RESUMO
The peptidoglycan fragments γ-d-glutamyl- meso-diaminopimelic acid (iE-DAP) and muramyl-dipeptide (MDP) are microbial-specific metabolites that activate intracellular pattern recognition receptors and stimulate immune signaling pathways. While extensive structure-activity studies have demonstrated that these bacterial cell wall metabolites trigger NOD1- and NOD2-dependent signaling, their direct binding to these innate immune receptors or other proteins in mammalian cells has not been established. To characterize these fundamental microbial metabolite-host interactions, we synthesized a series of peptidoglycan metabolite photoaffinity reporters and evaluated their cross-linking to NOD1 and NOD2 in mammalian cells. We show that active iE-DAP and MDP photoaffinity reporters selectively cross-linked NOD1 and NOD2, respectively, and not their inactive mutants. We also discovered MDP reporter cross-linking to Arf GTPases, which interacted most prominently with GTP-bound Arf6 and coimmunoprecipitated with NOD2 upon MDP stimulation. Notably, MDP binding to NOD2 and Arf6 was abrogated with loss-of-function NOD2 mutants associated with Crohn's disease. Our studies demonstrate peptidoglycan metabolite photoaffinity reporters can capture their cognate immune receptors in cells and reveal unpredicted ligand-induced interactions with other cellular cofactors. These photoaffinity reporters should afford useful tools to discover and characterize other peptidoglycan metabolite-interacting proteins.
Assuntos
Fatores de Ribosilação do ADP/metabolismo , Acetilmuramil-Alanil-Isoglutamina/metabolismo , Ácido Diaminopimélico/análogos & derivados , Peptidoglicano/metabolismo , Receptores de Reconhecimento de Padrão/metabolismo , Parede Celular/metabolismo , Citocinas/metabolismo , Ácido Diaminopimélico/metabolismo , Células HEK293 , Humanos , Ligantes , Proteínas Mutantes/metabolismo , Mutação , Proteína Adaptadora de Sinalização NOD1/metabolismo , Proteína Adaptadora de Sinalização NOD2/metabolismo , Ligação Proteica , Transdução de Sinais , Relação Estrutura-AtividadeRESUMO
ADP-ribosylation is a post-translational modification that is known to be involved in cellular homeostasis and stress but has been challenging to analyze biochemically. To facilitate the detection of ADP-ribosylated proteins, we show that an alkyne-adenosine analog, N6-propargyl adenosine (N6pA), is metabolically incorporated in mammalian cells and enables fluorescence detection and proteomic analysis of ADP-ribosylated proteins. Notably, our analysis of N6pA-labeled proteins that are upregulated by oxidative stress revealed differential ADP-ribosylation of small GTPases. We discovered that oxidative stress induced ADP-ribosylation of Hras on Cys181 and Cys184 in the C-terminal hypervariable region, which are normally S-fatty-acylated. Downstream Hras signaling is impaired by ADP-ribosylation during oxidative stress, but is rescued by ADP-ribosyltransferase inhibitors. Our study demonstrates that ADP-ribosylation of small GTPases not only is mediated by bacterial toxins but is endogenously regulated in mammalian cells. N6pA provides a useful tool to characterize ADP-ribosylated proteins and their regulatory mechanisms in cells.
Assuntos
Difosfato de Adenosina/metabolismo , Proteínas Monoméricas de Ligação ao GTP/química , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Estresse Oxidativo , Proteômica , Células Cultivadas , Células HEK293 , Humanos , Estrutura MolecularRESUMO
The rapid development of new small molecule drugs, nanomaterials, and genetic tools to modulate cellular function through cell surface manipulation has revolutionized the diagnosis, study, and treatment of disorders in human health. Since the cell membrane is a selective gateway barrier that serves as the first line of defense/offense and communication to its environment, new approaches that molecularly engineer or tailor cell membrane surfaces would allow for a new era in therapeutic design, therapeutic delivery, complex coculture tissue construction, and in situ imaging probe tracking technologies. In order to develop the next generation of multimodal therapies, cell behavior studies, and biotechnologies that focus on cell membrane biology, new tools that intersect the fields of chemistry, biology, and engineering are required. Herein, we develop a liposome fusion and delivery strategy to present a novel dual receptor and reporter system at cell surfaces without the use of molecular biology or metabolic biosynthesis. The cell surface receptor is based on bio-orthogonal functional groups that can conjugate a range of ligands while simultaneously reporting the conjugation through the emission of fluorescence. We demonstrate this dual receptor and reporter system by conjugating and tracking various cell surface ligands for temporal control of cell fluorescent signaling, cell-cell interaction, and tissue assembly construction.
