RESUMO
To eliminate infected and cancerous cells, antigen processing and presentation play a pivotal role through the recognition of antigenic peptides displayed on Major Histocompatibility Complex classâ I (MHCâ I) molecules. Here, we developed a photostimulated antigen release system that enables the temporal inception of antigen flux. Simple and effective photocaging of the human immunodeficiency virus (HIV)-Nef73-derived epitope, a representative high-affinity MHCâ I ligand, was provided by steric hindrance to block the recognition by the transporter associated with antigen processing (TAP) in the peptide loading complex (PLC). In response to light, a heteronomous release of antigens and subsequent translocation in various scenarios is demonstrated, including a TAP-related ATP-binding cassette (ABC) transporter reconstituted in liposomes and the native PLC in the endoplasmic reticulum (ER) membrane of human cells. The photochemically induced 'burst' of antigens opens new opportunities for a mechanistic analysis of the antigen translocation machinery and will help to provide insights into antigen processing pathways via an on-demand, subcellular pulse-chase release of antigens.
RESUMO
Membrane receptor clustering is fundamental to cell-cell communication; however, the physiological function of receptor clustering in cell signaling remains enigmatic. Here, we developed a dynamic platform to induce cluster formation of neuropeptide Y2 hormone receptors (Y2R) in situ by a chelator nanotool. The multivalent interaction enabled a dynamic exchange of histidine-tagged Y2R within the clusters. Fast Y2R enrichment in clustered areas triggered ligand-independent signaling as determined by an increase in cytosolic calcium and cell migration. Notably, the calcium and motility response to ligand-induced activation was amplified in preclustered cells, suggesting a key role of receptor clustering in sensitizing the dose response to lower ligand concentrations. Ligand-independent versus ligand-induced signaling differed in the binding of arrestin-3 as a downstream effector, which was recruited to the clusters only in the presence of the ligand. This approach allows in situ receptor clustering, raising the possibility to explore different receptor activation modalities.
Assuntos
Histidina , Neuropeptídeo Y , Neuropeptídeo Y/metabolismo , Cálcio/metabolismo , beta-Arrestina 2/metabolismo , Ligantes , Transdução de Sinais , Receptores de Neuropeptídeos/metabolismo , Quelantes , HormôniosRESUMO
With the advent of single-molecule methods, chemoselective and site-specific labeling of proteins evolved to become a central aspect in chemical biology as well as cell biology. Protein labeling demands high specificity, rapid as well as efficient conjugation, while maintaining low concentration and biocompatibility under physiological conditions. Generic methods that do not interfere with the function, dynamics, subcellular localization of proteins, and crosstalk with other factors are crucial to probe and image proteins inâ vitro and in living cells. Alternatives to enzyme-based tags or autofluorescent proteins are short peptide-based recognition tags. These tags provide high specificity, enhanced binding rates, bioorthogonality, and versatility. Here, we report on recent applications of multivalent chelator heads, recognizing oligohistidine-tagged proteins. The striking features of this system has facilitated the analysis of protein complexes by single-molecule approaches.
Assuntos
Quelantes/química , Proteínas/análise , Coloração e Rotulagem , Estrutura MolecularRESUMO
The photostability of fluorescent labels comprises one of the main limitations in single-molecule fluorescence (SMF) and super-resolution imaging. An attractive strategy to increase the photostability of organic fluorophores relies on their coupling to photostabilizers, e.g., triplet excited state quenchers, rendering self-healing dyes. Herein we report the self-healing properties of trisNTA-Alexa647 fluorophores (NTA, N-nitrilotriacetic acid). Primarily designed to specifically label biomolecules containing an oligohistidine tag, we hypothesized that the increased effective concentration of Ni(II) triplet state quenchers would lead to their improved photostability. We evaluated photon output, survival time, and photon count rate of different Alexa647-labeled trisNTA constructs differing in the length and rigidity of the fluorophore- trisNTA linker. Maximum photon output enhancements of 25-fold versus Alexa647-DNA were recorded for a short tetraproline linker, superseding the solution based photostabilization by Ni(II). Steady-state and time-resolved studies illustrate that trisNTA self-healing role is associated with a dynamic excited triplet state quenching by Ni(II). Here improved photophysical/photochemical properties require for a judicious choice of linker length and rigidity, and in turn a balance between rapid dynamic triplet excited state quenching versus dynamic/static singlet excited state quenching. TrisNTA fluorophores offer superior properties for SMF allowing specific labeling and increased photostability, making them ideal candidates for extended single-molecule imaging techniques.
