RESUMO
Local and systemic low-grade inflammation, mainly involving the innate immune system, plays an important role in the development of OA. A receptor playing a key role in initiation of this inflammation is the pattern-recognition receptor Toll-like receptor 4 (TLR4). In the joint, various ligands for TLR4, many of which are damage-associated molecular patterns (DAMPs), are present that can activate TLR4 signalling. This leads to the production of pro-inflammatory and catabolic mediators that cause joint damage. In this narrative review, we will first discuss the involvement of TLR4 ligands and signalling in OA. Furthermore, we will provide an overview of methods for inhibit, TLR4 signalling by RNA interference, neutralizing anti-TLR4 antibodies, small molecules and inhibitors targeting the TLR4 co-receptor MD2. Finally, we will focus on possible applications and challenges of these strategies in the dampening of inflammation in OA.
Assuntos
Osteoartrite , Receptor 4 Toll-Like , Humanos , Inflamação , Transdução de Sinais , AlarminasRESUMO
Glycans play a pivotal role in biology. However, because of the low-affinity of glycan-protein interactions, many interaction pairs remain unknown. Two important glycoproteins involved in B-cell biology are the B-cell receptor and its secreted counterpart, antibodies. It has been indicated that glycans expressed by these B-cell-specific molecules can modulate immune activation via glycan-binding proteins. In several autoimmune diseases, an increased prevalence of variable domain glycosylation of IgG autoantibodies has been observed. Especially, the hallmarking autoantibodies in rheumatoid arthritis, anti-citrullinated protein antibodies, carry a substantial amount of variable domain glycans. The variable domain glycans expressed by these autoantibodies are N-linked, complex-type, and α2-6 sialylated, and B-cell receptors carrying variable domain glycans have been hypothesized to promote selection of autoreactive B cells via interactions with glycan-binding proteins. Here, we use the anti-citrullinated protein antibody response as a prototype to study potential in solution and in situ B-cell receptor-variable domain glycan interactors. We employed SiaDAz, a UV-activatable sialic acid analog carrying a diazirine moiety that can form covalent bonds with proximal glycan-binding proteins. We show, using oligosaccharide engineering, that SiaDAz can be readily incorporated into variable domain glycans of both antibodies and B-cell receptors. Our data show that antibody variable domain glycans are able to interact with inhibitory receptor, CD22. Interestingly, although we did not detect this interaction on the cell surface, we captured CD79 ß glycan-B-cell receptor interactions. These results show the utility of combining photoaffinity labeling and oligosaccharide engineering for identifying antibody and B-cell receptor interactions and indicate that variable domain glycans appear not to be lectin cis ligands in our tested conditions.
Assuntos
Linfócitos B , Receptores de Antígenos de Linfócitos B , Receptores de Antígenos de Linfócitos B/metabolismo , Linfócitos B/metabolismo , Autoanticorpos , Polissacarídeos/química , Oligossacarídeos/metabolismoRESUMO
The click reaction between a functionalized trans-cyclooctene (TCO) and a tetrazine (Tz) is a compelling method for bioorthogonal conjugation in combination with payload releasing capabilities. However, the synthesis of difunctionalized TCOs remains challenging. As a result, these compounds are poorly accessible, which impedes the development of novel applications. In this work, the scalable and accessible synthesis of a new bioorthogonal difunctionalized TCO is reported in only four single selective high yielding steps starting from commercially available compounds. The TCO-Tz click reaction was assessed and revealed excellent kinetic rates and subsequently payload release was shown with various functionalized derivatives. Tetrazine triggered release of carbonate and carbamate payloads was demonstrated up to 100 % release efficiency and local drug release was shown in a cellular toxicity study which revealed a >20-fold increase in cytotoxicity.