Assuntos
Lipossomos/metabolismo , Receptores de Superfície Celular/química , Células 3T3 , Animais , Sobrevivência Celular , Citometria de Fluxo , Fluorescência , Humanos , Lipossomos/química , Camundongos , Modelos Biológicos , Engenharia de Proteínas , Receptores de Superfície Celular/metabolismoRESUMO
Kinetochore (KT) localization of mitotic checkpoint proteins is essential for their function during mitosis. hSpindly KT localization is dependent on the RZZ complex and hSpindly recruits the dynein-dynactin complex to KTs during mitosis, but the mechanism of hSpindly KT recruitment is unknown. Through domain-mapping studies we characterized the KT localization domain of hSpindly and discovered it undergoes farnesylation at the C-terminal cysteine residue. The N-terminal 293 residues of hSpindly are dispensable for its KT localization. Inhibition of farnesylation using a farnesyl transferase inhibitor (FTI) abrogated hSpindly KT localization without affecting RZZ complex, CENP-E, and CENP-F KT localization. We showed that hSpindly is farnesylated in vivo and farnesylation is essential for its interaction with the RZZ complex and hence KT localization. FTI treatment and hSpindly knockdown displayed the same mitotic phenotypes, indicating that hSpindly is a key FTI target in mitosis. Our data show a novel role of lipidation in targeting a checkpoint protein to KTs through protein-protein interaction.
Assuntos
Proteínas de Transporte/genética , Segregação de Cromossomos/genética , Cinetocoros/metabolismo , Pontos de Checagem da Fase M do Ciclo Celular/genética , Prenilação de Proteína/genética , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Farnesiltranstransferase/antagonistas & inibidores , Células HEK293 , Células HeLa , Humanos , Células MCF-7 , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Mutação , Estrutura Terciária de Proteína , Interferência de RNA , RNA Interferente Pequeno , Deleção de Sequência , Fuso Acromático/genéticaRESUMO
Host-pathogen protein interactions are fundamental to every microbial infection, yet their identification has remained challenging due to the lack of simple detection tools that avoid abundance biases while providing an open format for experimental modifications. Here, we applied the Nucleic Acid-Programmable Protein Array and a HaloTag-Halo ligand detection system to determine the interaction network of Legionella pneumophila effectors (SidM and LidA) with 10â¯000 unique human proteins. We identified known targets of these L. pneumophila proteins and potentially novel interaction candidates. In addition, we applied our Click chemistry-based NAPPA platform to identify the substrates for SidM, an effector with an adenylyl transferase domain that catalyzes AMPylation (adenylylation), the covalent addition of adenosine monophosphate (AMP). We confirmed a subset of the novel SidM and LidA targets in independent in vitro pull-down and in vivo cell-based assays, and provided further insight into how these effectors may discriminate between different host Rab GTPases. Our method circumvents the purification of thousands of human and pathogen proteins, and does not require antibodies against or prelabeling of query proteins. This system is amenable to high-throughput analysis of effectors from a wide variety of human pathogens that may bind to and/or post-translationally modify targets within the human proteome.
