RESUMO
MitoNEET is a [2Fe-2S] redox active mitochondrial protein belonging to the CDGSH iron-sulfur domain (CISD) family of proteins. MitoNEET has been implicated as a potential target for drug development to treat various disorders, including type-2 diabetes, cancer, and Parkinson's disease. However, the specific cellular function(s) for mitoNEET still remains to be fully elucidated, and this presents a significant roadblock in rational drug development. Here, we show that mitoNEET binds the enzymatic cofactor pyridoxal phosphate (PLP) specifically at only one of its 11 lysine residues, Lys55. Lys55 is part of the soluble portion of the protein and is in a hydrogen-bonding network with the histidine residue that ligates the [2Fe-2S] cluster. In the presence of mitoNEET, PLP catalyzes the transamination reaction of the amino acid cysteine and the alpha-keto acid 2-oxoglutarate to form 3-mercaptopyruvate and glutamate. This work identifies, for the first time, mitoNEET as an enzyme with cysteine transaminase activity.
Assuntos
Proteínas Ferro-Enxofre , Proteínas Ferro-Enxofre/química , Fosfato de Piridoxal/metabolismo , Histidina , Cisteína , Transaminases/metabolismo , Ácidos Cetoglutáricos , Lisina , Proteínas Mitocondriais/metabolismo , Ferro/metabolismo , Enxofre , Glutamatos , Hidrogênio/metabolismoRESUMO
Antibody-based therapeutics are the fastest-growing drug class on the market, used to treat aggressive forms of cancer, chronic autoimmune conditions, and numerous other disease states. Although the specificity, affinity, and versatility of therapeutic antibodies can provide an advantage over traditional small-molecule drugs, their development and optimization can be much more challenging and time-consuming. This is, in part, because the ideal formulation buffer systems used for in vitro characterization inadequately reflect the crowded biological environments (serum, endosomal lumen, etc.) that these drugs experience once administered to a patient. Such environments can perturb the binding of antibodies to their antigens and receptors, as well as homo- and hetero-aggregation, thereby altering therapeutic effect and disposition in ways that are incompletely understood. Although excluded volume effects are classically thought to favor binding, weak interactions with co-solutes in crowded conditions can inhibit binding. The second virial coefficient (B2) parameter quantifies such weak interactions and can be determined by a variety of techniques in dilute solution, but analogous methods in complex biological fluids are not well established. Here, we demonstrate that fluorescence correlation spectroscopy is able to measure diffusive B2-values directly in undiluted serum. Apparent second virial coefficient (B2,app) measurements of antibodies in serum reveal that changes in the balance between attractive and repulsive interactions can dramatically impact global nonideality. Furthermore, our findings suggest that the approach of isolating specific components and completing independent cross-term virial coefficient measurements may not be an effective approach to characterizing nonideality in serum. The approach presented here could enrich our understanding of the effects of biological environments on proteins in general and advance the development of therapeutic antibodies and other protein-based therapeutics.
Assuntos
Proteínas , Difusão , Humanos , SoluçõesRESUMO
Protein/lipid coassembly is an understudied phenomenon that is important to the function of antimicrobial peptides as well as the pathological effects of amyloid. Here, we study the coassembly process of PAP248-286, a seminal peptide that displays both amyloid-forming and antimicrobial activity. PAP248-286 is a peptide fragment of prostatic acid phosphatase and has been reported to form amyloid fibrils, known as semen-derived enhancer of viral infection (SEVI), that enhance the viral infectivity of human immunodeficiency virus. We find that in addition to forming amyloid, PAP248-286 much more readily assembles with lipid vesicles into peptide/lipid coaggregates that resemble amyloid fibrils in some important ways but are a distinct species. The formation of these PAP248-286/lipid coaggregates, which we term "messicles," is controlled by the peptide:lipid (P:L) ratio and by the lipid composition. The optimal P:L ratio is around 1:10, and at least 70% anionic lipid is required for coaggregate formation. Once formed, messicles are not disrupted by subsequent changes in P:L ratio. We propose that messicles form through a polyvalent assembly mechanism, in which a critical surface density of PAP248-286 on liposomes enables peptide-mediated particle bridging into larger species. Even at â¼50-fold lower PAP248-286 concentrations, messicles form at least 10-fold faster than amyloid fibrils. It is therefore possible that some or all of the biological activities assigned to SEVI, the amyloid form of PAP248-286, could instead be attributed to a PAP248-286/lipid coaggregate. More broadly speaking, this work could provide a potential framework for the discovery and characterization of nonamyloid peptide/lipid coaggregates by other amyloid-forming proteins and antimicrobial peptides.