RESUMO
Functionalized antibodies and antibody fragments have found applications in the fields of biomedical imaging, theranostics, and antibody-drug conjugates (ADC). In addition, therapeutic and theranostic approaches benefit from the possibility to deliver more than one type of cargo to target cells, further challenging stochastic labeling strategies. Thus, bioconjugation methods to reproducibly obtain defined homogeneous conjugates bearing multiple different cargo molecules, without compromising target affinity, are in demand. Here, we describe a straightforward CRISPR/Cas9-based strategy to rapidly engineer hybridoma cells to secrete Fab' fragments bearing two distinct site-specific labeling motifs, which can be separately modified by two different sortase A mutants. We show that sequential genetic editing of the heavy chain (HC) and light chain (LC) loci enables the generation of a stable cell line that secretes a dual tagged Fab' molecule (DTFab'), which can be easily isolated. To demonstrate feasibility, we functionalized the DTFab' with two distinct cargos in a site-specific manner. This technology platform will be valuable in the development of multimodal imaging agents, theranostics, and next-generation ADCs.
Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Hibridomas/química , Fragmentos Fab das Imunoglobulinas/química , Anticorpos Monoclonais/química , Imunoconjugados/química , Processos EstocásticosRESUMO
Bacterial infections are still one of the leading causes of death worldwide; despite the near-ubiquitous availability of antibiotics. With antibiotic resistance on the rise, there is an urgent need for novel classes of antibiotic drugs. One particularly troublesome class of bacteria are those that have evolved highly efficacious mechanisms for surviving inside the host. These contribute to their virulence by immune evasion, and make them harder to treat with antibiotics due to their residence inside intracellular membrane-limited compartments. This has sparked the development of new chemical reporter molecules and bioorthogonal probes that can be metabolically incorporated into bacteria to provide insights into their activity status. In this review, we provide an overview of several classes of metabolic labeling probes capable of targeting either the peptidoglycan cell wall, the mycomembrane of mycobacteria and corynebacteria, or specific bacterial proteins. In addition, we highlight several important insights that have been made using these metabolic labeling probes.
Assuntos
Proteínas de Bactérias/metabolismo , Parede Celular/metabolismo , Corynebacterium/metabolismo , Mycobacterium/metabolismo , Peptidoglicano/metabolismo , Proteínas de Bactérias/química , Parede Celular/química , Corynebacterium/química , Interações Hospedeiro-Patógeno , Humanos , Conformação Molecular , Mycobacterium/química , Peptidoglicano/químicaRESUMO
Activity-based protein profiling (ABPP) is a powerful technique to label and detect active enzyme species within cell lysates, cells, or whole animals. In the last two decades, a wide variety of applications and experimental read-out techniques have been pursued in order to increase our understanding of physiological and pathological processes, to identify novel drug targets, to evaluate selectivity of drugs, and to image probe targets in cells. Bioorthogonal chemistry has substantially contributed to the field of ABPP, as it allows the introduction of tags, which may be bulky or have unfavorable physicochemical properties, at a late stage in the experiment. In this review, we give an overview of the bioorthogonal reactions that have been implemented in ABPP, provide examples of applications of bioorthogonal chemistry in ABPP, and share some thoughts on future directions.
Assuntos
Enzimas/metabolismo , Corantes Fluorescentes/química , Animais , Enzimas/química , Humanos , Estrutura MolecularRESUMO
Bioorthogonal reactions can be performed selectively in the presence of any biological functional group and are widely used to achieve site-selective chemical modifications of biomolecules. The click-to-release reaction is a bioorthogonal bond-cleavage variant that has gained much interest over the last few years. The bioorthogonal reaction between tetrazines and trans-cyclooctenes or vinyl ethers, for example, initiates the release of a small molecule immediately after the cycloaddition with tetrazines. Recently, our group reported that vinylboronic acids (VBAs) give exceptionally high reaction rates in the bioorthogonal inverse electron-demand Diels-Alder reaction with tetrazines that are substituted with boron-coordinating ligands. In the present study, we show that VBAs can be used in a click-to-release variant and demonstrate its bioorthogonality with a VBA-protected doxorubicin prodrug. We show that the cytotoxicity of doxorubicin is silenced by the attachment of the VBA, and activity can be largely restored upon the reaction with a tetrazine, inducing cell death.
