RESUMO
Posttranslational modification (PTM), through the recruitment of effector proteins (i.e., "readers") that signal downstream events, plays key roles in regulating a variety of cellular processes. To understand how a PTM is recognized, it is necessary to find its readers and, importantly, the location of the binding pockets responsible for PTM recognition. Although various methods have been developed to identify PTM readers, it remains a challenge to directly map the PTM-binding regions, especially for intrinsically disordered domains. Here, we demonstrate a photo-crosslinkable, clickable, and cleavable tri-functional amino acid, ADdis-Cys, that when coupled with mass spectrometry (ADdis-Cys-MS) can not only identify PTM readers from complex proteomes but also simultaneously map their PTM-recognition modules. Using ADdis-Cys-MS, we successfully identify the binding sites of several reader-PTM interactions, among which we discover human C1QBP as a histone chaperone. This robust method should find wide applications in examining other histone or non-histone PTM-mediated protein-protein interactions.
Assuntos
Aminoácidos/química , Aminoácidos/metabolismo , Mapeamento de Interação de Proteínas/métodos , Aminoácidos/genética , Sítios de Ligação , Química Click/métodos , Reagentes de Ligações Cruzadas , Cisteína/análogos & derivados , Cisteína/síntese química , Cisteína/química , Histonas/metabolismo , Humanos , Espectrometria de Massas/métodos , Mapas de Interação de Proteínas/genética , Mapas de Interação de Proteínas/fisiologia , Processamento de Proteína Pós-Traducional/genética , Processamento de Proteína Pós-Traducional/fisiologia , Proteoma/metabolismo , Proteômica/métodosRESUMO
G-quadruplexes (G4s) are peculiar nucleic acid secondary structures formed by DNA or RNA and are considered as fundamental features of the genome. Many proteins can specifically bind to G4 structures. There is increasing evidence that G4-protein interactions involve in the regulation of important cellular processes, such as DNA replication, transcription, RNA splicing, and translation. Additionally, G4-protein interactions have been demonstrated to be potential targets for disease treatment. In order to unravel the detailed regulatory mechanisms of G4-binding proteins (G4BPs), biochemical methods for detecting G4-protein interactions with high specificity and sensitivity are highly demanded. Here, we review recent advances in screening and validation of new G4BPs and highlight both their features and limitations.
Assuntos
Quadruplex G , DNA/química , Replicação do DNA , RNA/químicaRESUMO
The epidermal growth factor receptor (EGFR) is an important target for cancer therapies. Many head and neck cancer (HNC) cells have been reported to overexpress EGFR; therefore, anti-EGFR therapies have been attempted in patients with HNC. However, its clinical efficacy is limited owing to the development of drug resistance. In this study, we developed an EGFR-targeting immunotoxin consisting of a clinically proven anti-EGFR IgG (cetuximab; CTX) and a toxin fragment (LR-LO10) derived from Pseudomonas exotoxin A (PE) using a novel site-specific conjugation technology (peptide-directed photo-crosslinking reaction), as an alternative option. The immunotoxin (CTX-LR-LO10) showed specific binding to EGFR and properties of a typical IgG, such as stability, interactions with receptors of immune cells, and pharmacokinetics, and inhibited protein synthesis via modification of elongation factor-2. Treatment of EGFR-positive HNC cells with the immunotoxin resulted in apoptotic cell death and the inhibition of cell migration and invasion. The efficacy of CTX-LR-LO10 was evaluated in xenograft mouse models, and the immunotoxin exhibited much stronger tumor suppression than CTX or LR-LO10. Transcriptome analyses revealed that the immunotoxins elicited immune responses and altered the expression of genes related to its mechanisms of action. These results support the notion that CTX-LR-LO10 may serve as a new therapeutic agent targeting EGFR-positive cancers.
