RESUMO
Targeted protein degradation (TPD) is an emerging therapeutic strategy that would benefit from new chemical entities with which to recruit a wider variety of ubiquitin E3 ligases to target proteins for proteasomal degradation. Here we describe a TPD strategy involving the recruitment of FBXO22 to induce degradation of the histone methyltransferase and oncogene NSD2. UNC8732 facilitates FBXO22-mediated degradation of NSD2 in acute lymphoblastic leukemia cells harboring the NSD2 gain-of-function mutation p.E1099K, resulting in growth suppression, apoptosis and reversal of drug resistance. The primary amine of UNC8732 is metabolized to an aldehyde species, which engages C326 of FBXO22 to recruit the SCFFBXO22 Cullin complex. We further demonstrate that a previously reported alkyl amine-containing degrader targeting XIAP is similarly dependent on SCFFBXO22. Overall, we present a potent NSD2 degrader for the exploration of NSD2 disease phenotypes and a new FBXO22-recruitment strategy for TPD.
RESUMO
Targeted protein degradation (TPD) is an emerging therapeutic strategy that would benefit from new chemical entities with which to recruit a wider variety of ubiquitin E3 ligases to target proteins for proteasomal degradation. Here, we describe a TPD strategy involving the recruitment of FBXO22 to induce degradation of the histone methyltransferase and oncogene NSD2. UNC8732 facilitates FBXO22-mediated degradation of NSD2 in acute lymphoblastic leukemia cells harboring the NSD2 gain of function mutation p.E1099K, resulting in growth suppression, apoptosis, and reversal of drug resistance. The primary amine of UNC8732 is metabolized to an aldehyde species, which engages C326 of FBXO22 in a covalent and reversible manner to recruit the SCF FBXO22 Cullin complex. We further demonstrate that a previously reported alkyl amine-containing degrader targeting XIAP is similarly dependent on SCF FBXO22 . Overall, we present a highly potent NSD2 degrader for the exploration of NSD2 disease phenotypes and a novel FBXO22-dependent TPD strategy.
RESUMO
The structurally conserved B-cell lymphoma 2 (Bcl-2) family of protein function to promote or inhibit apoptosis through an exceedingly complex web of specific, intrafamilial protein-protein interactions. The critical role of these proteins in lymphomas and other cancers has motivated a widespread interest in understanding the molecular mechanisms that drive specificity in Bcl-2 family interactions. However, the high degree of structural similarity among Bcl-2 homologues has made it difficult to rationalize the highly specific (and often divergent) binding behavior exhibited by these proteins using conventional structural arguments. In this work, we use time-resolved hydrogen deuterium exchange mass spectrometry to explore shifts in conformational dynamics associated with binding partner engagement in the Bcl-2 family proteins Bcl-2 and Mcl-1. Using this approach combined with homology modeling, we reveal that Mcl-1 binding is driven by a large-scale shift in conformational dynamics, while Bcl-2 complexation occurs primarily through a classical charge compensation mechanism. This work has implications for understanding the evolution of internally regulated biological systems composed of structurally similar proteins and for the development of drugs targeting Bcl-2 family proteins for promotion of apoptosis in cancer.
Assuntos
Proteínas Reguladoras de Apoptose , Proteínas Proto-Oncogênicas c-bcl-2 , Proteínas Proto-Oncogênicas c-bcl-2/química , Proteína de Sequência 1 de Leucemia de Células Mieloides/química , Ligação Proteica , ApoptoseRESUMO
Biological macromolecules, such as proteins, nucleic acids, and carbohydrates, contain heteroatom-bonded hydrogens that undergo exchange with solvent hydrogens on timescales ranging from microseconds to hours. In hydrogen-deuterium exchange mass spectrometry (HDX-MS), this exchange process is used to extract information about biomolecular structure and dynamics. This minireview focuses on millisecond timescale HDX-MS measurements, which, while less common than 'conventional' timescale (seconds to hours) HDX-MS, provide a unique window into weakly structured species, weak (or fast cycling) binding interactions, and subtle shifts in conformational dynamics. This includes intrinsically disordered proteins and regions (IDPs/IDRs) that are associated with cancer and amyloidotic neurodegenerative disease. For nucleic acids and carbohydrates, structures such as isomers, stems, and loops, can be elucidated and overall structural rigidity can be assessed. We will provide a brief overview of technical developments in rapid HDX followed by highlights of various applications, emphasising the importance of broadening the HDX timescale to improve throughput and to capture a wider range of function-relevant dynamic and structural shifts.
