RESUMO
Recently a report by Ruan et al. in Nature described how relatively simple random heteropolymers can replicate the properties of biological fluids. These polymers capture the segmental-level interactions between proteins and could enhance folding of membrane proteins, improve stability, and enable DNA sequestration in a chemistry specific manner.
Assuntos
Polímeros , Proteínas , Polímeros/química , Proteínas/química , DNARESUMO
The circadian clock is a timekeeping, homeostatic system that temporally coordinates all major cellular processes. The function of the circadian clock is compensated in the face of variable environmental conditions ranging from normal to stress-inducing conditions. Salinity is a critical environmental factor affecting plant growth, and plants have evolved the SALT OVERLY SENSITIVE (SOS) pathway to acquire halotolerance. However, the regulatory systems for clock compensation under salinity are unclear. Here, we show that the plasma membrane Na+/H+ antiporter SOS1 specifically functions as a salt-specific circadian clock regulator via GIGANTEA (GI) in Arabidopsis thaliana. SOS1 directly interacts with GI in a salt-dependent manner and stabilizes this protein to sustain a proper clock period under salinity conditions. SOS1 function in circadian clock regulation requires the salt-mediated secondary messengers cytosolic free calcium and reactive oxygen species, pointing to a distinct regulatory role for SOS1 in addition to its function as a transporter to maintain Na+ homeostasis. Our results demonstrate that SOS1 maintains homeostasis of the salt response under high or daily fluctuating salt levels. These findings highlight the genetic capacity of the circadian clock to maintain timekeeping activity over a broad range of salinity levels.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Ritmo Circadiano , Estresse Salino , Trocadores de Sódio-Hidrogênio , Arabidopsis/genética , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Estabilidade Proteica , Trocadores de Sódio-Hidrogênio/genética , Trocadores de Sódio-Hidrogênio/metabolismoRESUMO
Most multicellular organisms are freeze sensitive, but the ability to survive freezing of the extracellular fluids evolved in several vertebrate ectotherms, some plants, and many insects. Here, we test the coupled hypotheses that are perpetuated in the literature: that irreversible denaturation of proteins and loss of biological membrane integrity are two ultimate molecular mechanisms of freezing injury in freeze-sensitive insects and that seasonally accumulated small cryoprotective molecules (CPs) stabilize proteins and membranes against injury in freeze-tolerant insects. Using the drosophilid fly, Chymomyza costata, we show that seven different soluble enzymes exhibit no or only partial loss of activity upon lethal freezing stress applied in vivo to whole freeze-sensitive larvae. In contrast, the enzymes lost activity when extracted and frozen in vitro in a diluted buffer solution. This loss of activity was fully prevented by adding low concentrations of a wide array of different compounds to the buffer, including C. costata native CPs, other metabolites, bovine serum albumin (BSA), and even the biologically inert artificial compounds HistoDenz and Ficoll. Next, we show that fat body plasma membranes lose integrity when frozen in vivo in freeze-sensitive but not in freeze-tolerant larvae. Freezing fat body cells in vitro, however, resulted in loss of membrane integrity in both freeze-sensitive and freeze-tolerant larvae. Different additives showed widely different capacities to protect membrane integrity when added to in vitro freezing media. A complete rescue of membrane integrity in freeze-tolerant larvae was observed with a mixture of proline, trehalose, and BSA.
Assuntos
Soroalbumina Bovina , Trealose , Aclimatação , Animais , Membrana Celular/metabolismo , Crioprotetores/farmacologia , Ficoll , Congelamento , Insetos/metabolismo , Larva/metabolismo , Prolina/metabolismoRESUMO
Deubiquitinase-targeting chimera (DUBTAC) is a promising technology for inducing targeted protein stabilization (TPS). Despite its therapeutic potential, very few proteins have been stabilized by DUBTACs to date. The limited applicability of this technology is likely due to the modest DUBTAC-induced protein stabilization effect, and the scarcity of effective deubiquitinase ligands that can be harnessed for DUBTAC development. Here, we report the discovery of MS7829 and MS8588, the first-in-class DUBTACs of cGAS, a key component of the cGAS-STING pathway. While these DUBTACs are based on a cGAS inhibitor, they effectively stabilized cGAS and activated the cGAS/STING/IRF3 signaling. To develop these cGAS DUBTACs, we optimized EN523, an OTUB1 covalent ligand, into an improved ligand, MS5105. We validated MS5105 by generating a MS5105-based CFTR DUBTAC, which was approximately 10-fold more effective in stabilizing the ΔF508-CFTR mutant protein than the previously reported EN523-based CFTR DUBTAC. Overall, this work advances the DUBTAC technology for TPS.
