RESUMO
IMP dehydrogenase(IMPDH) is an essential enzyme that catalyzes the rate-limiting step in the guanine nucleotide pathway. In eukaryotic cells, GTP binding to the regulatory domain allosterically controls the activity of IMPDH by a mechanism that is fine-tuned by post-translational modifications and enzyme polymerization. Nonetheless, the mechanisms of regulation of IMPDH in bacterial cells remain unclear. Using biochemical, structural, and evolutionary analyses, we demonstrate that, in most bacterial phyla, (p)ppGpp compete with ATP to allosterically modulate IMPDH activity by binding to a, previously unrecognized, conserved high affinity pocket within the regulatory domain. This pocket was lost during the evolution of Proteobacteria, making their IMPDHs insensitive to these alarmones. Instead, most proteobacterial IMPDHs evolved to be directly modulated by the balance between ATP and GTP that compete for the same allosteric binding site. Altogether, we demonstrate that the activity of bacterial IMPDHs is allosterically modulated by a universally conserved nucleotide-controlled conformational switch that has divergently evolved to adapt to the specific particularities of each organism. These results reconcile the reported data on the crosstalk between (p)ppGpp signaling and the guanine nucleotide biosynthetic pathway and reinforce the essential role of IMPDH allosteric regulation on bacterial GTP homeostasis.
Assuntos
Nucleotídeos de Guanina , IMP Desidrogenase , Adenina , Trifosfato de Adenosina , Guanosina Pentafosfato , Guanosina Trifosfato/metabolismo , Homeostase , IMP Desidrogenase/genética , IMP Desidrogenase/metabolismo , Modelos MolecularesRESUMO
Inosine-5'-monophosphate dehydrogenase (IMPDH), a key regulatory enzyme in purine nucleotide biosynthesis, dynamically assembles filaments in response to changes in metabolic demand. Humans have two isoforms: IMPDH2 filaments reduce sensitivity to feedback inhibition, while IMPDH1 assembly remains uncharacterized. IMPDH1 plays a unique role in retinal metabolism, and point mutants cause blindness. Here, in a series of cryogenic-electron microscopy structures we show that human IMPDH1 assembles polymorphic filaments with different assembly interfaces in extended and compressed states. Retina-specific splice variants introduce structural elements that reduce sensitivity to GTP inhibition, including stabilization of the extended filament form. Finally, we show that IMPDH1 disease mutations fall into two classes: one disrupts GTP regulation and the other has no effect on GTP regulation or filament assembly. These findings provide a foundation for understanding the role of IMPDH1 in retinal function and disease and demonstrate the diverse mechanisms by which metabolic enzyme filaments are allosterically regulated.
Assuntos
IMP Desidrogenase/genética , Retina/enzimologia , Trifosfato de Adenosina/metabolismo , Regulação Alostérica , Sítios de Ligação , Domínio Catalítico , Guanosina Trifosfato/metabolismo , Células HEK293 , Humanos , IMP Desidrogenase/química , IMP Desidrogenase/ultraestrutura , Modelos Moleculares , NAD/metabolismo , Doenças Retinianas/genéticaRESUMO
We have structurally and functionally characterized Skl and Pal endolysins, the latter being the first endolysin shown to kill effectively Streptococcus pneumoniae, a leading cause of deathly diseases. We have proved that Skl and Pal are cysteine-amidases whose catalytic domains, from CHAP and Amidase_5 families, respectively, share an α3ß6-fold with papain-like topology. Catalytic triads are identified (for the first time in Amidase_5 family), and residues relevant for substrate binding and catalysis inferred from in silico models, including a calcium-binding site accounting for Skl dependence on this cation for activity. Both endolysins contain a choline-binding domain (CBD) with a ß-solenoid fold (homology modeled) and six conserved choline-binding loci whose saturation induced dimerization. Remarkably, Pal and Skl dimers display a common overall architecture, preserved in choline-bound dimers of pneumococcal lysins with other catalytic domains and bond specificities, as disclosed using small angle X-ray scattering (SAXS). Additionally, Skl is proved to be an efficient anti-pneumococcal agent that kills multi-resistant strains and clinical emergent-serotype isolates. Interestingly, Skl and Pal time-courses of pneumococcal lysis were sigmoidal, which might denote a limited access of both endolysins to target bonds at first stages of lysis. Furthermore, their DTT-mediated activation, of relevance for other cysteine-peptidases, cannot be solely ascribed to reversal of catalytic-cysteine oxidation.