Assuntos
Proteínas Intrinsicamente Desordenadas , Doenças Neurodegenerativas , Humanos , Deutério , Medição da Troca de Deutério/métodos , Hidrogênio/química , Proteínas Intrinsicamente Desordenadas/química , Conformação ProteicaRESUMO
Acetaminophen (APAP)-related toxicity is caused by the formation of N-acetyl p-benzoquinone imine (NAPQI), a reactive metabolite able to covalently bind to protein thiols. A targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, using multiple reaction monitoring (MRM), was developed to measure APAP binding on selected target proteins, including glutathione S-transferases (GSTs). In vitro incubations with CYP3A4 were performed to form APAP in the presence of different proteins, including four purified GST isozymes. A custom alkylation agent was used to prepare heavy labeled modified protein containing a structural isomer of APAP on all cysteine residues for isotope dilution. APAP incubations were spiked with heavy labeled protein, digested with either trypsin or pepsin, followed by peptide fractionation by HPLC prior to LC-MRM analysis. Relative site occupancy on the protein-level was used for comparing levels of modification of different sites in target proteins, after validation of protein and peptide-level relative quantitation using human serum albumin as a model system. In total, seven modification sites were quantified, namely Cys115 and 174 in GSTM2, Cys15, 48 and 170 in GSTP1, and Cys50 in human MGST1 and rat MGST1. In addition, APAP site occupancies of three proteins from liver microsomes were also quantified by using heavily labeled microsomes spiked into APAP microsomal incubations. A novel approach employing an isotope-labeled alkylation reagent was used to determine site occupancies on multiple protein thiols.
RESUMO
The success of bevacizumab (Avastin), a monoclonal antibody (mAb) anticancer drug targeting vascular endothelial growth factor A (VEGF-A), has motivated the development of biosimilars. Establishing target epitope similarity using epitope mapping is a critical step in preclinical mAb biosimilar development. Here we use time-resolved electrospray ionization hydrogen-deuterium exchange (HDX) mass spectrometry to rapidly compare the epitopes of commercial Avastin and a biosimilar in preclinical development (ApoBev) on an extended construct of VEGF-A. The Avastin and ApoBev epitopes determined in our experiments agree with each other and with the known epitope derived from the Avastin Fab domain/truncated VEGF co-crystal structure. However, subtly different allosteric effects observed exclusively at short (millisecond) HDX labeling times may reflect a slightly different binding mode for ApoBev.
Assuntos
Bevacizumab/imunologia , Medicamentos Biossimilares/farmacologia , Mapeamento de Epitopos , Espectrometria de Massa com Troca Hidrogênio-Deutério , Fator A de Crescimento do Endotélio Vascular/química , Humanos , Cinética , Microfluídica , Modelos MolecularesRESUMO
The dyshomeostasis of copper, iron and zinc ions in pathological conditions, which are critically involved in many brain activities, may result in an accumulation of them in the brain that has been reported for the patients with Alzheimer's disease. Conformational change is one of the consequences of metal-peptide interaction as we observed for the interaction of the Cu2+ with microtubule binding repeats of tau protein, which ultimately cause peptide aggregation. Herein, we show that interaction of Zn2+, Fe2+, and Fe3+ with full-length tau peptide R1 (tau244-274) and R4 (tau337-368), the first and fourth microtubule binding repeats of tau protein, lead to the conformational changes. And while the Electrospray ionization-mass spectrometry (ESI-MS) confirmed the complexation of Zn2+ and Fe2+ with both R1 and R4, there is no evidence for metalation of R1 or R4 with Fe3+.