RESUMO
Current antibody (Ab) therapies require development of stable formulations and an optimal delivery system. Here, we present a new strategy to create a single-administration long-lasting Ab-delivery microarray (MA) patch, which can carry high doses of thermally stabilized Abs. The MA fabricated by an additive three-dimensional manufacturing technology can be fully embedded into the skin via a single application to deliver doses of Abs at multiple programmable time points, thus sustaining Ab concentrations in systemic circulation. We developed an MA formulation that stabilized and delivered human immunoglobulins (hIg) in a time-controlled manner while maintaining their structure and functionality. As an example, the b12 Abâa broadly neutralizing Ab against HIV-1âmaintained antiviral activity in vitro after MA manufacturing and heat exposure. Pharmacokinetic studies of MA patch-delivered hIg in rats successfully provided a proof of concept for concurrent and time-delayed Ab delivery. These MA patches codeliver different Abs, providing a tool for expanded protection against viral infections or combination HIV therapy and prevention.
Assuntos
Anticorpos , Infecções por HIV , Humanos , Ratos , Animais , Pele , Infecções por HIV/tratamento farmacológico , Infecções por HIV/prevenção & controleRESUMO
The PARP1 (Poly (ADP-ribose) polymerase 1) enzyme is essential for single and double-strand break repair in humans. Alterations affecting PARP1 activity have severe consequences for human health and are associated with pathologies like cancer, and metabolic and neurodegenerative disorders. Here, we have developed a fast and easy procedure for the expression and purification of PARP1. Biologically active protein was purified to an apparent purity > 95%, with only two purification steps. A thermostability analysis revealed that PARP1 possessed improved stability in 50 mM Tris-HCl pH 8.0 (Tm = 44.2 ± 0.3 °C), thus this buffer was used throughout the whole purification procedure. The protein was shown to bind to DNA and has no inhibitor molecules bound to the active site. Finally, the yield of the purified PARP1 protein is sufficient for both biochemical, biophysical and structural analysis. The new protocol provides a fast and simple purification procedure while producing similar protein quantities to what has been described previously.
Assuntos
Reparo do DNA , DNA , Humanos , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , DNA/químicaRESUMO
G protein-coupled receptors (GPCRs) are an important family of membrane proteins responsible for many physiological functions in human body. High resolution GPCR structures are required to understand their molecular mechanisms and perform rational drug design, as GPCRs play a crucial role in a variety of diseases. That is difficult to obtain for the wild-type proteins because of their low stability. In this review, we discuss how this problem can be solved by using protein design strategies developed to obtain homogeneous stabilized GPCR samples for crystallization and cryoelectron microscopy.
Assuntos
Proteínas de Membrana , Receptores Acoplados a Proteínas G , Humanos , Microscopia Crioeletrônica , Modelos Moleculares , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , CristalizaçãoRESUMO
Detergents are widely used for membrane protein structural study. Many recently developed detergents contain multiple tail and head groups, which are typically connected via a small and branched linker. Due to their inherent compact structures, with small inter-alkyl chain distances, these detergents form micelles with high alkyl chain density in the interiors, a feature favorably associated with membrane-protein stability. A recent study on tandem triazine maltosides (TZMs) revealed a distinct trend; despite possession of an apparently large inter-alkyl chain distance, the TZM-Es were highly effective at stabilizing membrane proteins. Thanks to the incorporation of a flexible spacer between the two triazine rings in the linker region, these detergents are prone to folding into a compact architecture in micellar environments instead of adopting an extended conformation. Detergent foldability represents a new concept of novel detergent design with significant potential for future detergent development.