Assuntos
Corantes Fluorescentes/química , Ácido Nitrilotriacético/química , Imagem Óptica , Microscopia de Fluorescência , Estrutura MolecularRESUMO
Influenza viruses have a segmented viral RNA (vRNA) genome, which is replicated by the viral RNA-dependent RNA polymerase (RNAP). Replication initiates on the vRNA 3' terminus, producing a complementary RNA (cRNA) intermediate, which serves as a template for the synthesis of new vRNA. RNAP structures show the 3' terminus of the vRNA template in a pre-initiation state, bound on the surface of the RNAP rather than in the active site; no information is available on 3' cRNA binding. Here, we have used single-molecule Förster resonance energy transfer (smFRET) to probe the viral RNA conformations that occur during RNAP binding and initial replication. We show that even in the absence of nucleotides, the RNAP-bound 3' termini of both vRNA and cRNA exist in two conformations, corresponding to the pre-initiation state and an initiation conformation in which the 3' terminus of the viral RNA is in the RNAP active site. Nucleotide addition stabilises the 3' vRNA in the active site and results in unwinding of the duplexed region of the promoter. Our data provide insights into the dynamic motions of RNA that occur during initial influenza replication and has implications for our understanding of the replication mechanisms of similar pathogenic viruses.
Assuntos
Transferência Ressonante de Energia de Fluorescência , Vírus da Influenza A/genética , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas , RNA Viral/química , RNA Viral/genética , Transcrição Gênica , Microscopia Confocal , Modelos Biológicos , Modelos Moleculares , Ligação Proteica , RNA Polimerase Dependente de RNA/metabolismo , Iniciação da Transcrição Genética , Replicação ViralRESUMO
The peptide-loading complex plays a pivotal role in Ag processing and is thus central to the efficient immune recognition of virally and malignantly transformed cells. The underlying mechanism by which MHC class I (MHC I) molecules sample immunodominant peptide epitopes, however, remains poorly understood. In this article, we delineate the interaction between tapasin (Tsn) and MHC I molecules. We followed the process of peptide editing in real time after ultra-fast photoconversion to pseudoempty MHC I molecules. Tsn discriminates between MHC I loaded with optimal and MHC I bound to suboptimal cargo. This differential interaction is key to understanding the kinetics of epitope proofreading. To elucidate the underlying mechanism at the atomic level, we modeled the Tsn/MHC I complex using all-atom molecular dynamics simulations. We present a catalytic working cycle, in which Tsn binds to MHC I with suboptimal cargo and thereby adjusts the energy landscape in favor of MHC I complexes with immunodominant epitopes.