Assuntos
Ferro/química , Microtúbulos/química , Zinco/química , Proteínas tau/química , Humanos , Sequências Repetitivas de AminoácidosRESUMO
Collision induced unfolding (CIU) is increasingly used to characterize protein complexes in the gas phase and is often employed to detect ligand binding-induced conformational stabilization. However, the extent to which gas-phase conformational stabilities measured by CIU reflect analogous parameters in solution is not yet clear, particularly for systems where conformational and protein complex stability are modulated by point mutation. Here, we compare CIU-derived relative stabilities of four point mutants of the homotetramer pyruvate kinase to solution stabilities measured by differential scanning fluorimetry (DSF) and solution conformational dynamics measured by time-resolved electrospray ionization hydrogen-deuterium exchange (TRESI-HDX). Our results demonstrate that both destabilization of the tetrameric state and generally reduced conformational stability of the monomer in solution are well correlated to lower onset energies for specific unfolding transitions observed in CIU. However, this correlation not fully retained when comparing CIU to HDX data, where the latter measurement is strongly impacted by conformational dynamics within the tetramer.
Assuntos
Proteínas de Escherichia coli/química , Escherichia coli/química , Piruvato Quinase/química , Medição da Troca de Deutério , Estabilidade Enzimática , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Fluorometria , Gases/química , Modelos Moleculares , Mutação Puntual , Conformação Proteica , Multimerização Proteica , Desdobramento de Proteína , Piruvato Quinase/genética , Soluções , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Lcn2 is a host defense protein induced via the innate immune response to sequester iron-loaded bacterial siderophores. However, excess or prolonged elevation of Lcn2 levels can induce adverse cellular effects, including oxidative stress and inflammation. In this work, we use Hydrogen-Deuterium eXchange (HDX) and Isothermal Titration Calorimetry (ITC) to characterize the binding interaction between Lcn2 and siderophores enterobactin and 2,3-DHBA, in the presence and absence of iron. Our results indicate a rare "Type II" interaction in which binding of siderophores drives the protein conformational equilibrium toward an unfolded state. Linking our molecular model to cellular assays, we demonstrate that this "distorted binding mode" facilitates a deleterious cellular accumulation of reactive oxygen species that could represent the molecular origin of Lcn2 pathology. These results add important insights into mechanisms of Lcn2 action and have implications in Lcn2-mediated effects including inflammation.
Assuntos
Anti-Infecciosos/química , Proteínas de Bactérias/química , Deutério/química , Lipocalina-2/química , Sideróforos/química , Anti-Infecciosos/metabolismo , Proteínas de Bactérias/metabolismo , Linhagem Celular , Relação Dose-Resposta a Droga , Descoberta de Drogas , Enterobactina/química , Humanos , Hidroxibenzoatos/química , Imunidade Inata/efeitos dos fármacos , Ferro/química , Cinética , Lipocalina-2/metabolismo , Simulação de Acoplamento Molecular , Ligação Proteica , Conformação Proteica , Espécies Reativas de Oxigênio/metabolismo , Sideróforos/metabolismo , Coloração e Rotulagem , Relação Estrutura-AtividadeRESUMO
Acetaminophen (APAP)-induced hepatotoxicity is the most common cause of acute liver failure in the Western world. APAP is bioactivated to N-acetyl p-benzoquinone imine (NAPQI), a reactive metabolite, which can subsequently covalently bind to glutathione and protein thiols. In this study, we have used liquid chromatography-tandem mass spectrometry (LC-MS/MS) to characterize NAPQI binding to human glutathione S-transferases (GSTs) in vitro. GSTs play a crucial role in the detoxification of reactive metabolites and therefore are interesting target proteins to study in the context of APAP covalent binding. Recombinantly-expressed and purified GSTs were used to assess NAPQI binding in vitro. APAP biotransformation to NAPQI was achieved using rat liver microsomes or human cytochrome P450 Supersomes in the presence of GSTA1, M1, M2, or P1. Resulting adducts were analyzed using bottom-up proteomics, with or without LC fractionation prior to LC-MS/MS analysis on a quadrupole-time-of-flight instrument with data-dependent acquisition (DDA). Targeted methods using multiple reaction monitoring (MRM) on a triple quadrupole platform were also developed by quantitatively labeling all available cysteine residues with a labeling reagent yielding isomerically-modified peptides following enzymatic digestion. Seven modified cysteine sites were confirmed, including Cys112 in GSTA1, Cys78 in GSTM1, Cys115 and 174 in GSTM2, as well as Cys15, 48, and 170 in GSTP1. Most modified peptides could be detected using both untargeted (DDA) and targeted (MRM) approaches, however the latter yielded better detection sensitivity with higher signal-to-noise and two sites were uniquely found by MRM.
