RESUMO

ATP has been recognized as a hydrotrope in the phase separation process of intrinsically disordered proteins (IDPs). Surprisingly, when using the disordered RG/RGG-rich motif from HNRNPG protein as a model system, we discover a biphasic relationship between the ATP concentration and IDP phase separation. We show that at a relatively low ATP concentration, ATP dynamically interacts with the IDP, which neutralizes protein surface charges, promotes intermolecular interactions, and consequently promotes phase separation. We further demonstrate that ATP induces a compact conformation of the IDP, accounting for the reduced solvent exchange rate and lower compression ratio during phase separation. As ATP concentration increases, its hydrotropic properties emerge, leading to the dissolution of the phase-separated droplets. Our finding uncovers a complex mechanism by which ATP molecules modulate the structure, interaction, and phase separation of IDPs, and accounts for the distinct phase separation behaviors for the charge-rich RGG motif and other low-complexity IDPs.

RESUMO

Unlocking the intricacies of protein structures and interactions within the dynamic landscape of subcellular organelles presents a significant challenge. To address this, we introduce SPACX, a method for spatially resolved protein complex profiling via biocompatible chemical cross(x)-linking with subcellular isolation, designed to monitor protein conformation, interactions, and translocation in living cells. By rapidly capturing protein complexes in their native physiological state and efficiently enriching cross-linked peptides, SPACX allows comprehensive analysis of the protein interactome within living cells. Leveraging structure refinement with cross-linking restraints, we identify subcellular-specific conformation heterogeneity of PTEN, revealing dynamic differences in its dual specificity domains between the nucleus and cytoplasm. Furthermore, by discerning conformational disparities, we identify 83 cytoplasm-exclusive and 109 nucleus-exclusive PTEN-interacting proteins, each associated with distinct biological functions. Upon induction of ubiquitin-proteasome system stress, we observe dynamic alterations in PTEN assembly and its interacting partners during translocation. These changes, including the identification of components and interaction sites, are characterized using the SPACX approach. Notably, SPACX enables identification of unique interacting proteins specific to PTEN isoforms, including PTEN and PTEN-Long, through the determination of sequence-specific cross-linking interfaces. These findings underscore the potential of SPACX to elucidate the functional diversity of proteins within distinct subcellular sociology.

Assuntos

Reagentes de Ligações Cruzadas , PTEN Fosfo-Hidrolase , Conformação Proteica , PTEN Fosfo-Hidrolase/metabolismo , PTEN Fosfo-Hidrolase/química , Humanos , Reagentes de Ligações Cruzadas/química , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Ligação Proteica , Mapeamento de Interação de Proteínas

RESUMO

Herbal extracts are rich sources of active compounds that can be used for drug screening due to their diverse and unique chemical structures. However, traditional methods for screening these compounds are notably laborious and time-consuming. In this manuscript, we introduce a new high-throughput approach that combines nuclear magnetic resonance (NMR) spectroscopy with a tailored database and algorithm to rapidly identify bioactive components in herbal extracts. This method distinguishes characteristic signals and structural motifs of active constituents in the raw extracts through a relaxation-weighted technique, particularly utilizing the perfect echo Carr-Purcell-Meiboom-Gill (peCPMG) sequence, complemented by precise 2D spectroscopic strategies. The cornerstone of our approach is a customized database designed to filter potential compounds based on defined parameters, such as the presence of CHn segments and unique chemical shifts, thereby expediting the identification of promising compounds. This innovative technique was applied to identifying substances interacting with choline kinase α (ChoKα1), resulting in the discovery of four new inhibitors. Our findings demonstrate a powerful tool for unraveling the complex chemical landscape of herbal extracts, considerably facilitating the search for new pharmaceutical candidates. This approach offers an efficient alternative to traditional methods in the quest for drug discovery from natural sources.