Assuntos
Detergentes , Proteínas de Membrana , Detergentes/química , Proteínas de Membrana/química , Micelas , Estabilidade Proteica , TriazinasRESUMO
Integral membrane proteins pose considerable challenges to high resolution structural analysis. Maintaining membrane proteins in their native state during protein isolation is essential for structural study of these bio-macromolecules. Detergents are the most commonly used amphiphilic compounds for stabilizing membrane proteins in solution outside a lipid bilayer. We previously introduced a glyco-diosgenin (GDN) detergent that was shown to be highly effective at stabilizing a wide range of membrane proteins. This steroidal detergent has additionally gained attention due to its compatibility with membrane protein structure study via cryo-EM. However, synthetic inconvenience limits widespread use of GDN in membrane protein study. To improve its synthetic accessibility and to further enhance detergent efficacy for protein stabilization, we designed a new class of glyco-steroid-based detergents using three steroid units: cholestanol, cholesterol and diosgenin. These new detergents were efficiently prepared and showed marked efficacy for protein stabilization in evaluation with a few model membrane proteins including two G protein-coupled receptors. Some new agents were not only superior to a gold standard detergent, DDM (n-dodecyl-ß-d-maltoside), but were also more effective than the original GDN at preserving protein integrity long term. These agents represent valuable alternatives to GDN, and are likely to facilitate structural determination of challenging membrane proteins.
Assuntos
Detergentes , Proteínas de Membrana , Detergentes/química , Interações Hidrofóbicas e Hidrofílicas , Proteínas de Membrana/química , Estabilidade Proteica , EsteroidesRESUMO
Small ubiquitin-like modifier (SUMO) conjugation (SUMOylation) is a reversible post-translational modification associated with protein stability and activity, and modulates hormone signaling and stress responses in plants. Previously, we reported that the pepper dehydration-responsive homeobox domain transcription factor CaDRHB1 acts as a positive modulator of drought response. Here, we show that CaDRHB1 protein stability is enhanced by SUMO E3 ligase Capsicum annuum DRHB1-interacting SAP and Miz domain (SIZ1) (CaDSIZ1)-mediated SUMOylation in response to drought, thereby positively modulating abscisic acid (ABA) signaling and drought responses. Substituting lysine (K) 138 of CaDRHB1 with arginine reduced CaDSIZ1-mediated SUMOylation, indicating that K138 is the principal site for SUMO conjugation. Virus-induced silencing of CaDSIZ1 promoted CaDRHB1 degradation, suggesting that CaDSIZ1 is involved in drought-induced SUMOylation of CaDRHB1. CaDSIZ1 interacted with and facilitated SUMO conjugation of CaDRHB1. CaDRHB1, mainly localized in the nucleus, but also in the cytoplasm in the SUMOylation mimic state, suggesting that SUMOylation of CaDRHB1 promotes its nuclear export, leading to cytoplasmic accumulation. Moreover, CaDSIZ1-silenced pepper plants were less sensitive to ABA and considerably sensitive to drought stress, whereas CaDSIZ1-overexpressing plants displayed ABA-hypersensitive and drought-tolerant phenotypes. Collectively, our data indicate that CaDSIZ1-mediated SUMOylation of CaDRHB1 functions in ABA-mediated drought tolerance.
Assuntos
Arabidopsis , Ubiquitina-Proteína Ligases , Ácido Abscísico/metabolismo , Ácido Abscísico/farmacologia , Arabidopsis/genética , Secas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Estresse Fisiológico , Fatores de Transcrição/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Membrane proteins are of biological and pharmaceutical significance. However, their structural study is extremely challenging mainly due to the fact that only a small number of chemical tools are suitable for stabilizing membrane proteins in solution. Detergents are widely used in membrane protein study, but conventional detergents are generally poor at stabilizing challenging membrane proteins such as G protein-coupled receptors and protein complexes. In the current study, we prepared tandem triazine-based maltosides (TZMs) with two amphiphilic triazine units connected by different diamine linkers, hydrazine (TZM-Hs) and 1,2-ethylenediamine (TZM-Es). These TZMs were consistently superior to a gold standard detergent (DDM) in terms of stabilizing a few membrane proteins. In addition, the TZM-Es containing a long linker showed more general protein stabilization efficacy with multiple membrane proteins than the TZM-Hs containing a short linker. This result indicates that introduction of the flexible1,2-ethylenediamine linker between two rigid triazine rings enables the TZM-Es to fold into favourable conformations in order to promote membrane protein stability. The novel concept of detergent foldability introduced in the current study has potential in rational detergent design and membrane protein applications.