RESUMO
We report the in vivo regulation of Inosine-5´-monophosphate dehydrogenase 1 (IMPDH1) in the retina. IMPDH1 catalyzes the rate-limiting step in the de novo synthesis of guanine nucleotides, impacting the cellular pools of GMP, GDP and GTP. Guanine nucleotide homeostasis is central to photoreceptor cells, where cGMP is the signal transducing molecule in the light response. Mutations in IMPDH1 lead to inherited blindness. We unveil a light-dependent phosphorylation of retinal IMPDH1 at Thr159/Ser160 in the Bateman domain that desensitizes the enzyme to allosteric inhibition by GDP/GTP. When exposed to bright light, living mice increase the rate of GTP and ATP synthesis in their retinas; concomitant with IMPDH1 aggregate formation at the outer segment layer. Inhibiting IMPDH activity in living mice delays rod mass recovery. We unveil a novel mechanism of regulation of IMPDH1 in vivo, important for understanding GTP homeostasis in the retina and the pathogenesis of adRP10 IMPDH1 mutations.
Assuntos
Guanosina Trifosfato/biossíntese , IMP Desidrogenase/genética , Luz , Processamento de Proteína Pós-Traducional , Retina/metabolismo , Retina/efeitos da radiação , Trifosfato de Adenosina/biossíntese , Animais , Fenômenos Bioquímicos , Regulação da Expressão Gênica , Homeostase , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Fosforilação , Estimulação Luminosa , Células Fotorreceptoras/fisiologiaRESUMO
Microtubules (MTs) are hollow cylinders made of tubulin, a GTPase responsible for essential functions during cell growth and division, and thus, key target for anti-tumor drugs. In MTs, GTP hydrolysis triggers structural changes in the lattice, which are responsible for interaction with regulatory factors. The stabilizing GTP-cap is a hallmark of MTs and the mechanism of the chemical-structural link between the GTP hydrolysis site and the MT lattice is a matter of debate. We have analyzed the structure of tubulin and MTs assembled in the presence of fluoride salts that mimic the GTP-bound and GDPâ¢Pi transition states. Our results challenge current models because tubulin does not change axial length upon GTP hydrolysis. Moreover, analysis of the structure of MTs assembled in the presence of several nucleotide analogues and of taxol allows us to propose that previously described lattice expansion could be a post-hydrolysis stage involved in Pi release.
Assuntos
Microtúbulos/química , Modelos Moleculares , Conformação Molecular , Microscopia Crioeletrônica , Guanosina Difosfato/química , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/química , Ligação de Hidrogênio , Microtúbulos/metabolismo , Relação Estrutura-Atividade , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismoRESUMO
IMP dehydrogenase (IMPDH) is an essential enzyme that catalyzes the rate-limiting step in the de novo guanine nucleotide biosynthetic pathway. Because of its involvement in the control of cell division and proliferation, IMPDH represents a therapeutic for managing several diseases, including microbial infections and cancer. IMPDH must be tightly regulated, but the molecular mechanisms responsible for its physiological regulation remain unknown. To this end, we recently reported an important role of adenine and guanine mononucleotides that bind to the regulatory Bateman domain to allosterically modulate the catalytic activity of eukaryotic IMPDHs. Here, we have used enzyme kinetics, X-ray crystallography, and small-angle X-ray scattering (SAXS) methodologies to demonstrate that adenine/guanine dinucleoside polyphosphates bind to the Bateman domain of IMPDH from the fungus Ashbya gossypii with submicromolar affinities. We found that these dinucleoside polyphosphates modulate the catalytic activity of IMPDHs in vitro by efficiently competing with the adenine/guanine mononucleotides for the allosteric sites. These results suggest that dinucleoside polyphosphates play important physiological roles in the allosteric regulation of IMPDHs by adding an additional mechanism for fine-tuning the activities of these enzymes. We propose that these findings may have important implications for the design of therapeutic strategies to inhibit IMPDHs.