RESUMO
The ongoing shift from small molecule drugs to protein therapeutics in the pharmaceuticals industry presents a considerable challenge to generic drug developers who are increasingly required to demonstrate biosimilarity for biological macromolecules, a task that is decidedly more complex than doing the same for small molecule drugs. In this work, we demonstrate a multipronged mass-spectrometry-based workflow that allows rapid and facile molecular characterization of antibody-based protein therapeutics, applied to biosimilars development. Specifically, we use a combination of native mass spectrometry (MS), ion mobility spectrometry (IMS), and global time-resolved hydrogen deuterium exchange (HDX) to provide an unambiguous assessment of the structural, dynamic, and chemical similarity between Avastin (bevacizumab) and a biosimilar in the late stages of pre-clinical development. Minor structural and dynamic differences between the biosimilar and Avastin, and between lots of the biosimilar, were tested for functional relevance using Surface Plasmon Resonance-derived kinetic and equilibrium binding parameters.
Assuntos
Antineoplásicos Imunológicos/química , Bevacizumab/química , Medicamentos Biossimilares/química , Espectrometria de Massa com Troca Hidrogênio-Deutério/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Antineoplásicos Imunológicos/farmacologia , Bevacizumab/farmacologia , Medicamentos Biossimilares/farmacologia , Desenho de Equipamento , Humanos , Espectrometria de Massa com Troca Hidrogênio-Deutério/economia , Espectrometria de Massa com Troca Hidrogênio-Deutério/instrumentação , Espectrometria de Mobilidade Iônica/economia , Espectrometria de Mobilidade Iônica/instrumentação , Espectrometria de Mobilidade Iônica/métodos , Espectrometria de Massas por Ionização por Electrospray/economia , Espectrometria de Massas por Ionização por Electrospray/instrumentação , Fatores de Tempo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Lipocalin-2 (Lcn2), a critical component of the innate immune response which binds siderophores and limits bacterial iron acquisition, can elicit spillover adverse proinflammatory effects. Here we show that holo-Lcn2 (Lcn2-siderophore-iron, 1:3:1) increases mitochondrial reactive oxygen species (ROS) generation and attenuates mitochondrial oxidative phosphorylation in adult rat primary cardiomyocytes in a manner blocked by N-acetyl-cysteine or the mitochondria-specific antioxidant SkQ1. We further demonstrate using siderophores 2,3-DHBA (2,3-dihydroxybenzoic acid) and 2,5-DHBA that increased ROS and reduction in oxidative phosphorylation are direct effects of the siderophore component of holo-Lcn2 and not due to apo-Lcn2 alone. Extracellular apo-Lcn2 enhanced the potency of 2,3-DHBA and 2,5-DHBA to increase ROS production and decrease mitochondrial respiratory capacity, whereas intracellular apo-Lcn2 attenuated these effects. These actions of holo-Lcn2 required an intact plasma membrane and were decreased by inhibition of endocytosis. The hearts, but not serum, of Lcn2 knockout (LKO) mice contained lower levels of 2,5-DHBA compared with wild-type hearts. Furthermore, LKO mice were protected from ischemia/reperfusion-induced cardiac mitochondrial dysfunction. Our study identifies the siderophore moiety of holo-Lcn2 as a regulator of cardiomyocyte mitochondrial bioenergetics.