Assuntos
ADP Ribose Transferases , Receptores ErbB , Exotoxinas , Neoplasias de Cabeça e Pescoço , Imunoglobulina G , Imunotoxinas , Exotoxina A de Pseudomonas aeruginosa , Fatores de Virulência , Humanos , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Receptores ErbB/imunologia , Animais , Imunotoxinas/farmacologia , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/imunologia , Neoplasias de Cabeça e Pescoço/metabolismo , Camundongos , Imunoglobulina G/farmacologia , Linhagem Celular Tumoral , Exotoxinas/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto , Cetuximab/farmacologia , Camundongos Nus , Toxinas Bacterianas , Apoptose/efeitos dos fármacos , Camundongos Endogâmicos BALB C , Feminino , Movimento Celular/efeitos dos fármacos , Antineoplásicos/farmacologiaRESUMO
In conventional crosslinking mass spectrometry, proteins are crosslinked using a highly selective, bifunctional chemical reagent, which limits crosslinks to residues that are accessible and reactive to the reagent. Genetically incorporating a photoreactive amino acid offers two key advantages: any site can be targeted, including those that are inaccessible to conventional crosslinking reagents, and photoreactive amino acids can potentially react with a broad range of interaction partners. However, broad reactivity imposes additional challenges for crosslink identification. In this study, we incorporate benzoylphenylalanine (BPA), a photoreactive amino acid, at selected sites in an intrinsically disordered region of the human protein HSPB5. We report and characterize a workflow for identifying and visualizing residue-level interactions originating from BPA. We routinely identify 30 to 300 crosslinked peptide spectral matches with this workflow, which is up to ten times more than existing tools for residue-level BPA crosslink identification. Most identified crosslinks are assigned to a precision of one or two residues, which is supported by a high degree of overlap between replicate analyses. Based on these results, we anticipate that this workflow will support the more general use of genetically incorporated, photoreactive amino acids for characterizing the structures of proteins that have resisted high-resolution characterization.
Assuntos
Reagentes de Ligações Cruzadas , Fenilalanina , Fluxo de Trabalho , Fenilalanina/química , Fenilalanina/análogos & derivados , Reagentes de Ligações Cruzadas/química , Humanos , Aminoácidos/química , Aminoácidos/genética , Proteômica/métodos , Espectrometria de Massas/métodosRESUMO
Heat shock cognate protein 70 (Hsc70/HSPA8) belongs to the Hsp70 family of molecular chaperones. The fundamental functions of Hsp70 family molecular chaperones depend on ATP-dependent allosteric regulation of binding and release of hydrophobic polypeptide substrates. Hsc70 is also involved in various other cellular functions including selective pathways of protein degradation: chaperone-mediated autophagy (CMA) and endosomal microautophagy (eMI), in which Hsc70 recruits substrate proteins containing a KFERQ-like pentapeptide motif from the cytosol to lysosomes and late endosomes, respectively. However, whether the interaction between Hsc70 and the pentapeptide motif is direct or mediated by other molecules has remained unknown. In the present study, we introduced a photo-crosslinker near the KFERQ motif in a CMA/eMI model substrate and successfully detected its crosslinking with Hsc70, revealing the direct interaction between Hsc70 and the KFERQ motif for the first time. In addition, we demonstrated that the loss of the Hsc70 ATPase activity by the D10 N mutation appreciably reduced the crosslinking efficiency. Our present results suggested that the ATP allostery of Hsc70 is involved in the direct interaction of Hsc70 with the KFERQ-like pentapeptide.
RESUMO
This study focuses on the synthesis of fully renewable polycarbonates (PCs) starting from cellulose-based platform molecules levoglucosenone (LGO) and 2,5-bis(hydroxymethyl)furan (BHMF). These unique bio-based PCs are obtained through the reaction of a citronellol-containing triol (Triol-citro) derived from LGO, with a dimethyl carbonate derivative of BHMF (BHMF-DC). Solvent-free polymerizations are targeted to minimize waste generation and promote an eco-friendly approach with a favorable environmental factor (E-factor). The choice of metal catalyst during polymerization significantly influences the polymer properties, resulting in high molecular weight (up to 755 kDa) when Na2 CO3 is employed as an inexpensive catalyst. Characterization using nuclear magnetic resonance confirms the successful incorporation of the furan ring and the retention of the terminal double bond of the citronellol pendant chain. Furthermore, under UV irradiation, the presence of both citronellol and furanic moieties induces singular structural changes, triggering the formation of three distinct structures within the polymer network, a phenomenon herein occurs for the first time in this type of polymer. These findings pave the way to new functional materials prepared from renewable monomers with tunable properties.