Assuntos
Fosfatos de Dinucleosídeos/química , IMP Desidrogenase/química , Conformação Proteica , Domínios Proteicos/genética , Regulação Alostérica/genética , Infecções Bacterianas/genética , Infecções Bacterianas/microbiologia , Sítios de Ligação/genética , Catálise , Cristalografia por Raios X , Fosfatos de Dinucleosídeos/genética , Eremothecium/genética , Nucleotídeos de Guanina , Humanos , IMP Desidrogenase/genética , IMP Desidrogenase/ultraestrutura , Modelos Moleculares , Neoplasias/genética , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
Inosine 5'-monophosphate dehydrogenase (IMPDH) catalyzes the rate-limiting step in the de novo GTP biosynthetic pathway and plays essential roles in cell proliferation. As a clinical target, IMPDH has been studied for decades, but it has only been within the last years that we are starting to understand the complexity of the mechanisms of its physiological regulation. Here, we report structural and functional insights into how adenine and guanine nucleotides control a conformational switch that modulates the assembly of the two human IMPDH enzymes into cytoophidia and allosterically regulates their catalytic activity. In vitro reconstituted micron-length cytoophidia-like structures show catalytic activity comparable to unassembled IMPDH but, in turn, are more resistant to GTP/GDP allosteric inhibition. Therefore, IMPDH cytoophidia formation facilitates the accumulation of high levels of guanine nucleotides when the cell requires it. Finally, we demonstrate that most of the IMPDH retinopathy-associated mutations abrogate GTP/GDP-induced allosteric inhibition and alter cytoophidia dynamics.
Assuntos
IMP Desidrogenase/metabolismo , Nucleotídeos/metabolismo , Catálise , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Guanosina Trifosfato/metabolismo , Células HeLa , Humanos , Conformação Molecular , PolimerizaçãoRESUMO
Inosine-5'-monophosphate dehydrogenase (IMPDH) is an essential enzyme for nucleotide metabolism and cell proliferation. Despite IMPDH is the target of drugs with antiviral, immunosuppressive and antitumor activities, its physiological mechanisms of regulation remain largely unknown. Using the enzyme from the industrial fungus Ashbya gossypii, we demonstrate that the binding of adenine and guanine nucleotides to the canonical nucleotide binding sites of the regulatory Bateman domain induces different enzyme conformations with significantly distinct catalytic activities. Thereby, the comparison of their high-resolution structures defines the mechanistic and structural details of a nucleotide-controlled conformational switch that allosterically modulates the catalytic activity of eukaryotic IMPDHs. Remarkably, retinopathy-associated mutations lie within the mechanical hinges of the conformational change, highlighting its physiological relevance. Our results expand the mechanistic repertoire of Bateman domains and pave the road to new approaches targeting IMPDHs.
Assuntos
Nucleotídeos de Adenina/metabolismo , Nucleotídeos de Guanina/metabolismo , IMP Desidrogenase/metabolismo , Nucleotídeos de Adenina/química , Sítios de Ligação , Nucleotídeos de Guanina/química , IMP Desidrogenase/química , Modelos Moleculares , Conformação Molecular , SaccharomycetalesRESUMO
Vitamins and related compounds, such as provitamins, biopigments and antioxidants, belong to those few chemicals that appeal in a positive way to most people. These terms sound synonymous to vitality, good health and mental strenght, even to the layman. Everyone of us needs his/her daily intake of (pro)vitamins and antioxidants, normally provided by a balanced and varied diet. However, current food habits or preferences, food availabilities, as well as food processing, preservation or cooking methodologies and technologies, do not always assure a sufficient balanced natural daily (pro)vitamin supply to a healthy individual, and even more so for a stressed or sick human being. Today, modern society is seldom confronted with the notorious avitaminoses of the past, well known to the Western World, but they do still occur frequently in overpopulated, war-ridden, poverty- or famine-struck regions on our globe, as well as for surprisingly large population groups in developed countries.