Assuntos
Epitopos/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Peptídeos/metabolismo , Sequência de Aminoácidos , Epitopos/química , Epitopos/genética , Polarização de Fluorescência , Antígeno HLA-B44/química , Antígeno HLA-B44/genética , Antígeno HLA-B44/metabolismo , Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Epitopos Imunodominantes/química , Epitopos Imunodominantes/genética , Epitopos Imunodominantes/metabolismo , Cinética , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Simulação de Dinâmica Molecular , Mutação , Peptídeos/química , Peptídeos/genética , Ligação Proteica , Isomerases de Dissulfetos de Proteínas/química , Isomerases de Dissulfetos de Proteínas/genética , Isomerases de Dissulfetos de Proteínas/metabolismo , Estrutura Terciária de Proteína , TermodinâmicaRESUMO
Coevolution of viruses and their hosts represents a dynamic molecular battle between the immune system and viral factors that mediate immune evasion. After the abandonment of smallpox vaccination, cowpox virus infections are an emerging zoonotic health threat, especially for immunocompromised patients. Here we delineate the mechanistic basis of how cowpox viral CPXV012 interferes with MHC class I antigen processing. This type II membrane protein inhibits the coreTAP complex at the step after peptide binding and peptide-induced conformational change, in blocking ATP binding and hydrolysis. Distinct from other immune evasion mechanisms, TAP inhibition is mediated by a short ER-lumenal fragment of CPXV012, which results from a frameshift in the cowpox virus genome. Tethered to the ER membrane, this fragment mimics a high ER-lumenal peptide concentration, thus provoking a trans-inhibition of antigen translocation as supply for MHC I loading. These findings illuminate the evolution of viral immune modulators and the basis of a fine-balanced regulation of antigen processing.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Antígenos Virais/genética , Vírus da Varíola Bovina/genética , Mutação da Fase de Leitura/genética , Genoma Viral/genética , Transportadores de Cassetes de Ligação de ATP/imunologia , Transportadores de Cassetes de Ligação de ATP/fisiologia , Sequência de Aminoácidos , Animais , Antígenos Virais/imunologia , Antígenos Virais/fisiologia , Retículo Endoplasmático , Retroalimentação Fisiológica/fisiologia , Células HeLa , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Dados de Sequência Molecular , Células Sf9RESUMO
Protein labeling with synthetic fluorescent probes is a key technology in chemical biology and biomedical research. A sensitive and efficient modular labeling approach (SLAP) was developed on the basis of a synthetic small-molecule recognition unit (Ni-trisNTA) and the genetically encoded minimal protein His6-10 -tag. High-density protein tracing by SLAP was demonstrated. This technique allows super-resolution fluorescence imaging and fulfills the necessary sampling criteria for single-molecule localization-based imaging techniques. It avoids masking by large probes, for example, antibodies, and supplies sensitive, precise, and robust size analysis of protein clusters (nanodomains).
Assuntos
Actinas/química , Corantes Fluorescentes/química , Lamina Tipo A/química , Microscopia de Fluorescência/métodos , Imagem Molecular/métodos , Animais , Células CHO , Cricetulus , Fluorescência , Células HeLa , Humanos , NanotecnologiaRESUMO
Selective and fast labeling of proteins in living cells is a major challenge. Live-cell labeling techniques require high specificity, high labeling density, and cell permeability of the tagging molecule to target the protein of interest. Here we report on the site-specific, rapid, and efficient labeling of endogenous and recombinant histidine-tagged proteins in distinct subcellular compartments using cell-penetrating multivalent chelator carrier complexes. In vivo labeling was followed in real time in living cells, demonstrating a high specificity and high degree of colocalization in the crowded cellular environment.