Assuntos
Lipocalina-2/fisiologia , Mitocôndrias/patologia , Miócitos Cardíacos/patologia , Espécies Reativas de Oxigênio/metabolismo , Traumatismo por Reperfusão/patologia , Sideróforos/metabolismo , Animais , Gentisatos/farmacologia , Hidroxibenzoatos/farmacologia , Ferro/metabolismo , Masculino , Camundongos , Camundongos Knockout , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Fosforilação Oxidativa , Ratos , Ratos Wistar , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/metabolismoRESUMO
The incorporation of intrinsically disordered domains enables proteins to engage a wide variety of targets, with phosphorylation often modulating target specificity and affinity. Although phosphorylation can clearly act as a chemical driver of complexation in structured proteins, e.g., by abrogating or permitting new charge-charge interactions, the basis for enhancement of the hydrophobically driven interactions that are typical of disordered protein-target complexation is less clear. To determine how phosphorylation can positively impact target recruitment in disordered domains, we have examined the interaction between the disordered N-terminal transactivation domain (TAD) of p53 and the pleckstrin homology (PH) domain of p62. Using time-resolved electrospray ionization with hydrogen-deuterium exchange, we demonstrate that phosphorylation has little effect on the conformation of the p53 TAD when it is bound to the PH domain but instead increases the degree of conformational disorder in the unbound state. We propose that this increase in the degree of disorder creates a wider free energy gap between the free and bound states, providing a target-independent mechanism for enhanced binding when the phosphorylated and unphosphorylated p53-target complexes have similar free energies.
Assuntos
Proteína Supressora de Tumor p53/química , Medição da Troca de Deutério , Humanos , Domínios de Homologia à Plecstrina , Ligação Proteica , Estabilidade Proteica , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
The Signal Transducer and Activator of Transcription 3 (STAT3) oncogene is a master regulator of many human cancers, and a well-recognized target for therapeutic intervention. A well known STAT3 inhibitor, S3I-201 (NSC 74859), is hypothesized to block STAT3 function in cancer cells by binding the STAT3 SH2 domain and disrupt STAT3 protein complexation events. In this study, liquid chromatography tandem mass spectrometry analysis revealed that STAT3, in the presence of S3I-201, showed a minimum of five specific sites of modification, cysteine's 108, 259, 367, 542, and 687. Moreover, a prepared fluorescently labeled chemical probe of S3I-201 (DB-6-055) revealed that S3I-201 non-specifically and globally alkylated intracellular proteins at concentrations consistent with S3I-201's reported IC50. These data are consistent with the hypothesis that S3I-201 is a sub-optimal probe for interrogating STAT3-related cell biology.
Assuntos
Alquilantes/farmacologia , Benzenossulfonatos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Fator de Transcrição STAT3/antagonistas & inibidores , Ácidos Aminossalicílicos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Células Tumorais CultivadasRESUMO
Mutations in the BCR-ABL1 kinase domain are an established mechanism of tyrosine kinase inhibitor (TKI) resistance in Philadelphia chromosome-positive leukemia, but fail to explain many cases of clinical TKI failure. In contrast, it is largely unknown why some patients fail TKI therapy despite continued suppression of BCR-ABL1 kinase activity, a situation termed BCR-ABL1 kinase-independent TKI resistance. Here, we identified activation of signal transducer and activator of transcription 3 (STAT3) by extrinsic or intrinsic mechanisms as an essential feature of BCR-ABL1 kinase-independent TKI resistance. By combining synthetic chemistry, in vitro reporter assays, and molecular dynamics-guided rational inhibitor design and high-throughput screening, we discovered BP-5-087, a potent and selective STAT3 SH2 domain inhibitor that reduces STAT3 phosphorylation and nuclear transactivation. Computational simulations, fluorescence polarization assays and hydrogen-deuterium exchange assays establish direct engagement of STAT3 by BP-5-087 and provide a high-resolution view of the STAT3 SH2 domain/BP-5-087 interface. In primary cells from chronic myeloid leukemia (CML) patients with BCR-ABL1 kinase-independent TKI resistance, BP-5-087 (1.0 µM) restored TKI sensitivity to therapy-resistant CML progenitor cells, including leukemic stem cells. Our findings implicate STAT3 as a critical signaling node in BCR-ABL1 kinase-independent TKI resistance, and suggest that BP-5-087 has clinical utility for treating malignancies characterized by STAT3 activation.