Assuntos
HIV-1 , Fosfatase Ácida , Amiloide , Humanos , Lipídeos , Peptídeos , SêmenRESUMO
CYP4Z1 is an "orphan" cytochrome P450 (P450) enzyme that has provoked interest because of its hypothesized role in breast cancer through formation of the signaling molecule 20-hydroxyeicosatetraenoic acid (20-HETE). We expressed human CYP4Z1 in Saccharomyces cerevisiae and evaluated its catalytic capabilities toward arachidonic and lauric acids (AA and LA). Specific and sensitive mass spectrometry assays enabled discrimination of the regioselectivity of hydroxylation of these two fatty acids. CYP4Z1 generated 7-, 8-, 9-, 10-, and 11-hydroxy LA, whereas the 12-hydroxy metabolite was not detected. HET0016, the prototypic CYP4 inhibitor, only weakly inhibited laurate metabolite formation (IC50 â¼15 µM). CYP4Z1 preferentially oxidized AA to the 14(S),15(R)-epoxide with high regioselectivity and stereoselectivity, a reaction that was also insensitive to HET0016, but neither 20-HETE nor 20-carboxy-AA were detectable metabolites. Docking of LA and AA into a CYP4Z1 homology model was consistent with this preference for internal fatty acid oxidation. Thus, human CYP4Z1 has an inhibitor profile and product regioselectivity distinct from most other CYP4 enzymes, consistent with CYP4Z1's lack of a covalently linked heme. These data suggest that, if CYP4Z1 modulates breast cancer progression, it does so by a mechanism other than direct production of 20-HETE.
Assuntos
Neoplasias da Mama/metabolismo , Família 4 do Citocromo P450/metabolismo , Ácidos Hidroxieicosatetraenoicos/metabolismo , Ácidos Láuricos/metabolismo , Amidinas/farmacologia , Família 4 do Citocromo P450/antagonistas & inibidores , Família 4 do Citocromo P450/química , Família 4 do Citocromo P450/isolamento & purificação , Progressão da Doença , Humanos , Hidroxilação/efeitos dos fármacos , Quinases Associadas a Receptores de Interleucina-1 , Espectrometria de Massas , Microssomos Hepáticos , Simulação de Acoplamento Molecular , Oxirredução/efeitos dos fármacos , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiaeRESUMO
The ATP binding cassette transporter P-glycoprotein (ABCB1 or P-gp) plays a major role in cellular resistance to drugs and drug interactions. Experimental studies support a mechanism with nucleotide-dependent fluctuation between inward-facing and outward-facing conformations, which are coupled to nucleotide hydrolysis. However, detailed insight into drug-dependent modulation of these conformational ensembles is lacking. Different drugs likely occupy partially overlapping but distinct sites and are therefore variably coupled to nucleotide binding and hydrolysis. Many fluorescent drug analogues are used in cell-based transport models; however, their specific interactions with P-gp have not been studied, and this limits interpretation of transport assays in terms of molecular models. Here we monitor binding of the fluorescent probe substrates BODIPY-verapamil, BODIPY-vinblastine, and Flutax-2 at low occupancy to murine P-gp in lipid nanodiscs via fluorescence correlation spectroscopy, in variable nucleotide-bound states. Changes in affinity for the different nucleotide-dependent conformations are probe-dependent. For BODIPY-verapamil and BODIPY-vinblastine, there are 2-10-fold increases in KD in the nucleotide-bound or vanadate-trapped state, compared to that in the nucleotide-free state. In contrast, the affinity of Flutax-2 is unaffected by nucleotide or vanadate trapping. In further contrast to BODIPY-verapamil and BODIPY-vinblastine, Flutax-2 does not cause stimulation of ATP hydrolysis despite the fact that it is transported in vesicle-based transport assays. Whereas the established substrates verapamil, paclitaxel, and vinblastine displace BODIPY-verapamil or BODIPY-vinblastine from their high-affinity sites, the transport substrate Flutax-2 is not displaced by any of these substrates. The results demonstrate a unique binding site for Flutax-2 that allows for transport without stimulation of ATP hydrolysis.
Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Bicamadas Lipídicas/química , Modelos Moleculares , Subfamília B de Transportador de Cassetes de Ligação de ATP/química , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Trifosfato de Adenosina/metabolismo , Animais , Sítios de Ligação , Ligação Competitiva , Transporte Biológico , Compostos de Boro/metabolismo , Dimiristoilfosfatidilcolina/química , Corantes Fluorescentes/metabolismo , Humanos , Hidrólise , Cinética , Ligantes , Camundongos , Nanoestruturas/química , Conformação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Taxoides/metabolismo , Verapamil/análogos & derivados , Verapamil/metabolismo , Vimblastina/análogos & derivados , Vimblastina/metabolismoRESUMO
Bupropion is a widely used antidepressant and smoking cessation aid and a strong inhibitor of CYP2D6 in vivo. Bupropion is administered as a racemic mixture of R- and S-bupropion and has stereoselective pharmacokinetics. Four primary metabolites of bupropion, threo- and erythro-hydrobupropion and R,R- and S,S-OH-bupropion, circulate at higher concentrations than the parent drug and are believed to contribute to the efficacy and side effects of bupropion as well as to the CYP2D6 inhibition. However, bupropion and its metabolites are only weak inhibitors of CYP2D6 in vitro, and the magnitude of the in vivo drug-drug interactions (DDI) caused by bupropion cannot be explained by the in vitro data even when CYP2D6 inhibition by the metabolites is accounted for. The aim of this study was to quantitatively explain the in vivo CYP2D6 DDI magnitude by in vitro DDI data. Bupropion and its metabolites were found to inhibit CYP2D6 stereoselectively with up to 10-fold difference in inhibition potency between enantiomers. However, the reversible inhibition or active uptake into hepatocytes did not explain the in vivo DDIs. In HepG2 cells and in plated human hepatocytes bupropion and its metabolites were found to significantly downregulate CYP2D6 mRNA in a concentration dependent manner. The in vivo DDI was quantitatively predicted by significant down-regulation of CYP2D6 mRNA and reversible inhibition of CYP2D6 by bupropion and its metabolites. This study is the first example of a clinical DDI resulting from CYP down-regulation and first demonstration of a CYP2D6 interaction resulting from transcriptional regulation.
Assuntos
Bupropiona/farmacologia , Citocromo P-450 CYP2D6/metabolismo , Bupropiona/metabolismo , Cromatografia Líquida , Regulação para Baixo , Interações Medicamentosas , Células Hep G2 , Humanos , Técnicas In Vitro , Abandono do Hábito de Fumar , Espectrometria de Massas em TandemRESUMO
Islet amyloid polypeptide (IAPP) is a peptide hormone whose pathological self-assembly is a hallmark of the progression of type II diabetes. IAPP-membrane interactions catalyze its higher-order self-assembly and also underlie its toxic effects toward cells. While there is great interest in developing small molecule reagents capable of altering the structure and behavior of oligomeric, membrane-bound IAPP, the dynamic and heterogeneous nature of this ensemble makes it recalcitrant to traditional approaches. Here, we build on recent insights into the nature of membrane-bound states and develop a combined computational and experimental strategy to address this problem. The generalized structural approach efficiently identified diverse compounds from large commercial libraries with previously unrecognized activities toward the gain-of-function behaviors of IAPP. The use of appropriate computational prescreening reduced the experimental burden by orders of magnitude relative to unbiased high-throughput screening. We found that rationally targeting experimentally derived models of membrane-bound dimers identified several compounds that demonstrate the remarkable ability to enhance IAPP-membrane binding and one compound that enhances IAPP-mediated cytotoxicity. Taken together, these findings imply that membrane binding per se is insufficient to generate cytotoxicity; instead, enhanced sampling of rare states within the membrane-bound ensemble may potentiate IAPP's toxic effects.