RESUMO
Bioorthogonal chemistry can be used for the selective modification of biomolecules without interfering with any other functionality that might be present. Recent developments in the field include orthogonal bioorthogonal reactions to modify multiple biomolecules simultaneously. During our research, we observed that the reaction rates for the bioorthogonal inverse-electron-demand Diels-Alder (iEDDA) reactions between nonstrained vinylboronic acids (VBAs) and dipyridyl-s-tetrazines were exceptionally higher than those between VBAs and tetrazines bearing a methyl or phenyl substituent. As VBAs are mild Lewis acids, we hypothesised that coordination of the pyridyl nitrogen atom to the boronic acid promoted tetrazine ligation. Herein, we explore the molecular basis and scope of VBA-tetrazine ligation in more detail and benefit from its unique reactivity in the simultaneous orthogonal tetrazine labelling of two proteins modified with VBA and norbornene, a widely used strained alkene. We further show that the two orthogonal iEDDA reactions can be performed in living cells by labelling the proteasome by using a nonselective probe equipped with a VBA and a subunit-selective VBA bearing a norbornene moiety.
Assuntos
Alcenos/química , Ácidos Borônicos/química , Reação de Cicloadição/métodos , Norbornanos/química , Proteínas/química , Compostos de Vinila/química , Alcenos/síntese química , Ácidos Borônicos/síntese química , Proteínas de Fluorescência Verde/síntese química , Proteínas de Fluorescência Verde/química , Compostos Heterocíclicos com 1 Anel/síntese química , Compostos Heterocíclicos com 1 Anel/química , Humanos , Norbornanos/síntese química , Proteínas/síntese química , Albumina Sérica Humana/síntese química , Albumina Sérica Humana/química , Compostos de Vinila/síntese químicaRESUMO
Bioorthogonal reactions are selective transformations that are not affected by any biological functional group and are widely used for chemical modification of biomolecules. Recently, we reported that vinylboronic acids (VBAs) gave exceptionally high reaction rates in the bioorthogonal inverse electron-demand Diels-Alder (iEDDA) reaction with tetrazines bearing a boron-coordinating pyridyl moiety compared to tetrazines lacking such a substituent. In this integrated experimental and theoretical study, we show how the reaction rate of the VBA-tetrazine ligation can be accelerated by shifting the equilibrium from boronic acid to the boronate anion in the reaction mixture. Quantum chemical activation strain analyses reveal that this rate enhancement is a direct consequence of the excellent electron-donating capability of the boronate anion in which the π HOMO is pushed to a higher energy due to the net negative potential of this species. We have explored the second-order rate constants of several tetrazines containing potential VBA-coordinating hydroxyl substituents. We observed an increase in rate constants of several orders of magnitude compared to the tetrazines lacking a hydroxyl substituent. Furthermore, we find the hydroxyl-substituted tetrazines to be more selective toward VBAs than toward the commonly used bioorthogonal reactant norbornene, and more stable in aqueous environment than the previously studied tetrazines containing a pyridyl substituent.