Assuntos
Antioxidantes/metabolismo , Biotecnologia/métodos , Pigmentos Biológicos/biossíntese , Vitaminas/biossíntese , HumanosRESUMO
Integrin α6ß4 is a major component of hemidesmosomes that mediate the stable anchorage of epithelial cells to the underlying basement membrane. Integrin α6ß4 has also been implicated in cell proliferation and migration and in carcinoma progression. The third and fourth fibronectin type III domains (FnIII-3,4) of integrin ß4 mediate binding to the hemidesmosomal proteins BPAG1e and BPAG2, and participate in signalling. Here, it is demonstrated that X-ray crystallography, small-angle X-ray scattering and double electron-electron resonance (DEER) complement each other to solve the structure of the FnIII-3,4 region. The crystal structures of the individual FnIII-3 and FnIII-4 domains were solved and the relative arrangement of the FnIII domains was elucidated by combining DEER with site-directed spin labelling. Multiple structures of the interdomain linker were modelled by Monte Carlo methods complying with DEER constraints, and the final structures were selected against experimental scattering data. FnIII-3,4 has a compact and cambered flat structure with an evolutionary conserved surface that is likely to correspond to a protein-interaction site. Finally, this hybrid method is of general application for the study of other macromolecules and complexes.
Assuntos
Fibronectinas/química , Integrina beta4/química , Sequência de Aminoácidos , Cristalografia por Raios X , Fibronectinas/metabolismo , Humanos , Integrina beta4/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Estrutura Terciária de Proteína , Espalhamento a Baixo Ângulo , Alinhamento de Sequência , Difração de Raios XRESUMO
Microtubule-targeting agents are widely used for the treatment of cancer and as tool compounds to study the microtubule cytoskeleton. BAL27862 is a novel microtubule-destabilizing drug that is currently undergoing phase I clinical evaluation as the prodrug BAL101553. The drug is a potent inhibitor of tumor cell growth and shows a promising activity profile in a panel of human cancer models resistant to clinically relevant microtubule-targeting agents. Here, we evaluated the molecular mechanism of the tubulin-BAL27862 interaction using a combination of cell biology, biochemistry and structural biology methods. Tubulin-binding assays revealed that BAL27862 potently inhibited tubulin assembly at 37 °C with an IC50 of 1.4 µM and bound to unassembled tubulin with a stoichiometry of 1 mol/mol tubulin and a dissociation constant of 244±30 nM. BAL27862 bound to tubulin independently of vinblastine, without the formation of tubulin oligomers. The kinetics of BAL27862 binding to tubulin were distinct from those of colchicine, with evidence of competition between BAL27862 and colchicine for binding. Determination of the tubulin-BAL27862 structure by X-ray crystallography demonstrated that BAL27862 binds to the same site as colchicine at the intradimer interface. Comparison of crystal structures of tubulin-BAL27862 and tubulin-colchicine complexes shows that the binding mode of BAL27862 to tubulin is similar to that of colchicine. However, comparative analyses of the effects of BAL27862 and colchicine on the microtubule mitotic spindle and in tubulin protease-protection experiments suggest different outcomes of tubulin binding. Taken together, our data define BAL27862 as a potent, colchicine site-binding, microtubule-destabilizing agent with distinct effects on microtubule organization.