Assuntos
Monoterpenos Acíclicos , Compostos Bicíclicos Heterocíclicos com Pontes , Furaldeído/análogos & derivados , Glucose/análogos & derivados , Cimento de Policarboxilato , Polímeros , Polímeros/químicaRESUMO
Recent technological advances have expanded the annotated protein coding content of mammalian genomes, as hundreds of previously unidentified, short open reading frame (ORF)-encoded peptides (SEPs) have now been found to be translated. Although several studies have identified important physiological roles for this emerging protein class, a general method to define their interactomes is lacking. Here, we demonstrate that genetic incorporation of the photo-crosslinking noncanonical amino acid AbK into SEP transgenes allows for the facile identification of SEP cellular interaction partners using affinity-based methods. From a survey of seven SEPs, we report the discovery of short ORF-encoded histone binding protein (SEHBP), a conserved microprotein that interacts with chromatin-associated proteins, localizes to discrete genomic loci, and induces a robust transcriptional program when overexpressed in human cells. This work affords a straightforward method to help define the physiological roles of SEPs and demonstrates its utility by identifying SEHBP as a short ORF-encoded transcription factor.
Assuntos
Diazometano/metabolismo , Histonas/genética , Lisina/metabolismo , Fases de Leitura Aberta , Peptídeos/genética , Transcrição Gênica , Sequência de Aminoácidos , Animais , Bovinos , Cromatina/química , Cromatina/metabolismo , Diazometano/análogos & derivados , Regulação da Expressão Gênica , Loci Gênicos , Células HEK293 , Células HeLa , Histonas/metabolismo , Humanos , Células K562 , Lisina/análogos & derivados , Camundongos , Pan troglodytes , Peptídeos/metabolismo , Ligação Proteica/efeitos da radiação , Mapeamento de Interação de Proteínas , Ratos , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transcrição Gênica/efeitos da radiação , Transgenes , Raios UltravioletaRESUMO
We herein report a method for site-selective photo-crosslinking of a DNA duplex. A stilbene pair was introduced into a DNA duplex and a ruthenium complex was conjugated with a triplex-forming oligonucleotide. We demonstrated that [2+2] photocycloaddition of the stilbene pair occurred upon irradiation with visible light when the ruthenium complex was in close proximity due to triplex formation. No reaction occurred when the ruthenium complex was not in proximity to the stilbene pair. The wavelength of visible light used was of lower energy than the wavelength of UV light necessary for direct excitation of stilbene. Quantum chemical calculation indicated that ruthenium complex catalyzed the photocycloaddition via triplet-triplet energy transfer. Site selectivity of this photo-crosslinking system was evaluated using a DNA duplex bearing two stilbene pairs as a substrate; we showed that the site of crosslinking was precisely regulated by the sequence of the oligonucleotide linked to the ruthenium complex. Since this method does not require orthogonal photoresponsive molecules, it will be useful in construction of complex photoresponsive DNA circuits, nanodevices and biological tools.
Assuntos
Rutênio , Estilbenos , Rutênio/química , DNA/química , Luz , OligonucleotídeosRESUMO
Herein, we report the major factor for deamination reaction rate acceleration, i.e., hydrophilicity, by using various 5-substituted target cytosines and by carrying out deamination at high temperatures. Through substitution of the groups at the 5'-position of the cytosine, the effect of hydrophilicity was understood. It was then used to compare the various modifications of the photo-cross-linkable moiety as well as the effect of the counter base of the cytosine to edit both DNA and RNA. Furthermore, we were able to achieve cytosine deamination at 37 °C with a half-life in the order of a few hours.