Assuntos
Detergentes , Proteínas de Membrana , Detergentes/química , Interações Hidrofóbicas e Hidrofílicas , Proteínas de Membrana/química , Estabilidade Proteica , TriazinasRESUMO
Chemical biology and the application of small molecules has proven to be a potent perturbation strategy, especially for the functional elucidation of proteins, their networks, and regulators. In recent years, the cellular thermal shift assay (CETSA) and its proteome-wide extension, thermal proteome profiling (TPP), have proven to be effective tools for identifying interactions of small molecules with their target proteins, as well as off-targets in living cells. Here, we asked the question whether isothermal dose-response (ITDR) CETSA can be exploited to characterize secondary effects downstream of the primary binding event, such as changes in post-translational modifications or protein-protein interactions (PPI). By applying ITDR-CETSA to MAPK14 kinase inhibitor treatment of living HL-60 cells, we found similar dose-responses for the direct inhibitor target and its known interaction partners MAPKAPK2 and MAPKAPK3. Extension of the dose-response similarity comparison to the proteome wide level using TPP with compound concentration range (TPP-CCR) revealed not only the known MAPK14 interaction partners MAPKAPK2 and MAPKAPK3, but also the potentially new intracellular interaction partner MYLK. We are confident that dose-dependent small molecule treatment in combination with ITDR-CETSA or TPP-CCR similarity assessment will not only allow discrimination between primary and secondary effects, but will also provide a novel method to study PPI in living cells without perturbation by protein modification, which we named "small molecule arranged thermal proximity coaggregation" (smarTPCA).
Assuntos
Proteína Quinase 14 Ativada por Mitógeno , Proteoma , Humanos , Proteoma/metabolismoRESUMO
A novel severe acute respiratory syndrome (SARS)-like coronavirus (SARS-CoV-2) has emerged as a human pathogen, causing global pandemic and resulting in over 400 000 deaths worldwide. The surface spike protein of SARS-CoV-2 mediates the process of coronavirus entry into human cells by binding angiotensin-converting enzyme 2 (ACE2). Due to the critical role in viral-host interaction and the exposure of spike protein, it has been a focus of most vaccines' developments. However, the structural and biochemical studies of the spike protein are challenging because it is thermodynamically metastable. Here, we develop a new pipeline that automatically identifies mutants that thermodynamically stabilize the spike protein. Our pipeline integrates bioinformatics analysis of conserved residues, motion dynamics from molecular dynamics simulations, and other structural analysis to identify residues that significantly contribute to the thermodynamic stability of the spike protein. We then utilize our previously developed protein design tool, Eris, to predict thermodynamically stabilizing mutations in proteins. We validate the ability of our pipeline to identify protein stabilization mutants through known prefusion spike protein mutants. We finally utilize the pipeline to identify new prefusion spike protein stabilization mutants.