Assuntos
Benzimidazóis/farmacologia , Colchicina/metabolismo , Microtúbulos/química , Microtúbulos/efeitos dos fármacos , Oxidiazóis/farmacologia , Moduladores de Tubulina/farmacologia , Tubulina (Proteína)/química , Tubulina (Proteína)/efeitos dos fármacos , Animais , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Benzimidazóis/química , Benzimidazóis/metabolismo , Sítios de Ligação , Ligação Competitiva , Bovinos , Linhagem Celular Tumoral , Colchicina/farmacologia , Cristalografia por Raios X , Células HeLa , Humanos , Microscopia Eletrônica de Transmissão , Microtúbulos/metabolismo , Modelos Moleculares , Oxidiazóis/química , Oxidiazóis/metabolismo , Ligação Proteica/efeitos dos fármacos , Conformação Proteica/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacos , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/química , Moduladores de Tubulina/metabolismo , Vimblastina/metabolismoRESUMO
End binding protein 1 (EB1) and cytoplasmic linker protein of 170 kDa (CLIP-170) are two well-studied microtubule plus-end-tracking proteins (+TIPs) that target growing microtubule plus ends in the form of comet tails and regulate microtubule dynamics. However, the mechanism by which they regulate microtubule dynamics is not well understood. Using full-length EB1 and a minimal functional fragment of CLIP-170 (ClipCG12), we found that EB1 and CLIP-170 cooperatively regulate microtubule dynamic instability at concentrations below which neither protein is effective. By use of small-angle X-ray scattering and analytical ultracentrifugation, we found that ClipCG12 adopts a largely extended conformation with two noninteracting CAP-Gly domains and that it formed a complex in solution with EB1. Using a reconstituted steady-state mammalian microtubule system, we found that at a low concentration of 250 nM, neither EB1 nor ClipCG12 individually modulated plus-end dynamic instability. Higher concentrations (up to 2 µM) of the two proteins individually did modulate dynamic instability, perhaps by a combination of effects at the tips and along the microtubule lengths. However, when low concentrations (250 nM) of EB1 and ClipCG12 were present together, the mixture modulated dynamic instability considerably. Using a pulsing strategy with [γ(32)P]GTP, we further found that unlike EB1 or ClipCG12 alone, the EB1-ClipCG12 mixture partially depleted the microtubule ends of stably bound (32)P(i). Together, our results suggest that EB1 and ClipCG12 act cooperatively to regulate microtubule dynamics. They further indicate that stabilization of microtubule plus ends by the EB1-ClipCG12 mixture may involve modification of an aspect of the stabilizing cap.
Assuntos
Proteínas Associadas aos Microtúbulos/química , Microtúbulos/química , Proteínas de Neoplasias/química , Sítios de Ligação , Guanosina Trifosfato/metabolismo , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Proteínas de Neoplasias/metabolismo , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismoRESUMO
Peloruside is a microtubule-stabilizing agent that targets the same site as laulimalide. It binds to microtubules with a 1:1 stoichiometry and with a binding affinity in the low-muM range; thereby reducing the number of microtubular protofilaments in the same way as paclitaxel. Although the binding affinity of the compound is comparable to that of the low-affinity stabilizing agent sarcodictyin, peloruside is more active in inducing microtubule assembly and is more cytotoxic to tumor cells; this suggests that the peloruside site is a more effective site for stabilizing microtubules. Acetylation of the C24 hydroxyl group results in inactive compounds. According to molecular modeling, this substitution at the C24 hydroxyl group presumably disrupts the interaction of the side chain with Arg320 in the putative binding site on alpha-tubulin. The binding epitope of peloruside on microtubules has been studied by using NMR spectroscopic techniques, and is compatible with the same binding site.