Assuntos
Ácidos Borônicos/química , Compostos Heterocíclicos/química , Reação de Cicloadição , Teoria da Densidade Funcional , Elétrons , Concentração de Íons de Hidrogênio , CinéticaRESUMO
Bioorthogonal chemistry can be used for the selective modification of biomolecules without interfering with any other functionality present in the cell. The tetrazine ligation is very suitable as a bioorthogonal reaction because of its selectivity and high reaction rates with several alkenes and alkynes. Recently, we described vinylboronic acids (VBAs) as novel hydrophilic bioorthogonal moieties that react efficiently with dipyridyl- s-tetrazines and used them for protein modification in cell lysate. It is not clear, however, whether VBAs are suitable for labeling experiments in living cells because of the possible coordination with, for example, vicinal carbohydrate diols. Here, we evaluated VBAs as bioorthogonal reactants for labeling of proteins in living cells using an irreversible inhibitor of the proteasome and compared the reactivity to that of an inhibitor containing norbornene, a widely used reactant for the tetrazine ligation. No large differences were observed between the VBA and norbornene probes in a two-step labeling approach with a cell-penetrable fluorescent tetrazine, indicating that the VBA gives little or no side reactions with diols and can be used efficiently for protein labeling in living cells.
Assuntos
Compostos de Boro/química , Ácidos Borônicos/química , Corantes Fluorescentes/química , Proteínas/análise , Piridinas/química , Compostos de Vinila/química , Células HeLa , Humanos , Interações Hidrofóbicas e Hidrofílicas , Microscopia Confocal/métodos , Complexo de Endopeptidases do Proteassoma/análise , Coloração e Rotulagem/métodosRESUMO
Virus capsids, i.e., viruses devoid of their genetic material, are suitable nanocarriers for biomedical applications such as drug delivery and diagnostic imaging. For this purpose, the reliable encapsulation of cargo in such a protein nanocage is crucial, which can be accomplished by the covalent attachment of the compounds of interest to the protein domains positioned at the interior of the cage. This approach is particularly valid for the capsid proteins of the cowpea chlorotic mottle virus (CCMV), which have their N-termini located at the inside of the capsid structure. Here, we examined several site-selective modification methods for covalent attachment and encapsulation of cargo at the N-terminus of the CCMV protein. Initially, we explored approaches to introduce an N-terminal azide functionality, which would allow the subsequent bioorthogonal modification with a strained alkyne to attach the desired cargo. As these methods showed compatibility issues with the CCMV capsid proteins, a strategy based on 2-pyridinecarboxaldehydes for site-specific N-terminal protein modification was employed. This method allowed the successful modification of the proteins, and was applied for the introduction of a bioorthogonal vinylboronic acid moiety. In a subsequent reaction, the proteins could be modified further with a fluorophore using the tetrazine ligation. The application of capsid assembly conditions on the functionalized proteins led to successful particle formation, showing the potential of this covalent encapsulation strategy.
Assuntos
Nanoestruturas , Proteínas/química , Bromovirus/química , Capsídeo/química , Proteínas do Capsídeo/química , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Ciclização , Eletroforese em Gel de Poliacrilamida , Espectroscopia de Prótons por Ressonância Magnética , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Autoreactive B cells are thought to play a pivotal role in many autoimmune diseases. Rheumatoid arthritis (RA) is an autoimmune disease affecting â¼1% of the Western population and is hallmarked by the presence of anticitrullinated proteins antibodies (ACPA) produced by autoreactive B cells. We intend to develop a method to target and selectively eliminate these autoreactive B cells using a sequential antigen prodrug targeting strategy. As ACPA-expressing B cells are thought to play essential roles in RA-disease pathogenesis, we used this B cell response as a prototype to analyze the feasibility to generate a construct consisting of a biologically silenced, that is, blocked, antigen connected to a cytotoxic prodrug. Blocking of the antigen is considered relevant as it is anticipated that circulating autoantibodies will otherwise clear the antigen-prodrug before it can reach the target cell. The antigen-prodrug can only bind to the autoantigen-specific B cell receptor (BCR) upon enzymatic removal of the blocking group in close proximity of the B cell surface. BCR binding ultimately induces antigen-specific cytotoxicity after internalization of the antigen. We have synthesized a cyclic citrullinated peptide (CCP) antigen suitable for BCR binding and demonstrated that binding by ACPA was impaired upon introduction of a carboxy- p-nitrobenzyl (CNBz) blocking group at the side chain of the citrulline residue. Enzymatic removal of the CNBz moiety by nitroreductase fully restored citrulline-specific recognition by both ACPA and ACPA-expressing B cells and showed targeted cell death of CCP-recognizing B cells only. These results mark an important step toward antigen-specific B cell targeting in general and more specifically in RA, as successful blocking and activation of citrullinated antigens forms the basis for subsequent use of such construct as a prodrug in the context of autoimmune diseases.