RESUMO
Metabolites orchestrate cellular processes as either substrates, co-enzymes, inhibitors, or activators of cellular proteins such as enzymes and receptors. Although traditional biochemical and structural biology-based approaches have been successfully employed for the discovery of protein-metabolite interactions, they often fail to detect transient and low-affinity biomolecular relationships. Another limitation of these approaches is that they are performed under in vitro conditions lacking the physiological context. Recently developed mass spectrometry-based methodologies overcome both these shortcomings, and have resulted in the discovery of global protein-metabolite cellular interaction networks. Herein, we describe traditional and modern approaches for the discovery of protein-metabolite interactions, and discuss the impact of these discoveries on our understanding of cellular physiology and on drug development.
Assuntos
Proteínas de Transporte , Proteínas , Proteínas/química , Proteínas de Transporte/metabolismo , Espectrometria de Massas/métodos , Mapas de Interação de ProteínasRESUMO
Semaphorin-3A (Sema-3A) is a chemorepellant protein with various biological functions, including kidney development. It interacts with a protein complex consisting of the receptors neuropilin-1 (NRP-1) and plexin-A1. After acute kidney injury, Sema-3A is overexpressed and secreted, leading to a loss of kidney function. The development of peptide inhibitors is a promising approach to modulate the interaction of Sema-3A with its receptor NRP-1. Few interaction points between these binding partners are known. However, an immunoglobulin-like domain-derived peptide of Sema-3A has shown a positive effect on cell proliferation. To specify these interactions between the peptide inhibitor and the Sema-3A-NRP-1 system, the peptides were modified with the photoactivatable amino acids 4-benzoyl-l-phenylalanine or photo-l-leucine by solid-phase peptide synthesis. Activity was tested by an enzyme-linked immunosorbent-based binding assay, and crosslinking experiments were analyzed by Western blot and mass spectrometry, demonstrating a specific binding site of the peptide at Sema-3A. The observed signals for Sema-3A-peptide interaction were found in a defined area of the Sema domain, which was also demonstrated to be involved in NRP-1 binding. The presented data identified the interaction site for further development of therapeutic peptides to treat acute kidney injury by blocking the Sema-3A-NRP-1 interaction.
Assuntos
Injúria Renal Aguda , Semaforina-3A , Humanos , Semaforina-3A/metabolismo , Peptídeos , Neuropilina-1RESUMO
Inverse vulcanization techniques are used to fabricate thermodynamically stable, sulfur polymers. Sulfur-based polymers exhibit higher refractive indices and improved transparency in the mid-wave infrared region compared with most organic polymers. Herein, the postsynthetic modification of sulfur polymers created via inverse vulcanization to generate novel, inorganic/organic photoresists is discussed. Amine-containing sulfur resins are postfunctionalized with cross-linkable alkynes. The sulfur-based materials undergo rapid photo-crosslinking to generate patternable films within 10 min under UV irradiation (365 nm). The development of these resins enables sulfur polymers to be utilized in processes where spatial and hierarchical control is necessary. The generation of this class of materials also expands on sulfur-based organic polymer systems with optical applications.
Assuntos
Polímeros , Enxofre , Raios UltravioletaRESUMO
Chaperone-mediated autophagy (CMA) is a unique proteolytic pathway, in which cytoplasmic proteins recognized by heat shock cognate protein 70 (Hsc70/HSPA8) are transported into lysosomes for degradation. The substrate/chaperone complex binds to the cytosolic tail of the lysosomal-associated membrane protein type 2A (LAMP2A), but whether the interaction between Hsc70 and LAMP2A is direct or mediated by other molecules has remained to be elucidated. The structure of LAMP2A comprises a large lumenal domain composed of two domains, both with the ß-prism fold, a transmembrane domain and a short cytoplasmic tail. We previously reported the structural basis for the homophilic interaction of the lumenal domains of LAMP2A, using site-specific photo-crosslinking and/or steric hindrance within cells. In the present study, we introduced a photo-crosslinker into the cytoplasmic tail of LAMP2A and successfully detected its crosslinking with Hsc70, revealing this direct interaction for the first time. Furthermore, we demonstrated that the truncation of the membrane-distal domain within the lumenal domain of LAMP2A reduced the amount of Hsc70 that coimmunoprecipitated with LAMP2A. Our present results suggested that the two-domain architecture of the lumenal domains of LAMP2A underlies the interaction with Hsc70 at the cytoplasmic surface of the lysosome.