Assuntos
Histidina/química , Ácido Nitrilotriacético/química , Produtos do Gene tat do Vírus da Imunodeficiência Humana/química , Sequência de Aminoácidos , Sobrevivência Celular , Células HeLa , Humanos , Cinética , Ácido Nitrilotriacético/metabolismo , Fragmentos de Peptídeos/química , Proteínas Recombinantes/química , Coloração e RotulagemRESUMO
Virus-infected cells are eliminated by cytotoxic T lymphocytes, which recognize viral epitopes displayed on major histocompatibility complex class I molecules at the cell surface. Herpesviruses have evolved sophisticated strategies to escape this immune surveillance. During the lytic phase of EBV infection, the viral factor BNLF2a interferes with antigen processing by preventing peptide loading of major histocompatibility complex class I molecules. Here we reveal details of the inhibition mechanism of this EBV protein. We demonstrate that BNLF2a acts as a tail-anchored protein, exploiting the mammalian Asna-1/WRB (Get3/Get1) machinery for posttranslational insertion into the endoplasmic reticulum membrane, where it subsequently blocks antigen translocation by the transporter associated with antigen processing (TAP). BNLF2a binds directly to the core TAP complex arresting the ATP-binding cassette transporter in a transport-incompetent conformation. The inhibition mechanism of EBV BNLF2a is distinct and mutually exclusive of other viral TAP inhibitors.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Infecções por Vírus Epstein-Barr/metabolismo , Herpesvirus Humano 4/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas da Matriz Viral/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Apresentação de Antígeno/genética , ATPases Transportadoras de Arsenito/genética , ATPases Transportadoras de Arsenito/metabolismo , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/virologia , Infecções por Vírus Epstein-Barr/genética , Células HeLa , Herpesvirus Humano 4/genética , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Complexos Multiproteicos/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Spodoptera , Proteínas da Matriz Viral/genéticaRESUMO
Recent waves of COVID-19 correlate with the emergence of the Delta and the Omicron variant. We report that the Spike trimer acts as a highly dynamic molecular caliper, thereby forming up to three tight bonds through its RBDs with ACE2 expressed on the cell surface. The Spike of both Delta and Omicron (B.1.1.529) Variant enhance and markedly prolong viral attachment to the host cell receptor ACE2, as opposed to the early Wuhan-1 isolate. Delta Spike shows rapid binding of all three Spike RBDs to three different ACE2 molecules with considerably increased bond lifetime when compared to the reference strain, thereby significantly amplifying avidity. Intriguingly, Omicron (B.1.1.529) Spike displays less multivalent bindings to ACE2 molecules, yet with a ten time longer bond lifetime than Delta. Delta and Omicron (B.1.1.529) Spike variants enhance and prolong viral attachment to the host, which likely not only increases the rate of viral uptake, but also enhances the resistance of the variants against host-cell detachment by shear forces such as airflow, mucus or blood flow. We uncover distinct binding mechanisms and strategies at single-molecule resolution, employed by circulating SARS-CoV-2 variants to enhance infectivity and viral transmission.
Assuntos
Enzima de Conversão de Angiotensina 2 , COVID-19 , SARS-CoV-2 , Imagem Individual de Molécula , Glicoproteína da Espícula de Coronavírus , Humanos , Enzima de Conversão de Angiotensina 2/metabolismo , COVID-19/virologia , SARS-CoV-2/metabolismo , SARS-CoV-2/fisiologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Ligação ViralRESUMO
Silaffins are highly charged proteins which are one of the major contributing compounds that are thought to be responsible for the formation of the hierarchically structured silica-based cell walls of diatoms. Here we describe the synthesis of an oligo-propyleneamine substituted lysine derivative and its incorporation into the KXXK peptide motif occurring repeatedly in silaffins. N(ε)-alkylation of lysine was achieved by a Mitsunobu reaction to obtain a protected lysine derivative which is convenient for solid phase peptide synthesis. Quantitative silica precipitation experiments together with structural information about the precipitated silica structures gained by scanning electron microscopy revealed a dependence of the amount and form of the silica precipitates on the peptide structure.
Assuntos
Peptídeos/química , Dióxido de Silício/química , Motivos de Aminoácidos , Parede Celular/química , Diatomáceas/química , Lisina/química , Estrutura MolecularRESUMO
Antigen presentation via major histocompatibility complex class I (MHC I) molecules is essential to mount an adaptive immune response against pathogens and cancerous cells. To this end, the transporter associated with antigen processing (TAP) delivers snippets of the cellular proteome, resulting from proteasomal degradation, into the ER lumen. After peptide loading and editing by the peptide-loading complex (PLC), stable peptide-MHC I complexes are released for cell surface presentation. Since the process of MHC I trafficking is poorly defined, we established an approach to control antigen presentation by introduction of a photo-caged amino acid in the catalytic ATP-binding site of TAP. By optical control, we initiate TAP-dependent antigen translocation, thus providing new insights into TAP function within the PLC and MHC I trafficking in living cells. Moreover, this versatile approach has the potential to be applied in the study of other cellular pathways controlled by P-loop ATP/GTPases.