RESUMO

BACKGROUND: In-cell NMR is a valuable technique for investigating protein structure and function in cellular environments. However, challenges arise due to highly crowded cellular environment, where nonspecific interactions between the target protein and other cellular components can lead to signals broadening or disappearance in NMR spectra. RESULTS: We implemented chemical reduction methylation to selectively modify lysine residues on protein surfaces aiming to weaken charge interactions and recover obscured NMR signals. This method was tested on six proteins varying in molecular size and lysine content. While methylation did not disrupt the protein's native conformation, it successful restored some previously obscured in-cell NMR signals, particularly for proteins with high isoelectric points that decreased post-methylation. SIGNIFICANCE: This study affirms lysine methylation as a feasible approach to enhance the sensitivity of in-cell NMR spectra for protein studies. By mitigating signal loss due to nonspecific interactions, this method expands the utility of in-cell NMR for investigating proteins in their natural cellular environment, potentially leading to more accurate structural and functional insights.

Assuntos

Lisina , Ressonância Magnética Nuclear Biomolecular , Lisina/química , Lisina/análise , Metilação , Proteínas/química , Proteínas/análise , Humanos

RESUMO

Infections of many viruses induce caspase activation to regulate multiple cellular pathways, including programmed cell death, immune signaling and etc. Characterizations of caspase cleavage sites and substrates are important for understanding the regulation mechanisms of caspase activation. Here, we identified and analyzed a novel caspase cleavage motif AEAD, and confirmed its caspase dependent cleavage activity in natural substrate, such as nitric oxide-associated protein 1 (NOA1). Fusing the enhanced green fluorescent protein (EGFP) with the mitochondrial marker protein Tom20 through the AEAD motif peptide localized EGFP to the mitochondria. Upon the activation of caspase triggered by Sendai virus (SeV) or herpes simplex virus type 1 (HSV-1) infection, EGFP diffusely localized to the cell due to the caspase-mediated cleavage, thus allowing visual detection of the virus-induced caspase activation. An AEAD peptide-derived inhibitor Z-AEAD-FMK were developed, which significantly inhibited the activities of caspases-1, -3, -6, -7, -8 and -9, exhibiting a broad caspase inhibition effect. The inhibitor further prevented caspases-mediated cleavage of downstream substrates, including BID, PARP1, LMNA, pro-IL-1ß, pro-IL-18, GSDMD and GSDME, protecting cells from virus-induced apoptotic and pyroptotic cell death. Together, our findings provide a new perspective for the identification of novel caspase cleavage motifs and the development of new caspase inhibitors and anti-inflammatory drugs.

RESUMO

The stability of protein folded states is crucial for its function, yet the relationship with the protein sequence remains poorly understood. Prior studies have focused on the amino acid composition and thermodynamic couplings within a single folded conformation, overlooking the potential contribution of protein dynamics. Here, we address this gap by systematically analyzing the impact of alanine mutations in the C-terminal ß-strand (ß5) of ubiquitin, a model protein exhibiting millisecond timescale interconversion between two conformational states differing in the ß5 position. Our findings unveil a negative correlation between millisecond dynamics and thermal stability, with alanine substitutions at seemingly flexible C-terminal residues significantly enhancing thermostability. Integrating spectroscopic and computational approaches, we demonstrate that the thermally unfolded state retains a substantial secondary structure but lacks ß5 engagement, recapitulating the transition state for millisecond dynamics. Thus, alanine mutations that modulate the stabilities of the folded states with respect to the partially unfolded state impact both the dynamics and stability. Our findings underscore the importance of conformational dynamics with implications for protein engineering and design.

RESUMO

Insects detect and discriminate a diverse array of chemicals using odorant receptors (ORs), which are ligand-gated ion channels comprising a divergent odorant-sensing OR and a conserved odorant receptor co-receptor (Orco). In this work, we report structures of the ApOR5-Orco heterocomplex from the pea aphid Acyrthosiphon pisum alone and bound to its known activating ligand, geranyl acetate. In these structures, three ApOrco subunits serve as scaffold components that cannot bind the ligand and remain relatively unchanged. Upon ligand binding, the pore-forming helix S7b of ApOR5 shifts outward from the central pore axis, causing an asymmetrical pore opening for ion influx. Our study provides insights into odorant recognition and channel gating of the OR-Orco heterocomplex and offers structural resources to support development of innovative insecticides and repellents for pest control.