Assuntos
Ácidos Aminossalicílicos/farmacologia , Proteínas de Fusão bcr-abl/genética , Regulação Leucêmica da Expressão Gênica , Leucócitos Mononucleares/efeitos dos fármacos , Células-Tronco Neoplásicas/efeitos dos fármacos , Fator de Transcrição STAT3/genética , Bibliotecas de Moléculas Pequenas/farmacologia , Sulfonamidas/farmacologia , Ácidos Aminossalicílicos/síntese química , Ácidos Aminossalicílicos/química , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Benzamidas/farmacologia , Linhagem Celular Tumoral , Dasatinibe , Descoberta de Drogas , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Proteínas de Fusão bcr-abl/metabolismo , Genes Reporter , Humanos , Mesilato de Imatinib , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/patologia , Luciferases/genética , Luciferases/metabolismo , Simulação de Acoplamento Molecular , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Fosforilação , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Estrutura Terciária de Proteína , Pirimidinas/farmacologia , Fator de Transcrição STAT3/antagonistas & inibidores , Fator de Transcrição STAT3/química , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Sulfonamidas/síntese química , Sulfonamidas/química , Tiazóis/farmacologiaRESUMO
The activity of the transcription factor signal transducer and activator of transcription 3 (STAT3) is dysregulated in a number of hematological and solid malignancies. Development of pharmacological STAT3 Src homology 2 (SH2) domain interaction inhibitors holds great promise for cancer therapy, and a novel class of salicylic acid-based STAT3 dimerization inhibitors that includes orally bioavailable drug candidates has been recently developed. The compounds SF-1-066 and BP-1-102 are predicted to bind to the STAT3 SH2 domain. However, given the highly unstructured and dynamic nature of the SH2 domain, experimental confirmation of this prediction was elusive. We have interrogated the protein-ligand interaction of STAT3 with these small molecule inhibitors by means of time-resolved electrospray ionization hydrogen-deuterium exchange mass spectrometry. Analysis of site-specific evolution of deuterium uptake induced by the complexation of STAT3 with SF-1-066 or BP-1-102 under physiological conditions enabled the mapping of the in silico predicted inhibitor binding site to the STAT3 SH2 domain. The binding of both inhibitors to the SH2 domain resulted in significant local decreases in dynamics, consistent with solvent exclusion at the inhibitor binding site and increased rigidity of the inhibitor-complexed SH2 domain. Interestingly, inhibitor binding induced hot spots of allosteric perturbations outside of the SH2 domain, manifesting mainly as increased deuterium uptake, in regions of STAT3 important for DNA binding and nuclear localization.
Assuntos
Simulação de Dinâmica Molecular , Fator de Transcrição STAT3/química , Fator de Transcrição STAT3/metabolismo , Domínios de Homologia de src , Sequência de Aminoácidos , Ácidos Aminossalicílicos/química , Ácidos Aminossalicílicos/metabolismo , Ácidos Aminossalicílicos/farmacologia , Animais , Sítios de Ligação , Medição da Troca de Deutério , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Ligação Proteica , Multimerização Proteica/efeitos dos fármacos , Fator de Transcrição STAT3/genética , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Espectrometria de Massas por Ionização por Electrospray , Sulfonamidas/química , Sulfonamidas/metabolismo , Sulfonamidas/farmacologiaRESUMO
Transthyretin (TTR) is a cerebrospinal fluid and serum protein that undergoes ordered aggregation (amyloidogenesis) in familial amyloidotic polyneuropathy (FAP) and senile systemic amyloidosis (SSA). It is now widely accepted that dissociation of the native TTR tetramer is a precondition for amyloidogenesis; thus, molecules that stabilize the tetramer have received much attention as potential TTR amyloidosis inhibitors. Many of these inhibitors bind to the thyroxine (T(4)) binding pocket and interact specifically with a section of the TTR sequence, corresponding to residues 105-115, that is implicated in amyloidogenic propensity. In this work, we study the effects of "stabilizing" inhibitors on ordered aggregation of TTR(105-115) peptide. We show that molecules known to bind full-length TTR at the T(4) site are potent, specific inhibitors of ordered aggregation, while molecules that do not interact with TTR exhibit milder, nonspecific disruption through a "hyperbundling" effect. Our results suggest that, in addition to annealing the native tetramer, "stabilizing" inhibitors may also directly disrupt amyloidogenic aggregation of TTR monomers through specific interactions with the exposed TTR(105-115) sequence.