Assuntos
Membrana Celular/metabolismo , Citotoxinas/toxicidade , Polipeptídeo Amiloide das Ilhotas Pancreáticas/toxicidade , Linhagem Celular Tumoral , Citotoxinas/química , Citotoxinas/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Humanos , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Ligação Proteica , Estrutura Quaternária de Proteína , Relação Estrutura-AtividadeRESUMO
Islet amyloid polypeptide (IAPP) is a hormone cosecreted with insulin by pancreatic ß cells. Upon contact with lipid bilayers, it is stabilized into a heterogeneous ensemble of structural states. These processes are associated with gains of function, including catalysis of ß sheet-rich amyloid formation, cell membrane penetration, loss of membrane integrity, and cytotoxicity. These contribute to the dysfunction of ß cells, a central component in the pathology and treatment of diabetes. To gain mechanistic insight into these phenomena, a related series of substituted oligoquinolines were designed. These inhibitors are unique in that they have the capacity to affect both solution- and phospholipid bilayer-catalyzed IAPP self-assembly. Importantly, we show that this activity is associated with the oligoquinoline's capacity to irreversibly adopt a noncovalent fold. This suggests that compact foldamer scaffolds, such as oligoquinoline, are an important paradigm for conformational manipulation of disordered protein state.
Assuntos
Polipeptídeo Amiloide das Ilhotas Pancreáticas/antagonistas & inibidores , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Quinolinas/química , Quinolinas/farmacologia , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Cinética , Modelos Moleculares , Conformação Molecular , Tamanho da Partícula , Propriedades de SuperfícieRESUMO
Several widespread and severe degenerative diseases are characterized by the deposition of amyloid protein aggregates in affected tissues. While there is great interest in the complete description of the aggregation pathway of the proteins involved, a molecular level understanding is hindered by the complexity of the self-assembly process. In particular, the early stages of aggregation, where dynamic, heterogeneous and often toxic intermediates are populated, are resistant to high-resolution structural characterization. Fluorescence spectroscopy is a powerful and versatile tool for such analysis. In this review, we survey its application to provide residue-specific information about amyloid intermediate states for three selected proteins: IAPP, α-synuclein, and tau.
Assuntos
Amiloide/química , Transferência Ressonante de Energia de Fluorescência/métodos , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , alfa-Sinucleína/química , Proteínas tau/química , Amiloide/metabolismo , Humanos , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Modelos Moleculares , Conformação Proteica , Estrutura Secundária de Proteína , Termodinâmica , alfa-Sinucleína/metabolismo , Proteínas tau/metabolismoRESUMO
Nanodiscs are a new class of model membranes that are being used to solubilize and study a range of integral membrane proteins and membrane-associated proteins. Unlike other model membranes, the Nanodisc bilayer is bounded by a scaffold protein coat that confers enhanced stability and a narrow particle size distribution. The bilayer diameter can be precisely controlled by changing the diameter of the protein coat. All these properties make Nanodiscs excellent model membranes for single-molecule fluorescence applications. In this chapter, we describe our work using Nanodiscs to apply total internal reflection fluorescence microscopy (TIRFM), fluorescence correlation spectroscopy (FCS), and Förster resonance energy transfer (FRET) to study the integral membrane protein cytochrome P450 3A4 and the peripheral membrane-binding proteins islet amyloid polypeptide (IAPP) and alpha-synuclein, respectively. The monodisperse size distribution of Nanodiscs enhances control over the oligomeric state of the membrane protein of interest, and facilitates accurate solution-based measurements as well. Nanodiscs also comprise an excellent system to stably immobilize integral membrane proteins in a bilayer without covalent modification, enabling a range of surface-based experiments where accurate localization of the protein of interest is required.