Assuntos

Acetatos , Afídeos , Proteínas de Insetos , Receptores Odorantes , Receptores Odorantes/química , Receptores Odorantes/metabolismo , Receptores Odorantes/genética , Animais , Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , Proteínas de Insetos/genética , Afídeos/química , Acetatos/química , Acetatos/metabolismo , Ligantes , Terpenos/química , Terpenos/metabolismo , Odorantes/análise , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Ativação do Canal Iônico , Microscopia Crioeletrônica , Monoterpenos Acíclicos

RESUMO

The WRKY transcription factors play essential roles in a variety of plant signaling pathways associated with biotic and abiotic stress response. The transcriptional activity of many WRKY members are regulated by a class of intrinsically disordered VQ proteins. While it is known that VQ proteins interact with the WRKY DNA-binding domains (DBDs), also termed as the WRKY domains, structural information regarding VQ-WRKY interaction is lacking and the regulation mechanism remains unknown. Herein we report a solution NMR study of the interaction between Arabidopsis WRKY33 and its regulatory VQ protein partner SIB1. We uncover a SIB1 minimal sequence neccessary for forming a stable complex with WRKY33 DBD, which comprises not only the consensus "FxxhVQxhTG" VQ motif but also its preceding region. We demonstrate that the ßN-strand and the extended ßN-ß1 loop of WRKY33 DBD form the SIB1 docking site, and build a structural model of the complex based on the NMR paramagnetic relaxation enhancement and mutagenesis data. Based on this model, we further identify a cluster of positively-charged residues in the N-terminal region of SIB1 to be essential for the formation of a SIB1-WRKY33-DNA ternary complex. These results provide a framework for the mechanism of SIB1-enhanced WRKY33 transcriptional activity.

Assuntos

Proteínas de Arabidopsis , Arabidopsis , Fatores de Transcrição , Sequência de Aminoácidos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/química , Regulação da Expressão Gênica de Plantas , Modelos Moleculares , Ligação Proteica , Domínios Proteicos , Fatores de Transcrição/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fator sigma/genética , Fator sigma/metabolismo

RESUMO

The validity of protein structures and interactions, whether determined under ideal laboratory conditions or predicted by AI tools such as Alphafold2, to precisely reflect those found in living cells remains to be examined. Moreover, understanding the changes in protein structures and interactions in response to stimuli within living cells, under both normal and disease conditions, is key to grasping proteins' functionality and cellular processes. Nevertheless, achieving high-resolution identification of these protein structures and interactions within living cells presents a technical challenge. In this Perspective, we summarize the recent advancements in in-cell nuclear magnetic resonance (NMR) and in vivo cross-linking mass spectrometry (XL-MS) for studying protein structures and interactions within a cellular context. Additionally, we discuss the challenges, opportunities, and potential benefits of integrating in-cell NMR and in vivo XL-MS in future research to offer an exhaustive approach to studying proteins in their natural habitat.