Assuntos
Reagentes de Ligações Cruzadas/metabolismo , Citoplasma/metabolismo , Proteínas de Choque Térmico HSC70/metabolismo , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Lisossomos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Proteínas de Choque Térmico HSC70/química , Humanos , Proteína 2 de Membrana Associada ao Lisossomo/químicaRESUMO
A novel in situ chemical upcycling strategy for plastic waste is proposed by the customized diphenylacetylene monomer with dual photo-response. That is, diphenylacetylene reactive monomers are in situ inserted into the macromolecular chain of polyethylene terephthalate (PET) plastics/fibers through one-pot transesterification of slight-depolymerization and re-polymerization. On the one hand, the diphenylacetylene group absorbs short-wave high-energy UV rays and then releases long-wave low-energy harmless fluorescence. On the other hand, the UV-induced photo-crosslinking reaction among diphenylacetylene groups produces extended π-conjugated structure, resulting in a red-shift (due to decreased HOMO-LUMO separation) in the UV absorption band and locked crosslink points between PET chains. Therefore, with increasing UV exposure time, the upcycled PET plastics exhibit reverse enhanced UV resistance and mechanical strength (superior to original performance), instead of serious UV-photodegradation and damaged performance. This upcycling strategy at oligomer-scale not only provides a new idea for traditional plastic recycling, but also solves the common problem of gradual degradation of polymer performance during use.
RESUMO
DNA-encoded chemical libraries (DEL) have attracted substantial attention due to the infinite possibility for hit discovery in both pharmaceutical companies and academia. The encoding method is the initial step of DEL construction and one of the cornerstones of DEL applications. Classified by the DNA format, the existing DEL encoding strategies were categorized into single-stranded DNA-based strategies and double-stranded DNA-based strategies. The two DEL formats have their unique advantages but are usually incompatible with each other. To address this issue, we propose the concept of interconversion between double- and single-stranded DEL based on the "reversible covalent headpiece (RCHP)" design, which combines maximum robustness of synthesis with extraordinary flexibility of applications in distinct setups. Future opportunities in this field are also proposed to advance DEL technology to a comprehensive drug discovery platform.
Assuntos
DNA de Cadeia Simples , Bibliotecas de Moléculas Pequenas , DNA/genética , Descoberta de Drogas , Biblioteca GênicaRESUMO
The use of a proper encoding methodology is one of the most important aspects when practicing DEL technology. A "headpiece"-based double-stranded DEL encoding method is currently the most widely used for productive DEL. However, the robustness of double-stranded DEL construction conflicts with the versatility presented by single-stranded DEL applications. We here report a novel encoding method, which is based on a "reversible covalent headpiece (RCHP)". The RCHP allows reversible interconversion between double- and single-stranded DNA formats, providing an avenue to robust synthesis and allowing for the applications in distinct setups. We have validated the versatility of this encoding method with encoded self-assembled chemical library and DNA-encoded dynamic library technology. Notably, based on the RCHP-settled library construction, a unique "ternary covalent complex" mediating ligand isolation methodology against non-immobilized targets was developed.
RESUMO
Multiple reports over the past 2 years have provided the first complete structural analyses for the essential yeast chromatin remodeler, RSC, providing elaborate molecular details for its engagement with the nucleosome. However, there still remain gaps in resolution, particularly within the many RSC subunits that harbor histone binding domains.Solving contacts at these interfaces is crucial because they are regulated by posttranslational modifications that control remodeler binding modes and function. Modifications are dynamic in nature often corresponding to transcriptional activation states and cell cycle stage, highlighting not only a need for enriched spatial resolution but also temporal understanding of remodeler engagement with the nucleosome. Our recent work sheds light on some of those gaps by exploring the binding interface between the RSC catalytic motor protein, Sth1, and the nucleosome, in the living nucleus. Using genetically encoded photo-activatable amino acids incorporated into histones of living yeast we are able to monitor the nucleosomal binding of RSC, emphasizing the regulatory roles of histone modifications in a spatiotemporal manner. We observe that RSC prefers to bind H2B SUMOylated nucleosomes in vivo and interacts with neighboring nucleosomes via H3K14ac. Additionally, we establish that RSC is constitutively bound to the nucleosome and is not ejected during mitotic chromatin compaction but alters its binding mode as it progresses through the cell cycle. Our data offer a renewed perspective on RSC mechanics under true physiological conditions.