Assuntos
Bicamadas Lipídicas/química , Nanoestruturas/química , Fosfolipídeos/química , Espectrometria de Fluorescência , Amiloide/química , Amiloide/metabolismo , Citocromo P-450 CYP3A/química , Citocromo P-450 CYP3A/metabolismo , Humanos , Processamento de Imagem Assistida por Computador , Polipeptídeo Amiloide das Ilhotas Pancreáticas , Lipoproteínas HDL/química , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Modelos Moleculares , Conformação Molecular , Tamanho da Partícula , Ligação Proteica , Espectrometria de Fluorescência/instrumentação , Espectrometria de Fluorescência/métodos , Propriedades de Superfície , alfa-Sinucleína/química , alfa-Sinucleína/metabolismoRESUMO
Human c-myc is critical for cell homeostasis and growth but is a potent oncogenic factor if improperly regulated. The c-myc far-upstream element (FUSE) melts into single-stranded DNA upon active transcription, and the noncoding strand FUSE recruits an activator [the FUSE-binding protein (FBP)] and a repressor [the FBP-interacting repressor (FIR)] to fine-tune c-myc transcription in a real-time manner. Despite detailed biological experiments describing this unique mode of transcriptional regulation, quantitative measurements of the physical constants regulating the protein-DNA interactions remain lacking. Here, we first demonstrate that the two FUSE strands adopt different conformations upon melting, with the noncoding strand DNA in an extended, linear form. FBP binds to the linear noncoding FUSE with a dissociation constant in the nanomolar range. FIR binds to FUSE more weakly, having its modest dissociation constants in the low micromolar range. FIR is monomeric under near-physiological conditions but upon binding of FUSE dimerizes into a 2:1 FIR(2)-FUSE complex mediated by the RRMs. In the tripartite interaction, our analysis suggests a stepwise addition of FIR onto an activating FBP-FUSE complex to form a quaternary FIR(2)-FBP-FUSE inhibitory complex. Our quantitative characterization enhances understanding of DNA strand preference and the mechanism of the stepwise complex formation in the FUSE-FBP-FIR regulatory system.
Assuntos
Proteínas de Transporte/metabolismo , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Repressoras/metabolismo , Transativadores/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Proteínas de Transporte/química , Proteínas de Transporte/fisiologia , DNA Helicases/química , DNA Helicases/fisiologia , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/fisiologia , Dimerização , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Ligação Proteica , Proteínas Proto-Oncogênicas c-myc/química , Proteínas Proto-Oncogênicas c-myc/fisiologia , Fatores de Processamento de RNA , Proteínas de Ligação a RNA , Proteínas Repressoras/química , Proteínas Repressoras/fisiologia , Fatores de Troca de Nucleotídeo Guanina Rho , Soluções , Transativadores/química , Transativadores/fisiologiaRESUMO
Catalytic promiscuity is a widespread, but poorly understood, phenomenon among enzymes with particular relevance to the evolution of new functions, drug metabolism, and in vitro biocatalyst engineering. However, there is at present no way to quantitatively measure or compare this important parameter of enzyme function. Here we define a quantitative index of promiscuity (I) that can be calculated from the catalytic efficiencies of an enzyme toward a defined set of substrates. A weighted promiscuity index (J) that accounts for patterns of similarity and dissimilarity among the substrates in the set is also defined. Promiscuity indices were calculated for three different enzyme classes: eight serine and cysteine proteases, two glutathione S-transferase (GST) isoforms, and three cytochrome P450 (CYP) isoforms. The proteases ranged from completely specific (granzyme B, J = 0.00) to highly promiscuous (cruzain, J = 0.83). The four drug-metabolizing enzymes studied (GST A1-1 and the CYP isoforms) were highly promiscuous, with J values between 0.72 and 0.92; GST A4-4, involved in the clearance of lipid peroxidation products, is moderately promiscuous (J = 0.37). Promiscuity indices also allowed for studies of correlation between substrate promiscuity and an enzyme's activity toward its most-favored substrate, for each of the three enzyme classes.