Assuntos
Artrite Reumatoide/tratamento farmacológico , Linfócitos B/efeitos dos fármacos , Epitopos de Linfócito B/administração & dosagem , Peptídeos Cíclicos/imunologia , Pró-Fármacos/administração & dosagem , Anticorpos Antiproteína Citrulinada/imunologia , Anticorpos Antiproteína Citrulinada/metabolismo , Artrite Reumatoide/sangue , Artrite Reumatoide/imunologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Linhagem Celular , Epitopos de Linfócito B/química , Estudos de Viabilidade , Humanos , Terapia de Alvo Molecular/métodos , Pró-Fármacos/química , Receptores de Antígenos de Linfócitos B/antagonistas & inibidores , Receptores de Antígenos de Linfócitos B/imunologia , Receptores de Antígenos de Linfócitos B/metabolismoRESUMO
Numerous molecular components have been identified that regulate the directed migration of eukaryotic cells toward sources of chemoattractant. However, how the components of this system are wired together to coordinate multiple aspects of the response, such as directionality, speed, and sensitivity to stimulus, remains poorly understood. Here we developed a method to shape chemoattractant gradients optically and analyze cellular chemotaxis responses of hundreds of living cells per well in 96-well format by measuring speed changes and directional accuracy. We then systematically characterized migration and chemotaxis phenotypes for 285 siRNA perturbations. A key finding was that the G-protein Giα subunit selectively controls the direction of migration while the receptor and Gß subunit proportionally control both speed and direction. Furthermore, we demonstrate that neutrophils chemotax persistently in response to gradients of fMLF but only transiently in response to gradients of ATP. The method we introduce is applicable for diverse chemical cues and systematic perturbations, can be used to measure multiple cell migration and signaling parameters, and is compatible with low- and high-resolution fluorescence microscopy.
Assuntos
Quimiotaxia , Raios Ultravioleta , Trifosfato de Adenosina/farmacologia , Linhagem Celular Tumoral , Fatores Quimiotáticos/farmacologia , Quimiotaxia/efeitos dos fármacos , Relação Dose-Resposta a Droga , Desenho de Equipamento , Fluoresceínas/análise , Corantes Fluorescentes/análise , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/fisiologia , Humanos , Processamento de Imagem Assistida por Computador , Leucemia Mieloide Aguda/patologia , Microscopia de Fluorescência/métodos , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Proteínas de Neoplasias/fisiologia , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Fotoquímica , RNA Interferente Pequeno/farmacologia , Imagem com Lapso de TempoRESUMO
Bioorthogonal reactions are widely used for the chemical modification of biomolecules. The application of vinylboronic acids (VBAs) as non-strained, synthetically accessible and water-soluble reaction partners in a bioorthogonal inverse electron-demand Diels-Alder (iEDDA) reaction with 3,6-dipyridyl-s-tetrazines is described. Depending on the substituents, VBA derivatives give second-order rate constants up to 27â m(-1) s(-1) in aqueous environments at room temperature, which is suitable for biological labeling applications. The VBAs are shown to be biocompatible, non-toxic, and highly stable in aqueous media and cell lysate. Furthermore, VBAs can be used orthogonally to the strain-promoted alkyne-azide cycloaddition for protein modification, making them attractive complements to the bioorthogonal molecular toolbox.