RESUMO

The influence of distance restraints from chemical cross-link mass spectroscopy (XL-MS) on the quality of protein structures modeled with the coarse-grained UNRES force field was assessed by using a protocol based on multiplexed replica exchange molecular dynamics, in which both simulated and experimental cross-link restraints were employed, for 23 small proteins. Six cross-links with upper distance boundaries from 4 Å to 12 Å (azido benzoic acid succinimide (ABAS), triazidotriazine (TATA), succinimidyldiazirine (SDA), disuccinimidyl adipate (DSA), disuccinimidyl glutarate (DSG), and disuccinimidyl suberate (BS3)) and two types of restraining potentials ((i) simple flat-bottom Lorentz-like potentials dependent on side chain distance (all cross-links) and (ii) distance- and orientation-dependent potentials determined based on molecular dynamics simulations of model systems (DSA, DSG, BS3, and SDA)) were considered. The Lorentz-like potentials with properly set parameters were found to produce a greater number of higher-quality models compared to unrestrained simulations than the MD-based potentials, because the latter can force too long distances between side chains. Therefore, the flat-bottom Lorentz-like potentials are recommended to represent cross-link restraints. It was also found that significant improvement of model quality upon the introduction of cross-link restraints is obtained when the sum of differences of indices of cross-linked residues exceeds 150.

Assuntos

Simulação de Dinâmica Molecular , Proteínas , Conformação Proteica , Proteínas/química

RESUMO

Abnormal fatty acid metabolism is recognized as a key driver of tumor development and progression. Although numerous inhibitors have been developed to target this pathway, finding drugs with high specificity that do not disrupt normal cellular metabolism remains a formidable challenge. In this paper, we introduced a novel real-time NMR-based drug screening technique that operates within living cells. This technique provides a direct way to putatively identify molecular targets involved in specific metabolic processes, making it a powerful tool for cell-based drug screening. Using 2-13C acetate as a tracer, combined with 3D cell clusters and a bioreactor system, our approach enables real-time detection of inhibitors that target fatty acid metabolism within living cells. As a result, we successfully demonstrated the initial application of this method in the discovery of traditional Chinese medicines that specifically target fatty acid metabolism. Elucidating the mechanisms behind herbal medicines remains challenging due to the complex nature of their compounds and the presence of multiple targets. Remarkably, our findings demonstrate the significant inhibitory effect of P. cocos on fatty acid synthesis within cells, illustrating the potential of this approach in analyzing fatty acid metabolism events and identifying drug candidates that selectively inhibit fatty acid synthesis at the cellular level. Moreover, this systematic approach represents a valuable strategy for discovering the intricate effects of herbal medicine.

RESUMO

Cross-linkers play a critical role in capturing protein dynamics in chemical cross-linking mass spectrometry techniques. Various types of cross-linkers with different backbone features are widely used in the study of proteins. However, it is still not clear how the cross-linkers' backbone affect their own structure and their interactions with proteins. In this study, we systematically characterized and compared methylene backbone and polyethylene glycol (PEG) backbone cross-linkers in terms of capturing protein structure and dynamics. The results indicate the cross-linker with PEG backbone have a better ability to capture the inter-domain dynamics of calmodulin, adenylate kinase, maltodextrin binding protein and dual-specificity protein phosphatase. We further conducted quantum chemical calculations and all-atom molecular dynamics simulations to analyze thermodynamic and kinetic properties of PEG backbone and methylene backbone cross-linkers. Solution nuclear magnetic resonance was employed to validate the interaction interface between proteins and cross-linkers. Our findings suggest that the polarity distribution of PEG backbone enhances the accessibility of the cross-linker to the protein surface, facilitating the capture of sites located in dynamic regions. By comprehensively benchmarking with disuccinimidyl suberate (DSS)/bis-sulfosuccinimidyl-suberate(BS3), bis-succinimidyl-(PEG)2 revealed superior advantages in protein dynamic conformation analysis in vitro and in vivo, enabling the capture of a greater number of cross-linking sites and better modeling of protein dynamics. Furthermore, our study provides valuable guidance for the development and application of PEG backbone cross-linkers.