Assuntos
Imunidade Adaptativa/imunologia , Apresentação de Antígeno/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Proteínas de Membrana Transportadoras/química , Luz , Transporte ProteicoRESUMO
Cell-cell communication relies on the assembly of receptor-ligand complexes at the plasma membrane. The spatiotemporal receptor organization has a pivotal role in evoking cellular responses. We studied the clustering of heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) and established a photoinstructive matrix with ultrasmall lock-and-key interaction pairs to control lateral membrane organization of hormone neuropeptide Y2 receptors in living cells by light. Within seconds, receptor clustering was modulated in size, location, and density. After in situ confinement, changes in cellular morphology, motility, and calcium signaling revealed ligand-independent receptor activation. This approach may enhance the exploration of mechanisms in cell signaling and mechanotransduction.
Assuntos
Membrana Celular/metabolismo , Receptores de Neuropeptídeo Y/metabolismo , Transdução de Sinais , Cálcio/metabolismo , Sinalização do Cálcio , Linhagem Celular , Movimento Celular , Citosol/metabolismo , Difusão , Humanos , Lasers , Ligantes , Lipídeos/análise , Modelos Biológicos , Neuropeptídeo Y/farmacologia , Agregação de Receptores , Receptores de Neuropeptídeo Y/agonistas , Receptores de Neuropeptídeo Y/antagonistas & inibidoresRESUMO
Organic compounds isolated from diatoms contain long-chain polyamines with a propylamine backbone and variable methylation levels and chain lengths. These long-chain polyamines are thought to be one of the important classes of molecules that are responsible for the formation of the hierarchically structured silica-based cell walls of diatoms. Here we describe a synthetic route based on solid-phase peptide synthesis from which well-defined long-chain polyamines with different chain lengths, methylation patterns, and subunits can be obtained. Quantitative silica precipitation experiments together with structural information about the precipitated silica structures gained by scanning and transmission electron microscopy revealed a distinct dependence of the amount, size, and form of the silica precipitates on the molecular structure of the polyamine. Moreover, the influence of the phosphate concentration was elucidated, revealing the importance of divalent anions for the precipitation procedure. We were able to derive further insights into the precipitation properties of long-chain polyamines as functions of their hydrophobicity, protonation state, and phosphate concentration, which may pave the way for better control of the formation of nanostructured silica under ambient conditions.
Assuntos
Materiais Biomiméticos/química , Poliaminas/química , Dióxido de Silício/química , Diatomáceas/química , Estrutura Molecular , Nanoestruturas/química , Nitrogênio/química , Tamanho da Partícula , Poliaminas/síntese química , Propriedades de SuperfícieRESUMO
Ether lipids with alkyl chains of uniform length and varying amine headgroups were synthesized and assembled into bilayer structures in aqueous solution, which served as templates for the formation of silica in two and three dimensions produced under ambient conditions. Dynamic light scattering revealed that unilamellar vesicles of the aminolipids are formed by the extrusion method. The alkylation of the polar amine headgroup was systematically increased from a primary, secondary, and tertiary amine to a quaternary ammonium salt, and the amount of silica was quantified by the beta-silicomolybdate method as a function of the headgroup. A lysinol-connected ether lipid harboring two primary amine groups was also investigated. This variation enabled us to compare the influence of the headgroup on the properties of the precipitated silica in detail. By spreading of unilamellar aminolipid vesicles onto planar silicon substrates, two-dimensional planar bilayers can be produced. By means of ellipsometry in conjunction with atomic force microscopy, we were able to demonstrate that very thin silica layers with a thickness of a few nanometers are formed within minutes on the surface of the aminolipid bilayers. All layers are composed of silica nanospheres, and the thickness turned out to be independent of the amine headgroup.