RESUMO
Citrullination is the conversion of peptidylarginine to peptidylcitrulline, which is catalyzed by peptidylarginine deiminases. This conversion is involved in different physiological processes and is associated with several diseases, including cancer and rheumatoid arthritis. A common method to detect citrullinated proteins relies on anti-modified citrulline antibodies directed to a specific chemical modification of the citrulline side chain. Here, we describe a versatile, antibody-independent method for the detection of citrullinated proteins on a membrane, based on the selective reaction of phenylglyoxal with the ureido group of citrulline under highly acidic conditions. The method makes use of 4-azidophenylglyoxal, which, after reaction with citrullinated proteins, can be visualized with alkyne-conjugated probes. The sensitivity of this procedure, using an alkyne-biotin probe, appeared to be comparable to the antibody-based detection method and independent of the sequence surrounding the citrulline.
Assuntos
Western Blotting , Citrulina/química , Fenilglioxal/química , Proteínas/química , Animais , Western Blotting/métodos , Catálise , Humanos , Hidrolases/metabolismo , Indicadores e Reagentes/química , Desiminases de Arginina em Proteínas , Proteínas/metabolismo , Coloração e RotulagemRESUMO
Bioorthogonal click chemistry has played a transformative role in many research fields, including chemistry, biology, and medicine. Click reactions are crucial to produce increasingly complex bioconjugates, to visualize and manipulate biomolecules in living systems and for various applications in bioengineering and drug delivery. As biological (model) systems grow more complex, researchers have an increasing need for using multiple orthogonal click reactions simultaneously. In this review, we will introduce the most common bioorthogonal reactions and discuss their orthogonal use on the basis of their mechanism and electronic or steric tuning. We provide an overview of strategies to create reaction orthogonality and show recent examples of mutual orthogonal chemistry used for simultaneous biomolecule labeling. We end by discussing some considerations for the type of chemistry needed for labeling biomolecules in a system of choice.
Assuntos
Química Click , Proteínas/químicaRESUMO
The ability to rapidly regulate the functions of specific proteins in living cells is a valuable tool for biological research. Here we describe a new technique by which the degradation of a specific protein is induced by a small molecule. A protein of interest is fused to a ligand-induced degradation (LID) domain, resulting in the expression of a stable and functional fusion protein. The LID domain is comprised of the FK506- and rapamycin-binding protein (FKBP) and a 19-amino-acid degron fused to the C terminus of FKBP. In the absence of the small molecule Shield-1, the degron is bound to the FKBP fusion protein and the protein is stable. When present, Shield-1 binds tightly to FKBP, displacing the degron and inducing rapid and processive degradation of the LID domain and any fused partner protein. Structure-function studies of the 19-residue peptide showed that a 4-amino-acid sequence within the peptide is responsible for degradation.
Assuntos
Desnaturação Proteica , Proteínas de Ligação a Tacrolimo/química , Sequência de Aminoácidos , Animais , Proteínas de Bactérias , Domínio Catalítico , Linhagem Celular , Clonagem Molecular , Regulação da Expressão Gênica/fisiologia , Proteínas Luminescentes , Camundongos , Modelos Moleculares , Morfolinas/farmacologia , Peptídeos/metabolismo , Estrutura Terciária de Proteína , Proteínas de Ligação a Tacrolimo/genética , Proteínas de Ligação a Tacrolimo/metabolismoRESUMO
Profiling the nascent cellular proteome and capturing early proteomic changes in response to external stimuli provides valuable insights into cellular physiology. Existing metabolic protein labeling approaches based on bioorthogonal methionine- or puromycin analogs allow for the selective visualization and enrichment of newly synthesized proteins. However, their applications are limited as they often require methionine-free conditions, auxotrophic cells and/or are toxic to cells. Here, we introduce THRONCAT, a threonine-derived non-canonical amino acid tagging method based on the bioorthogonal threonine analog ß-ethynylserine (ßES) that enables efficient labeling of the nascent proteome in complete growth media within minutes. We use THRONCAT for the visualization and enrichment of nascent proteins in bacteria, mammalian cells and Drosophila melanogaster. We profile immediate proteome dynamics of B-cells in response to B-cell receptor activation simply by adding ßES to the culture medium, demonstrating the ease-of-use of the method and its potential to address diverse biological questions. In addition, using a Drosophila model of Charcot-Marie-Tooth peripheral neuropathy, we show that THRONCAT enables visualization and quantification of relative protein synthesis rates in specific cell types in vivo.