Assuntos

Polietilenoglicóis , Proteínas , Polietilenoglicóis/química , Proteínas/química , Espectrometria de Massas , Conformação Proteica , Simulação de Dinâmica Molecular

RESUMO

OBJECTIVE@#To investigate the clinical effect of unilateral percutaneous vertebroplasty (PVP) combined with 3D printing technology for the treatment of thoracolumbar osteoporotic compression fracture.@*METHODS@#A total of 77 patients with thoracolumbar osteoporotic compression fractures from October 2020 to April 2022 were included in the study, all of which were vertebral body compression fractures caused by trauma. According to different treatment methods, they were divided into experimental group and control group. Thirty-two patients used 3D printing technology to improve unilateral transpedicle puncture vertebroplasty in the experimental group, there were 5 males and 27 females, aged from 63 to 91 years old with an average of (77.59±8.75) years old. Forty-five patients were treated with traditional bilateral pedicle puncture vertebroplasty, including 7 males and 38 females, aged from 60 to 88 years old with an average of(74.89±7.37) years old. Operation time, intraoperative C-arm X-ray times, anesthetic dosage, bone cement injection amount, bone cement diffusion good and good rate, complications, vertebral height, kyphotic angle (Cobb angle), visual analogue scale(VAS), Oswestry disability index (ODI) and other indicators were recorded before and after surgery, and statistically analyzed.@*RESULTS@#All patients were followed up for 6 to 23 months, with preoperative imaging studies, confirmed for thoracolumbar osteoporosis compression fractures, two groups of patients with postoperative complications, no special two groups of patients' age, gender, body mass index (BMI), time were injured, the injured vertebral distribution had no statistical difference(P>0.05), comparable data. Two groups of patients with bone cement injection, bone cement dispersion rate, preoperative and postoperative vertebral body height, protruding after spine angle(Cobb angle), VAS, ODI had no statistical difference(P>0.05). The operative time, intraoperative fluoroscopy times and anesthetic dosage were statistically different between the two groups(P<0.05). Compared with the traditional bilateral puncture group, the modified unilateral puncture group combined with 3D printing technology had shorter operation time, fewer intraoperative fluoroscopy times and less anesthetic dosage. The height of anterior vertebral edge, kyphosis angle (Cobb angle), VAS score and ODI of the affected vertebrae were statistically different between two groups at each time point after surgery(P<0.05).@*CONCLUSION@#In the treatment of thoracolumbar osteoporotic compression fractures, 3D printing technology is used to improve unilateral puncture PVP, which is convenient and simple, less trauma, short operation time, fewer fluoroscopy times, satisfactory distribution of bone cement, vertebral height recovery and kyphotic Angle correction, and good functional improvement.

Assuntos

Masculino , Feminino , Humanos , Pessoa de Meia-Idade , Idoso , Idoso de 80 Anos ou mais , Fraturas por Compressão/cirurgia , Fraturas da Coluna Vertebral/cirurgia , Cimentos Ósseos , Resultado do Tratamento , Vertebroplastia/métodos , Cifose/cirurgia , Punções , Impressão Tridimensional , Tecnologia , Fraturas por Osteoporose/cirurgia , Anestésicos , Estudos Retrospectivos , Cifoplastia/métodos

RESUMO

Designing inhibitors for RNA is still challenging due to the bottleneck of maintaining the binding interaction of inhibitor-RNA accompanied by subtle RNA flexibility. Thus, the current approach usually needs to screen thousands of candidate inhibitors for binding. Here, we propose a dynamic geometry design approach to enrich the hits with only a tiny pool of designed geometrically compatible scaffold candidates. First, our method uses graph-based tree decomposition to explore the complementarity rigid binding cyclic peptide and design the amino acid side chain length and charge to fit the RNA pocket. Then, we perform an energy-based dynamical network algorithm to optimize the inhibitor-RNA hydrogen bonds. Dynamic geometry-guided design yields successful inhibitors with low micromolar binding affinity scaffolds and experimentally competes with the natural RNA chaperone. The results indicate that the dynamic geometry method yields higher efficiency and accuracy than traditional methods. The strategy could be further optimized to design the length and chirality by adopting nonstandard amino acids and facilitating RNA engineering for biological or medical applications.