Assuntos
Antineoplásicos/química , Compostos Bicíclicos Heterocíclicos com Pontes/química , Lactonas/química , Microtúbulos/química , Tubulina (Proteína)/química , Antineoplásicos/farmacologia , Sítios de Ligação , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Linhagem Celular Tumoral , Humanos , Lactonas/farmacologia , Espectroscopia de Ressonância Magnética , Microtúbulos/metabolismo , Modelos Moleculares , Relação Estrutura-Atividade , Termodinâmica , Tubulina (Proteína)/metabolismoRESUMO
Microtubule plus-end tracking proteins (+TIPs) are involved in many microtubule-based processes. End binding (EB) proteins constitute a highly conserved family of +TIPs. They play a pivotal role in regulating microtubule dynamics and in the recruitment of diverse +TIPs to growing microtubule plus ends. Here we used a combination of methods to investigate the dimerization properties of the three human EB proteins EB1, EB2, and EB3. Based on Förster resonance energy transfer, we demonstrate that the C-terminal dimerization domains of EBs (EBc) can readily exchange their chains in solution. We further document that EB1c and EB3c preferentially form heterodimers, whereas EB2c does not participate significantly in the formation of heterotypic complexes. Measurements of the reaction thermodynamics and kinetics, homology modeling, and mutagenesis provide details of the molecular determinants of homo- versus heterodimer formation of EBc domains. Fluorescence spectroscopy and nuclear magnetic resonance studies in the presence of the cytoskeleton-associated protein-glycine-rich domains of either CLIP-170 or p150(glued) or of a fragment derived from the adenomatous polyposis coli tumor suppressor protein show that chain exchange of EBc domains can be controlled by binding partners. Extension of these studies of the EBc domains to full-length EBs demonstrate that heterodimer formation between EB1 and EB3, but not between EB2 and the other two EBs, occurs both in vitro and in cells as revealed by live cell imaging. Together, our data provide molecular insights for rationalizing the dominant negative control by C-terminal EB domains and form a basis for understanding the functional role of heterotypic chain exchange by EBs in cells.
Assuntos
Proteínas Associadas aos Microtúbulos/metabolismo , Modelos Moleculares , Multimerização Proteica/fisiologia , Animais , Células CHO , Linhagem Celular , Cricetinae , Cricetulus , Complexo Dinactina , Humanos , Cinética , Espectroscopia de Ressonância Magnética , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/genética , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometria de FluorescênciaRESUMO
Microtubules are filamentous polymers essential for cell viability. Microtubule plus-end tracking proteins (+TIPs) associate with growing microtubule plus ends and control microtubule dynamics and interactions with different cellular structures during cell division, migration, and morphogenesis. EB1 and its homologs are highly conserved proteins that play an important role in the targeting of +TIPs to microtubule ends, but the underlying molecular mechanism remains elusive. By using live cell experiments and in vitro reconstitution assays, we demonstrate that a short polypeptide motif, Ser-x-Ile-Pro (SxIP), is used by numerous +TIPs, including the tumor suppressor APC, the transmembrane protein STIM1, and the kinesin MCAK, for localization to microtubule tips in an EB1-dependent manner. Structural and biochemical data reveal the molecular basis of the EB1-SxIP interaction and explain its negative regulation by phosphorylation. Our findings establish a general "microtubule tip localization signal" (MtLS) and delineate a unifying mechanism for this subcellular protein targeting process.
Assuntos
Proteínas Associadas aos Microtúbulos/química , Microtúbulos/química , Sinais Direcionadores de Proteínas , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Cristalografia por Raios X , Humanos , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Fosforilação , Alinhamento de SequênciaRESUMO
BACKGROUND INFORMATION: Hsp90 (90 kDa heat-shock protein) plays a key role in the folding and activation of many client proteins involved in signal transduction and cell cycle control. The cycle of Hsp90 has been intimately associated with large conformational rearrangements, which are nucleotide-binding-dependent. However, up to now, our understanding of Hsp90 conformational changes derives from structural information, which refers to the crystal states of either recombinant Hsp90 constructs or the prokaryotic homologue HtpG (Hsp90 prokaryotic homologue). RESULTS AND DISCUSSION: Here, we present the first nucleotide-free structures of the entire eukaryotic Hsp90 (apo-Hsp90) obtained by small-angle X-ray scattering and single-particle cryo-EM (cryo-electron microscopy). We show that, in solution, apo-Hsp90 is in a conformational equilibrium between two open states that have never been described previously. By comparing our cryo-EM maps with HtpG and known Hsp90 structures, we establish that the structural changes involved in switching between the two Hsp90 apo-forms require large movements of the NTD (N-terminal domain) and MD (middle domain) around two flexible hinge regions. CONCLUSIONS: The present study shows, for the first time, the structure of the entire eukaryotic apo-Hsp90, along with its intrinsic flexibility. Although large structural rearrangements, leading to partial closure of the Hsp90 dimer, were previously attributed to the binding of nucleotides, our results reveal that they are in fact mainly due to the intrinsic flexibility of Hsp90 dimer. Taking into account the preponderant role of the dynamic nature of the structure of Hsp90, we reconsider the Hsp90 ATPase cycle.