Assuntos
Proteoma , Treonina , Animais , Proteoma/metabolismo , Drosophila melanogaster/metabolismo , Proteômica , Aminoácidos/metabolismo , Metionina/metabolismo , Mamíferos/metabolismoRESUMO
Small heat shock proteins (sHSPs) are essential ATP-independent chaperones that protect the cellular proteome. These proteins assemble into polydisperse oligomeric structures, the composition of which dramatically affects their chaperone activity. The biomolecular consequences of variations in sHSP ratios, especially inside living cells, remain elusive. Here, we study the consequences of altering the relative expression levels of HspB2 and HspB3 in HEK293T cells. These chaperones are partners in a hetero-oligomeric complex, and genetic mutations that abolish their mutual interaction are associated with myopathic disorders. HspB2 displays three distinct phenotypes when co-expressed with HspB3 at varying ratios. Expression of HspB2 alone leads to formation of liquid nuclear condensates, while shifting the stoichiometry towards HspB3 resulted in the formation of large solid-like aggregates. Only cells co-expressing HspB2 with a limited amount of HspB3 formed fully soluble complexes that were distributed homogeneously throughout the nucleus. Strikingly, both condensates and aggregates were reversible, as shifting the HspB2:HspB3 balance in situ resulted in dissolution of these structures. To uncover the molecular composition of HspB2 condensates and aggregates, we used APEX-mediated proximity labelling. Most proteins interact transiently with the condensates and were neither enriched nor depleted in these cells. In contrast, we found that HspB2:HspB3 aggregates sequestered several disordered proteins and autophagy factors, suggesting that the cell is actively attempting to clear these aggregates. This study presents a striking example of how changes in the relative expression levels of interacting proteins affects their phase behavior. Our approach could be applied to study the role of protein stoichiometry and the influence of client binding on phase behavior in other biomolecular condensates and aggregates.
Assuntos
Proteínas de Choque Térmico Pequenas , Proteínas de Choque Térmico , Humanos , Proteínas de Choque Térmico/metabolismo , Proteínas de Choque Térmico Pequenas/genética , Células HEK293 , Proteínas de Choque Térmico HSP27/química , Núcleo Celular/metabolismo , Agregados ProteicosRESUMO
Multivalent scaffolds that carry multiple molecules with immunophenotyping or immunomodulatory properties are invaluable tools for studying and modulating specific functions of human immune responses. So far, streptavidin-biotin-based tetramers have been widely used for B-cell immunophenotyping purposes. However, the utility of these tetramers is limited by their tetravalency, the inherent immunogenicity of streptavidin (a bacterial protein that can potentially be recognized by B cells), and the limited feasibility to functionalize these reagents. This has rendered tetramers suboptimal for studying rare, in particular, antigen-specific B-cell populations in the context of clinical applications. Here, we used polyisocyanopeptides (PICs), multivalent polymeric scaffolds functionalized with around 50 peptide antigens, to detect autoreactive B cells in the peripheral blood of patients with rheumatoid arthritis. To explore the potential immunomodulatory functionalities, we functionalized PICs with autoantigenic peptides and a trisaccharide CD22 ligand to inhibit autoreactive B-cell activation through interference with the B-cell receptor activation pathway, as evidenced by reduced phospho-Syk expression upon PIC binding. Given the possibilities to functionalize PICs, our data demonstrate that the modular and versatile character of PIC scaffolds makes them promising candidates for future clinical applications in B-cell-mediated diseases.