Assuntos

Peptídeos Cíclicos , RNA , Peptídeos Cíclicos/química , Aminoácidos

RESUMO

Protein dynamics play a crucial role in their diverse functions. The intracellular environment significantly influences protein dynamics, particularly for intrinsically disordered proteins (IDPs). To comprehensively capture structural information from various proteins within cells and characterize protein dynamics, chemical cross-linking mass spectrometry was employed. In this study, we introduce a hierarchical decoding strategy that enables the investigation of protein dynamics in vivo. Computational analysis based on distance restraints derived from cross-links is used to infer protein dynamics in cells. To facilitate this analysis, we leverage the prior structure obtained from AlphaFold2. By employing this strategy, we can characterize the full-length structure of multi-domain proteins taking into account their distinct dynamic features. Furthermore, by combining restraint sampling with an unbiased sampling and evaluation approach, we can provide a comprehensive description of the intrinsic motion of IDPs. Consequently, the hierarchical strategy we propose holds significant potential in advancing our understanding of the molecular mechanisms that undelie protein functions in cells.

Assuntos

Proteínas Intrinsicamente Desordenadas , Proteínas Intrinsicamente Desordenadas/química , Espectrometria de Massas , Conformação Proteica , Simulação de Dinâmica Molecular

RESUMO

Chemical cross-linking coupled with mass spectrometry (XL-MS) is an important technique for the structural analysis of protein complexes where the coverage of amino acids and the identification of cross-linked sites are crucial. Photo-cross-linking has multisite reactivity and is valuable for the structural analysis of chemical cross-linking. However, a high degree of heterogeneity results from this multisite reactivity, which results in samples with higher complexity and lower abundance. Additionally, the applicability of photo-cross-linking is limited to purified protein complexes. In this work, we demonstrate a photo-cross-linker, alkynyl-succinimidyl-diazirine (ASD) with the reactive groups of N-hydroxysuccinimide ester and diazirine, as well as the click-enrichable alkyne group. Photo-cross-linkers can provide higher site reactivity for proteins that contain only a small number of lysine residues, thereby complementing the more commonly used lysine-targeting cross-linkers. By systematically analyzing proteins with differing lysine contents and differing flexibilities, we demonstrated clear enhancement in structure elucidation for proteins containing less lysine and with high flexibility. In addition, enrichment approaches of alkynyl-azide click chemistry conjugated with biotin-streptavidin purification (coinciding with parallel orthogonal digestion) improved the identification coverage of cross-links. We show that this photo-cross-linking approach can be used for membrane proteome-wide complex analysis. This method led to the identification of a total of 14066 lysine-X cross-linked site pairs from a total of 2784 proteins. Thus, this cross-linker is a valuable addition to a photo-cross-linking toolkit and improves the identification coverage of XL-MS in functional structure analysis.

Assuntos

Diazometano , Lisina , Lisina/química , Aminoácidos/química , Espectrometria de Massas/métodos , Proteoma , Reagentes de Ligações Cruzadas/química

RESUMO

The clinically used androgen receptor (AR) antagonists for the treatment of prostate cancer (PCa) are all targeting the AR ligand binding pocket (LBP), resulting in various drug-resistant problems. Therefore, a new strategy to combat PCa is urgently needed. Enlightened by the gain-of-function mutations of androgen insensitivity syndrome, we discovered for the first time small-molecule antagonists toward a prospective pocket on the AR dimer interface named the dimer interface pocket (DIP) via molecular dynamics (MD) simulation, structure-based virtual screening, structure-activity relationship exploration, and bioassays. The first-in-class antagonist M17-B15 targeting the DIP is capable of effectively disrupting AR self-association, thereby suppressing AR signaling. Furthermore, M17-B15 exhibits extraordinary anti-PCa efficacy in vitro and also in mouse xenograft tumor models, demonstrating that AR dimerization disruption by small molecules targeting the DIP is a novel and valid strategy against PCa.

RESUMO

Proteins and RNAs are primary biomolecules that are involved in most biological processes [...].