers have been uncovered, including phosphate and tensin homolog (PTEN), cyclic adenosine monophosphate-dependent protein kinase (PKA), and nonstructural protein 1. Interactions with PI3K regulators modulate the intrinsic membrane affinity and either the rate of phosphoryl transfer or product release. Crystal structures for the class I and class III PI3Ks in complexes with associated regulators and inhibitors have contributed to developing isoform-specific inhibitors and have shed light on the numerous regulatory mechanisms controlling PI3K activation and inhibition.


Assuntos
Fosfatidilinositol 3-Quinases/química , Multimerização Proteica , Estrutura Terciária de Proteína , Ativação Enzimática/efeitos dos fármacos , Humanos , Indazóis/farmacologia , Modelos Moleculares , Neoplasias/enzimologia , Neoplasias/patologia , Neoplasias/prevenção & controle , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/farmacologia
10.
Structure ; 19(8): 1127-37, 2011 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-21827948

RESUMO

Phosphoinositide 3-kinase δ is upregulated in lymphocytic leukemias. Because the p85-regulatory subunit binds to any class IA subunit, it was assumed there is a single universal p85-mediated regulatory mechanism; however, we find isozyme-specific inhibition by p85α. Using deuterium exchange mass spectrometry (DXMS), we mapped regulatory interactions of p110δ with p85α. Both nSH2 and cSH2 domains of p85α contribute to full inhibition of p110δ, the nSH2 by contacting the helical domain and the cSH2 via the C terminus of p110δ. The cSH2 inhibits p110ß and p110δ, but not p110α, implying that p110α is uniquely poised for oncogenic mutations. Binding RTK phosphopeptides disengages the SH2 domains, resulting in exposure of the catalytic subunit. We find that phosphopeptides greatly increase the affinity of the heterodimer for PIP2-containing membranes measured by FRET. DXMS identified regions decreasing exposure at membranes and also regions gaining exposure, indicating loosening of interactions within the heterodimer at membranes.


Assuntos
Classe Ia de Fosfatidilinositol 3-Quinase/química , Lipídeos de Membrana/química , Fosfatidilinositol 3-Quinases/química , Substituição de Aminoácidos , Animais , Classe I de Fosfatidilinositol 3-Quinases , Classe Ia de Fosfatidilinositol 3-Quinase/genética , Medição da Troca de Deutério , Humanos , Lipossomos/química , Camundongos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Fragmentos de Peptídeos , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Receptores do Fator de Crescimento Derivado de Plaquetas/química , Propriedades de Superfície
11.
J Biol Chem ; 282(17): 13011-21, 2007 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-17298948

RESUMO

Cryptochromes are flavoproteins that are evolutionary related to the DNA photolyases but lack DNA repair activity. Drosophila cryptochrome (dCRY) is a blue light photoreceptor that is involved in the synchronization of the circadian clock with the environmental light-dark cycle. Until now, spectroscopic and structural studies on this and other animal cryptochromes have largely been hampered by difficulties in their recombinant expression. We have therefore established an expression and purification scheme that enables us to purify mg amounts of monomeric dCRY from Sf21 insect cell cultures. Using UV-visible spectroscopy, mass spectrometry, and reversed phase high pressure liquid chromatography, we show that insect cell-purified dCRY contains flavin adenine dinucleotide in its oxidized state (FAD(ox)) and residual amounts of methenyltetrahydrofolate. Upon blue light irradiation, dCRY undergoes a reversible absorption change, which is assigned to the conversion of FAD(ox) to the red anionic FAD(.) radical. Our findings lead us to propose a novel photoreaction mechanism for dCRY, in which FAD(ox) corresponds to the ground state, whereas the FAD(.) radical represents the light-activated state that mediates resetting of the Drosophila circadian clock.


Assuntos
Ritmo Circadiano , Proteínas de Drosophila/química , Flavina-Adenina Dinucleotídeo/química , Flavoproteínas/química , Células Fotorreceptoras de Invertebrados/química , Animais , Linhagem Celular , Criptocromos , Reparo do DNA , Desoxirribodipirimidina Fotoliase/química , Drosophila/química , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Flavoproteínas/metabolismo , Oxirredução , Fotoquímica , Células Fotorreceptoras de Invertebrados/metabolismo
12.
Mol Cell ; 17(1): 69-82, 2005 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-15629718

RESUMO

PERIOD proteins are central components of the Drosophila and mammalian circadian clock. Their function is controlled by daily changes in synthesis, cellular localization, phosphorylation, degradation, as well as specific interactions with other clock components. Here we present the crystal structure of a Drosophila PERIOD (dPER) fragment comprising two tandemly organized PAS (PER-ARNT-SIM) domains (PAS-A and PAS-B) and two additional C-terminal alpha helices (alphaE and alphaF). Our analysis reveals a noncrystallographic dPER dimer mediated by intermolecular interactions of PAS-A with PAS-B and helix alphaF. We show that alphaF is essential for dPER homodimerization and that the PAS-A-alphaF interaction plays a crucial role in dPER clock function, as it is affected by the 29 hr long-period perL mutation.


Assuntos
Proteínas de Drosophila/química , Proteínas Nucleares/química , Sequência de Aminoácidos , Animais , Ritmo Circadiano , Cristalografia por Raios X , Dimerização , Drosophila/química , Drosophila/genética , Drosophila/fisiologia , Proteínas de Drosophila/genética , Proteínas de Drosophila/fisiologia , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/fisiologia , Proteínas Circadianas Period , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Sequências Repetitivas de Aminoácidos , Homologia de Sequência de Aminoácidos , Eletricidade Estática
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