Assuntos
Proteínas de Choque Térmico HSP90/química , Adenosina Trifosfatases/química , Adenosina Trifosfatases/ultraestrutura , Animais , Cristalografia por Raios X , Proteínas de Choque Térmico HSP90/ultraestrutura , Microscopia Eletrônica , Modelos Moleculares , Conformação Proteica , Dobramento de Proteína , Soluções , SuínosRESUMO
Cyclostreptin (1), a natural product from Streptomyces sp. 9885, irreversibly stabilizes cellular microtubules, causes cell cycle arrest, evades drug resistance mediated by P-glycoprotein in a tumor cell line and potently inhibits paclitaxel binding to microtubules, yet it only weakly induces tubulin assembly. In trying to understand this paradox, we observed irreversible binding of synthetic cyclostreptin to tubulin. This results from formation of covalent crosslinks to beta-tubulin in cellular microtubules and microtubules formed from purified tubulin in a 1:1 total stoichiometry distributed between Thr220 (at the outer surface of a pore in the microtubule wall) and Asn228 (at the lumenal paclitaxel site). Unpolymerized tubulin was only labeled at Thr220. Thus, the pore region of beta-tubulin is an undescribed binding site that (i) elucidates the mechanism by which taxoid-site compounds reach the kinetically unfavorable lumenal site and (ii) explains how taxoid-site drugs induce microtubule formation from dimeric and oligomeric tubulin.
Assuntos
Microtúbulos/metabolismo , Compostos Policíclicos/metabolismo , Tubulina (Proteína)/metabolismo , Alcanos/metabolismo , Sequência de Aminoácidos , Asparagina/metabolismo , Sítios de Ligação , Ligação Competitiva , Carbamatos/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Docetaxel , Epotilonas/metabolismo , Humanos , Concentração Inibidora 50 , Lactonas/metabolismo , Espectrometria de Massas , Microtúbulos/química , Microtúbulos/efeitos dos fármacos , Modelos Químicos , Modelos Moleculares , Dados de Sequência Molecular , Paclitaxel/metabolismo , Paclitaxel/farmacologia , Compostos Policíclicos/química , Compostos Policíclicos/farmacologia , Ligação Proteica , Pironas/metabolismo , Taxoides/metabolismo , Taxoides/farmacologia , Treonina/metabolismo , Tubulina (Proteína)/química , Moduladores de Tubulina/química , Moduladores de Tubulina/metabolismo , Moduladores de Tubulina/farmacologiaRESUMO
The combination of farnesyltransferase inhibitors (FTIs) and taxanes has been shown to result in potent antiproliferative and antimitotic synergy. Recent phase I and II clinical trials have shown that this combination shows clinical activity in taxane-refractory or taxane-resistant cancer patients. To understand the mechanism behind these clinical observations, we used a cancer cell model of paclitaxel resistance and showed that the FTI/taxane combination retains potent antiproliferative, antimitotic, and proapoptotic activity against the paclitaxel-resistant cells, at doses where each drug alone has little or no activity. To probe the mechanistic basis of these observations, paclitaxel activity was monitored in living cells using the fluorescently conjugated paclitaxel, Flutax-2. We observed that all FTIs tested increase the amount of microtubule-bound Flutax-2 and the number of microtubules labeled with Flutax-2 in both paclitaxel-resistant and paclitaxel-sensitive cells. Importantly, we observed a consequential increase in microtubule stability and tubulin acetylation with the combination of the two drugs, even in paclitaxel-resistant cells, confirming that the enhanced taxane binding in the presence of FTI affects microtubule function. Furthermore, this mechanism is dependent on the function of the tubulin deacetylase, HDAC6, because in cells overexpressing a catalytically inactive HDAC6, FTIs are incapable of enhancing Flutax-2-microtubule binding. Similar results were obtained by using an FTI devoid of farnesyltransferase inhibitory activity, indicating that functional inhibition of farnesyltransferase is also required. Overall, these studies provide a new insight into the functional relationship between HDAC6, farnesyltransferase, and microtubules, and support clinical data showing that the FTI/taxane combination is effective in taxane-refractory patients.