Assuntos
Amiloide/química , Amiloidose/metabolismo , Pré-Albumina/química , Amiloide/metabolismo , Neuropatias Amiloides Familiares/metabolismo , Humanos , Cinética , Peptídeos/química , Peptídeos/metabolismo , Pré-Albumina/antagonistas & inibidores , Pré-Albumina/metabolismo , Dobramento de ProteínaRESUMO
Amyloidogenic aggregation has been the subject of intense research over the past few decades, but the mechanisms underlying the early stages of amyloidogenesis remain elusive. Here we demonstrate for the first time manipulation of amyloidogenesis based on an atomic level map of peptide-fibril interactions in early- and late-stage ordered aggregation. Several point mutants with specific amyloidogenic properties are introduced, including one that "stalls" early in the aggregation process, forming early-stage fibrillar aggregates, but not mature fibrils.
Assuntos
Peptídeos/química , Interações Medicamentosas , Peptídeos/genética , Mutação PuntualRESUMO
One of the central aims of amyloid research is to identify chemical and structural features that confer amyloidogenic propensity. In this study, we use Saturation Transfer Difference (STD) NMR spectroscopy to acquire an atom-specific map of the interactions between soluble and aggregated Transthyretin peptide (TTR(105-115)) in early- and late-stage amyloidogenesis. Atomic Force Microscopy (AFM) was used to monitor the transition of early-stage samples, containing protofilaments, to late-stage samples composed of fully-mature fibrils. Progressive aggregation was accompanied by an increase in the correlation time tau(c) of soluble TTR(105-115) as indicated by (1)H NMR line broadening, but no significant change in the (1)H chemical shifts. The STD profile of backbone amide protons is in good agreement with an earlier computational study predicting hydrogen bonding propensity for each residue in small TTR(105-115) aggregates (Paci et al., J. Mol. Biol. (2004) 555-569). The STD profiles of C(alpha) and C(beta) protons identify a central aliphatic region of the peptide, Ala108-Leu111, that plays a crucial, but different role in early- and late-stage amyloidogenesis. In general, the STD profiles of early and fully-mature samples are dissimilar, suggesting different mechanisms of self-assembly in protofilaments and mature amyloid fibrils. The early-stage mechanism appears to be more dependent on main-chain hydrogen bonding, while the late-stage mechanism involves an increased number of interactions between bulky side chains.
Assuntos
Amiloide/química , Pré-Albumina/química , Ligação de Hidrogênio , Espectroscopia de Ressonância Magnética , Microscopia de Força Atômica , Peptídeos/química , Ligação ProteicaRESUMO
It remains an open question as to whether experiments involving collision-induced dissociation (CID) can provide a viable approach for monitoring spatially resolved deuteration levels in electrosprayed polypeptide ions. A number of laboratories reported the successful application of CID following solution-phase H/D exchange (HDX), whereas others found that H/D scrambling precluded site-specific measurements. The aim of the current work is to help clarify the general feasibility of HDX-CID methods, using a 22-residue zinc-bound protein domain (Zn-ZBD) as model system. Metal binding in Zn-ZBD should confer structural rigidity, and the presence of several basic residues should sequester mobile charge carriers in the gas phase. Both of these factors were expected to suppress the extent of scrambling. HDX was carried out by employing rapid on-line mixing, thereby mimicking conditions typically encountered in kinetic pulse-labeling studies. Quadrupole time-of-flight MS/MS of pulse-labeled Zn-ZBD provides high sequence coverage. However, the measured fragment deuteration levels do not correlate with the known H-bonding pattern of Zn-ZBD, suggesting the occurrence of extensive scrambling. Instead of showing a uniform distribution, the fragment ions reveal a distinct nonrandom pattern of deuteration levels. In the absence of prior information, these data could erroneously be ascribed to the presence of protected sites. However, the observed patterns clearly originate from other factors; possibly they are caused by modulations of the amide CID efficiency by kinetic isotope effects. It is concluded that scrambling does not represent the only conceptual problem in HDX-CID studies and that control experiments on uniformly labeled samples are essential for ruling out interpretation artifacts.