Assuntos
COVID-19/virologia , Mutagênese , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/genética , Enzima de Conversão de Angiotensina 2/metabolismo , Sítios de Ligação , Biologia Computacional , Humanos , Simulação de Dinâmica Molecular , Mutação , Ligação Proteica , Domínios Proteicos , TermodinâmicaRESUMO
The hypoxia-inducible factor (HIF-1α) functions as a master regulator of oxygen homeostasis. Oxygen-dependent hydroxylation of HIF-1α is tightly regulated by prolyl hydroxylase domain containing proteins (PHD1, PHD2, and PHD3). The prolyl hydroxylation facilitates the recruitment of the von Hippel-Lindau (VHL) protein, leading to ubiquitination and degradation of HIF-1α by the proteasomes. Besides prolyl hydroxylation, phosphorylation of HIF-1α is another central post-translational modification, which regulates its stability under hypoxic conditions as well as normoxic conditions. By use of LC/MS/MS-based analysis, we were able to identify a specific serine residue (Ser451) of HIF-1α phosphorylated under hypoxic conditions. Using plasmids expressing wild type (WT), non-phosphorylatable mutant HIF-1α (S451A), and phosphomimetic mutant HIF-1α (S451E), we demonstrated that the phosphorylation at Ser451 is important in maintaining the HIF-1α protein stability. Notably, phosphorylation at S451 interrupts the interaction of HIF-1α with PHD and pVHL. A phosphomimetic construct of HIF-1α at Ser451 (S451E) is significantly more stable than WT HIF-1α under normoxic conditions. Cells transfected with unphosphorylatable HIF-1α exhibited significantly lower HIF-1 transcriptional activity than WT cells and markedly reduced tumor cell migration. Further, tumors derived from the phosphomimetic mutant cells grew faster, whereas the tumors derived from non-phosphorylatable mutant cells grew slower than the control tumors, suggesting that the phosphorylation of HIF-1α at the Ser451 site is critical to promote tumor growth in vivo. Taken together, our data suggest an alternative mechanism responsible for the regulation of HIF-1α stability.
Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/química , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Substituição de Aminoácidos , Materiais Biomiméticos/química , Materiais Biomiméticos/metabolismo , Hipóxia Celular , Células HCT116 , Células HEK293 , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Modelos Biológicos , Mutagênese Sítio-Dirigida , Peptidilprolil Isomerase de Interação com NIMA/metabolismo , Fosforilação , Prolil Hidroxilases/química , Prolil Hidroxilases/metabolismo , Domínios e Motivos de Interação entre Proteínas , Processamento de Proteína Pós-Traducional , Estabilidade Proteica , Serina/química , Proteína Supressora de Tumor Von Hippel-Lindau/química , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismoRESUMO
Polysorbates (PSs, Tweens) are widely used surfactant products consisting of a sorbitan ring connecting up to four ethylene oxide (EO) chains of variable lengths, one or more of which are esterified with fatty acids of variable lengths and saturation degrees. Pharmaceutical applications include the stabilization of biologicals in solutions and the solubilization of poorly water soluble, active ingredients. This study characterizes the complex association behavior of compendial PSs PS20 and PS80, which is fundamentally different from that of single-component surfactants. To this end, a series of demicellization experiments of isothermal titration calorimetry with different PS concentrations are evaluated. Their experiment-dependent heats of titration are converted into a common function of the state of a sample, the micellar enthalpy Qm(c). These functions demonstrate that initial micelles are already present at the lowest concentrations investigated, 2 µM for PS20 and 10 µM for PS80. Initial micelles consist primarily of the surfactant species with the lowest individual critical micelle concentration (cmc). With increasing concentration, the other PS species gradually enter these micelles in the sequence of increasing individual cmc's and hydrophilic-lipophilic balance. Concentration ranges with pronounced slopes of Qm(c) can be tentatively assigned to the uptake of the major components of the PS products. Micellization and the variation of the micelle properties progress up to at least 10 mM PS. That means the published cmc values or ranges of PS20 and PS80 may be related to certain, major components being incorporated into and forming specific micelles but must not be interpreted in terms of an absence of micelles below and constant properties, e.g., the surface activity, of the micelles above these ranges. The micellization enthalpy curves differ quite substantially between PS20 and PS80 and, in a subtler fashion, between individual quality grades such as high purity, pure lauric acid/pure oleic acid, super-refined, and China grade.