Assuntos
Antineoplásicos/uso terapêutico , Hidrocarbonetos Aromáticos com Pontes/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Inibidores Enzimáticos/uso terapêutico , Farnesiltranstransferase/antagonistas & inibidores , Taxoides/uso terapêutico , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Feminino , Humanos , Mitose/efeitos dos fármacos , Neoplasias Ovarianas , Taxoides/farmacocinética , Tubulina (Proteína)/efeitos dos fármacos , Tubulina (Proteína)/metabolismoRESUMO
The interactions of microtubules with most compounds described as stabilizing agents have been studied. Several of them (lonafarnib, dicumarol, lutein, and jatrophane polyesters) did not show any stabilizing effect on microtubules. Taccalonolides A and E show paclitaxel-like effects in cells, but they were not able to modulate in vitro tubulin assembly or to bind microtubules, which suggests that other factors are involved in their cellular effects. The binding constants of epothilones, eleutherobin, discodermolide, sarcodictyins, 3,17beta-diacetoxy-2-ethoxy-6-oxo-B-homo-estra-1,3,5(10)-triene, and dictyostatin to the paclitaxel site; the critical concentrations of ligand-induced assembly; and their cytotoxicity in carcinoma cells have been measured, and correlations between these parameters have been determined. The inhibition of cell proliferation correlates better with the binding enthalpy change than with the binding constants, suggesting that large, favorable enthalpic contribution to the binding is desired to design paclitaxel site drugs with higher cytotoxicity.
Assuntos
Antineoplásicos/química , Antineoplásicos/metabolismo , Microtúbulos/química , Microtúbulos/metabolismo , Paclitaxel/química , Paclitaxel/metabolismo , Antineoplásicos/farmacologia , Ligação Competitiva/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Desenho de Fármacos , Humanos , Ligantes , Microtúbulos/efeitos dos fármacos , Estrutura Molecular , Paclitaxel/farmacologia , Valor Preditivo dos Testes , Ligação Proteica/efeitos dos fármacos , Termodinâmica , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina , Células Tumorais CultivadasRESUMO
Cyclostreptin (FR182877), a bacterial natural product, was reported to have weak paclitaxel-like activity with tubulin but antitumor activity in vivo. We used synthetic cyclostreptin in studies of its mechanism of action. Although less potent than paclitaxel in several human cancer cell lines, cyclostreptin was active against cells resistant to paclitaxel and epothilone A. At equitoxic concentrations with paclitaxel, cyclostreptin was more effective in arresting MCF-7 cells in mitosis and equivalent in bundling microtubules in PtK(2) cells. Tubulin assembly with paclitaxel occurs at low temperatures and in the absence of GTP or microtubule-associated proteins (MAPs). Brisk assembly with cyclostreptin required MAPs, GTP, and higher reaction temperatures. On the basis of turbidimetry, cyclostreptin-induced microtubules were more stable in the cold than the paclitaxel-induced polymer. Moreover, at 37 degrees C cyclostreptin was a strong competitive inhibitor of the binding of radiolabeled paclitaxel to tubulin polymer, with an apparent K(i) value of 88 nM. Competition studies versus a fluorescent taxoid across a temperature range, in comparison with paclitaxel and docetaxel, showed that only the binding of cyclostreptin to microtubules was markedly reduced at 4 degrees C versus temperatures over 30 degrees C. The binding of cyclostreptin to microtubules was characterized by a relatively greater endothermic and entropic profile as compared with those of the taxoid binding reactions, which are characterized more by exothermic and enthalpic interactions. Molecular modeling showed that cyclostreptin formed a pharmacophore with taxoids but formed hydrogen bonds only with the S9-S10 and M loops in the taxoid site. Initial studies also indicate that, relative to paclitaxel, cyclostreptin is more deficient in nucleation than elongation of polymer.