Assuntos
Proteínas de Ciclo Celular/genética , Proteínas de Ligação a DNA/genética , Histonas/genética , Proteínas Nucleares/genética , Nucleossomos/genética , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Acetilação , Cromatina/genética , Montagem e Desmontagem da Cromatina/genética , Processamento de Proteína Pós-Traducional/genética , Saccharomyces cerevisiae/genética , Sumoilação/genéticaRESUMO
The calcium signaling protein calmodulin regulates numerous intracellular processes. We introduce a sensitive microchip assay to separate and detect calmodulin binding proteins. The assay utilizes an optimized microchip electrophoresis protein separation platform with laser-induced fluorescence detection. Fluorescence-labeled calmodulin modified with a photoreactive diazirine crosslinker allowed selective detection of calmodulin binding proteins. We demonstrate successful in-vitro crosslinking of calmodulin with two calmodulin binding proteins, calcineurin and nitric oxide synthase. We compare the efficacy of commonly applied electrophoretic separation modes: microchip capillary zone electrophoresis, microchip micellar electrokinetic chromatography/gel electrophoresis, and nanoparticle colloidal arrays. Out of the methods tested, polydymethylsiloxane/glass chips with microchip zone electrophoresis gave the poorest separation, whereas sieving methods in which electro-osmotic flow was suppressed gave the best separation of photoproducts of calmodulin conjugated with calmodulin binding proteins.
Assuntos
Proteínas de Ligação a Calmodulina/análise , Eletroforese em MicrochipRESUMO
We synthesized the first multifunctionalized phosphoinositide polyphosphate derivatives featuring a photo-removable protecting group ("cage"), a photo-crosslinkable diazirine group, and a terminal alkyne group useful for click chemistry. We demonstrate that the lipid derivatives readily enter cells. After photo-crosslinking, cell fixation and fluorescent tagging via click chemistry, we determined the intracellular location of the lipid derivatives before and after uncaging of the lipids. We find that there is rapid trafficking of PI(3,4)P2 and PI(3,4,5)P3 derivatives to the plasma membrane, opening the intriguing possibility that there is active transport of these lipids involved. We employed the photo-crosslinking and click chemistry functions to analyze the proteome of PI(3,4,5)P3 -binding proteins. From the latter, we validated by RNAi that the putative lipid binding proteins ATP11A and MPP6 are involved in the transport of PI(3,4,5)P3 to the plasma membrane.
Assuntos
Fosfatidilinositóis/metabolismo , Polifosfatos/metabolismo , Células HeLa , Humanos , Estrutura Molecular , Fosfatidilinositóis/síntese química , Fosfatidilinositóis/química , Polifosfatos/síntese química , Polifosfatos/químicaRESUMO
Developing endogenous photo-activated theranostic platforms to overcome the limitation of low tissue-penetration from external light sources is highly significant for cancer diagnosis and treatment. We report a H2 O2 -initiated chemiluminescence (CL)-triggered nanoparticle aggregation strategy to activate theranostic functions of gold nanoparticles (AuNPs) for effective tumor imaging and therapy. Two types of AuNPs (tAuNP & mAuNP) were designed and fabricated by conjugating 2,5-diphenyltetrazole and methacrylic acid onto the surface of AuNPs, respectively. Luminol was adsorbed onto the mAuNPs to afford self-illuminating mAuNP/Lu NPs that could produce strong CL by reaction with H2 O2 in the tumor microenvironment, which triggers significant aggregation of AuNPs resulting in enhanced accumulation and retention of AuNPs for activated photoacoustic imaging and photothermal therapy of tumors. We thus believe that this approach may offer a promising tool for effective tumor treatment.