Assuntos
Micelas , Polissorbatos/química , Tensoativos/química , Calorimetria/métodos , Química Farmacêutica/métodos , Composição de Medicamentos/métodos , Estabilidade de Medicamentos , Difusão Dinâmica da Luz/métodos , Ésteres/química , Excipientes/química , Ácidos Graxos/química , Temperatura Alta , Interações Hidrofóbicas e Hidrofílicas , Ácidos Láuricos/química , Ácido Oleico/química , Estabilidade Proteica , SolubilidadeRESUMO
Mutations in hydroxymethylbilane synthase (HMBS) cause acute intermittent porphyria (AIP), an autosomal dominant disease where typically only one HMBS allele is mutated. In AIP, the accumulation of porphyrin precursors triggers life-threatening neurovisceral attacks and at long-term, entails an increased risk of hepatocellular carcinoma, kidney failure, and hypertension. Today, the only cure is liver transplantation, and a need for effective mechanism-based therapies, such as pharmacological chaperones, is prevailing. These are small molecules that specifically stabilize a target protein. They may be developed into an oral treatment, which could work curatively during acute attacks, but also prophylactically in asymptomatic HMBS mutant carriers. With the use of a 10,000 compound library, we identified four binders that further increased the initially very high thermal stability of wild-type HMBS and protected the enzyme from trypsin digestion. The best hit and a selected analog increased steady-state levels and total HMBS activity in human hepatoma cells overexpressing HMBS, and in an Hmbs-deficient mouse model with a low-expressed wild-type-like allele, compared to untreated controls. Moreover, the concentration of porphyrin precursors decreased in liver of mice treated with the best hit. Our findings demonstrate the great potential of these hits for the development of a pharmacological chaperone-based corrective treatment of AIP by enhancing wild-type HMBS function independently of the patients' specific mutation.
Assuntos
Biomarcadores , Descoberta de Drogas , Porfiria Aguda Intermitente/metabolismo , Animais , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Humanos , Camundongos , Camundongos Knockout , Terapia de Alvo Molecular , Porfiria Aguda Intermitente/etiologia , Porfiria Aguda Intermitente/terapia , Dobramento de Proteína , Proteínas/antagonistas & inibidores , Proteínas/química , Proteínas/metabolismo , Reprodutibilidade dos Testes , Bibliotecas de Moléculas Pequenas , Relação Estrutura-AtividadeRESUMO
The phytohormone ethylene regulates many aspects of plant growth and development. EIN2 is the central regulator of ethylene signaling, and its turnover is crucial for triggering ethylene responses. Here, we identified a stabilizer of OsEIN2 through analysis of the rice ethylene-response mutant mhz3. Loss-of-function mutations lead to ethylene insensitivity in etiolated rice seedlings. MHZ3 encodes a previously uncharacterized membrane protein localized to the endoplasmic reticulum. Ethylene induces MHZ3 gene and protein expression. Genetically, MHZ3 acts at the OsEIN2 level in the signaling pathway. MHZ3 physically interacts with OsEIN2, and both the N- and C-termini of MHZ3 specifically associate with the OsEIN2 Nramp-like domain. Loss of mhz3 function reduces OsEIN2 abundance and attenuates ethylene-induced OsEIN2 accumulation, whereas MHZ3 overexpression elevates the abundance of both wild-type and mutated OsEIN2 proteins, suggesting that MHZ3 is required for proper accumulation of OsEIN2 protein. The association of MHZ3 with the Nramp-like domain is crucial for OsEIN2 accumulation, demonstrating the significance of the OsEIN2 transmembrane domains in ethylene signaling. Moreover, MHZ3 negatively modulates OsEIN2 ubiquitination, protecting OsEIN2 from proteasome-mediated degradation. Together, these results suggest that ethylene-induced MHZ3 stabilizes OsEIN2 likely by binding to its Nramp-like domain and impeding protein ubiquitination to facilitate ethylene signal transduction. Our findings provide insight into the mechanisms of ethylene signaling.