Assuntos
Dióxido de Silício/química , Lipossomas Unilamelares/química , Microscopia de Força Atômica , Microscopia Eletrônica de Varredura , Microscopia de Fluorescência , Nanosferas/química , Nanosferas/ultraestrutura , Compostos de Amônio Quaternário/químicaRESUMO
Atomic force microscope (AFM) based single molecule force spectroscopy (SMFS) and a quartz crystal microbalance (QCM) were respectively employed to probe interfacial characteristics of fibronectin fragment FNIII8-14 and full-length fibronectin (FN) on CH3-, OH-, COOH-, and NH2-terminated alkane-thiol self-assembled monolayers (SAMs). Force-distance curves acquired between hexahistidine-tagged FNIII8-14 immobilised on trisNTA-Ni2+ functionalized AFM cantilevers and the OH and COOH SAM surfaces were predominantly 'loop-like' (76% and 94% respectively), suggesting domain unfolding and preference for 'end-on' oriented binding, while those generated with NH2 and CH3 SAMs were largely 'mixed type' (81% and 86%, respectively) commensurate with unravelling and desorption, and 'side-on' binding. Time-dependent binding of FN to SAM-coated QCM crystals occurred in at least two phases: initial rapid coverage over the first 5 min; and variably diminishing adsorption thereafter (5-70 min). Loading profiles and the final hydrated surface concentrations reached (~ 950, ~ 1200, ~ 1400, ~ 1500 ng cm-2 for CH3, OH, COOH and NH2 SAMs) were consistent with: space-filling 'side-on' orientation and unfolding on CH3 SAM; greater numbers of FN molecules arranged 'end-on' on OH and especially COOH SAMs; and initial 'side-on' contact, followed by either (1) gradual tilting to a space-saving 'end-on' configuration, or (2) bi-/multi-layer adsorption on NH2 SAM.
Assuntos
Fibronectinas/química , Imagem Individual de Molécula , Adsorção , Ouro/química , Humanos , Propriedades de SuperfícieRESUMO
How membrane proteins oligomerize determines their function. Superresolution microscopy can report on protein clustering and extract quantitative molecular information. Here, we evaluate the blinking kinetics of four photoactivatable fluorescent proteins for quantitative single-molecule microscopy. We identified mEos3.2 and mMaple3 to be suitable for molecular quantification through blinking histogram analysis. We designed synthetic and genetic dimers of mEos3.2 as well as fusion proteins of monomeric and dimeric membrane proteins as reference structures, and we demonstrate their versatile use for quantitative superresolution imaging in vitro and in situ. We further found that the blinking behavior of mEos3.2 and mMaple3 is modified by a reducing agent, offering the possibility to adjust blinking parameters according to experimental needs.
Assuntos
Dimerização , Membrana Celular/metabolismo , Células HeLa , Humanos , Proteínas Luminescentes/metabolismo , Probabilidade , Imagem Individual de MoléculaRESUMO
The interaction between the T4 bacteriophage gp37 adhesin and the bacterial lipopolysaccharide (LPS) is a well-studied system, however, the affinity and strength of the interaction haven't been analyzed so far. Here, we use atomic force microscopy to determine the strength of the interaction between the adhesin and its receptor, namely LPS taken from a wild strain of E. coli B. As negative controls we used LPSs of E. coli O111:B and Hafnia alvei. To study the interaction an AFM tip modified with the gp37 adhesin was used to scan surfaces of mica covered with one of the three different LPSs. Using the correlation between the surface topography images and the tip-surface interaction we could verify the binding between the specific LPS and the tip in contrast to the very weak interaction between the tip and the non-binding LPSs. Using force spectroscopy we could then measure the binding strength by pulling on the AFM tip until it lifted off from the surface. The force necessary to break the interaction between gp37 and LPS from E. coli B, LPS from E. coli O111:B and LPS from H. alvei were measured to be 70 ± 29 pN, 46 ± 13 pN and 45 ± 14 pN, respectively. The latter values are likely partially due to non-specific interaction between the gp37 and the solid surface, as LPS from E. coli O111:B and LPS from H. alvei have been shown to not bind to gp37, which is confirmed by the low correlation between binding and topography for these samples.