Assuntos
Proteínas de Membrana/metabolismo , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Etilenos/metabolismo , Estiolamento , Proteínas de Membrana/química , Proteínas de Membrana/genética , Mutação , Oryza/genética , Reguladores de Crescimento de Plantas/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genética , Domínios Proteicos , Plântula/genética , Plântula/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/fisiologiaRESUMO
Membraneless organelles are aggregates of disordered proteins that form spontaneously to promote specific cellular functions in vivo. The possibility of synthesizing membraneless organelles out of cells will therefore enable fabrication of protein-based materials with functions inherent to biological matter. Since random copolymers contain various compositions and sequences of solvophobic and solvophilic groups, they are expected to function in nonbiological media similarly to a set of disordered proteins in membraneless organelles. Interestingly, the internal environment of these organelles has been noted to behave more like an organic solvent than like water. Therefore, an adsorbed layer of random copolymers that mimics the function of disordered proteins could, in principle, protect and enhance the proteins' enzymatic activity even in organic solvents, which are ideal when the products and/or the reactants have limited solubility in aqueous media. Here, we demonstrate via multiscale simulations that random copolymers efficiently incorporate proteins into different solvents with the potential to optimize their enzymatic activity. We investigate the key factors that govern the ability of random copolymers to encapsulate proteins, including the adsorption energy, copolymer average composition, and solvent selectivity. The adsorbed polymer chains have remarkably similar sequences, indicating that the proteins are able to select certain sequences that best reduce their exposure to the solvent. We also find that the protein surface coverage decreases when the fluctuation in the average distance between the protein adsorption sites increases. The results herein set the stage for computational design of random copolymers for stabilizing and delivering proteins across multiple media.
Assuntos
Simulação por Computador , Composição de Medicamentos/métodos , Modelos Químicos , Polímeros/química , Proteínas/química , Adsorção , Proteínas de Bactérias/química , Hidrolases de Éster Carboxílico/química , Desenho de Fármacos , Proteínas Fúngicas/química , Interações Hidrofóbicas e Hidrofílicas , Lipase/química , Modelos Moleculares , Compostos Orgânicos , Elastase Pancreática/química , Conformação Proteica , Solubilidade , Solventes , Subtilisina/química , Ubiquitina/químicaRESUMO
G-protein coupled receptors (GPCRs) are known for their low stability and large conformational changes upon transitions between multiple states. A widely used method for stabilizing these receptors is to make chimeric receptors by fusing soluble proteins (i.e., fusion partner proteins) into the intracellular loop 3 (ICL3) connecting the transmembrane helices 5 and 6 (TM5 and TM6). However, this fusion approach requires experimental trial and error to identify appropriate soluble proteins, residue positions, and linker lengths for making the fusion. Moreover, this approach has not provided state-targeting stabilization of GPCRs. Here, to rationally stabilize a class A GPCR, adenosine A2A receptor (A2AR) in a target state, we carried out the custom-made de novo design of α-helical fusion partner proteins, which can fix the conformation of TM5 and TM6 to that in an inactive state of A2AR through straight helical connections without any kinks or intervening loops. The chimeric A2AR fused with one of the designs (FiX1) exhibited increased thermal stability. Moreover, compared with the wild type, the binding affinity of the chimera against the agonist NECA was significantly decreased, whereas that against the inverse agonist ZM241385 was similar, indicating that the inactive state was selectively stabilized. Our strategy contributes to the rational state-targeting stabilization of GPCRs.
Assuntos
Agonistas do Receptor A2 de Adenosina/metabolismo , Proteínas/metabolismo , Receptor A2A de Adenosina/química , Receptor A2A de Adenosina/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Adenosina/metabolismo , Agonistas do Receptor A2 de Adenosina/química , Humanos , Ligantes , Modelos Moleculares , Simulação de Dinâmica Molecular , Conformação Proteica em alfa-Hélice , Proteínas/química , Proteínas Recombinantes de Fusão/químicaRESUMO
The transcription factor NRF2 plays a key role in the protection against environmental stress and maintaining cellular homeostasis. The acetyltransferase p300 is a known component of the NRF2 transcriptional complex and promotes its transcriptional activity. In this study we describe a novel mechanism by which p300 facilitates NRF2 activity. p300 physically interacts with NRF2 and interferes with NRF2-KEAP1 complex formation. In particular, p300 increases NRF2 protein abundance and stability, thereby promoting NRF2 nuclear localization. Notably, the acetyltransferase activity of p300 was indispensable for the stabilizing effects towards NRF2. Furthermore, overexpression of p300 protected HEK293T cells from oxidative stress and increased viability. Together our study uncovers a link between p300 and control of NRF2-KEAP1 signaling via regulation of NRF2 stability and this may act as a novel checkpoint on the adaptation to oxidative stress.