Assuntos
Proteínas de Bactérias/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Interações Hospedeiro-Patógeno/fisiologia , Legionella pneumophila/metabolismo , Análise Serial de Proteínas/métodos , Proteínas/metabolismo , Primers do DNA/genética , DNA Complementar/genética , Humanos , Microscopia de Fluorescência , Plasmídeos/genética , Especificidade da Espécie , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
Bacterial pathogens secrete protein toxins and effectors that hijack metabolites to covalently modify host proteins and interfere with their function during infection. Adenosine metabolites, such as nicotinamide adenine dinucleotide (NAD) and adenosine triphosphate (ATP), have in particular been coopted by these secreted virulence factors to reprogram host pathways. While some host targets for secreted virulence factors have been identified, other toxin and effector substrates have been elusive, which require new methods for their characterization. In this review, we focus on chemical reporters based on NAD and ATP that should facilitate the discovery and characterization of adenosine diphosphate (ADP)-ribosylation and adenylylation/AMPylation in bacterial pathogenesis and cell biology.
Assuntos
Monofosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Interações Hospedeiro-Patógeno , Sondas Moleculares , ADP Ribose Transferases/metabolismo , Adenosina/química , Animais , Humanos , NAD/metabolismo , Nucleotidiltransferases/metabolismoRESUMO
A new strategy to create a dynamic scaffold for three-dimensional (3D) cell experiments based on a photo-activated cell adhesive peptide ligand is described. After polymerization, the inert matrix becomes cell adhesive by chemoselective modification through the conjugation of oxyamine-terminated ligands. Furthermore, spatial and temporal control of cell culture within the 3D matrix was achieved by the use of a biospecific photoprotected peptide and visualized by confocal microscopy.
Assuntos
Técnicas de Cultura de Células/métodos , Fibroblastos/fisiologia , Luz , Técnicas de Cultura de Órgãos/métodos , Peptídeos/metabolismo , Alicerces Teciduais , Animais , Células Cultivadas , Fibroblastos/efeitos dos fármacos , CamundongosRESUMO
AMPylation (adenylylation) is a recently discovered mechanism employed by infectious bacteria to regulate host cell signaling. However, despite significant effort, only a few host targets have been identified, limiting our understanding of how these pathogens exploit this mechanism to control host cells. Accordingly, we developed a novel nonradioactive AMPylation screening platform using high-density cell-free protein microarrays displaying human proteins produced by human translational machinery. We screened 10,000 unique human proteins with Vibrio parahaemolyticus VopS and Histophilus somni IbpAFic2, and identified many new AMPylation substrates. Two of these, Rac2, and Rac3, were confirmed in vivo as bona fide substrates during infection with Vibrio parahaemolyticus. We also mapped the site of AMPylation of a non-GTPase substrate, LyGDI, to threonine 51, in a region regulated by Src kinase, and demonstrated that AMPylation prevented its phosphorylation by Src. Our results greatly expanded the repertoire of potential host substrates for bacterial AMPylators, determined their recognition motif, and revealed the first pathogen-host interaction AMPylation network. This approach can be extended to identify novel substrates of AMPylators with different domains or in different species and readily adapted for other post-translational modifications.
Assuntos
Monofosfato de Adenosina/química , Proteínas de Bactérias/metabolismo , Química Click/métodos , Reação de Cicloadição , Processamento de Proteína Pós-Traducional/fisiologia , Sequência de Bases , Cobre/química , Interações Hospedeiro-Patógeno , Humanos , Pasteurellaceae/metabolismo , Análise Serial de Proteínas , Estrutura Terciária de Proteína , Vibrioses/patologia , Vibrio parahaemolyticus/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Inibidor beta de Dissociação do Nucleotídeo Guanina rho/metabolismo , Proteína RAC2 de Ligação ao GTPRESUMO
Proper cell polarization and division are critical for a developing organism and a number of downstream biological processes including cancer metastasis, cell migration, and organelle organization. Both cell behaviors are complex and influenced by a number of external factors including, the extracellular matrix (ECM), physical-mechanical and hydrodynamic forces. In particular, the ECM functions as a dynamic 3-dimensional scaffold support for tissue segregation and cell adhesion. Although cells are microns in size, they sense and respond to dynamic nanoarchitecture changes of the ECM. To further understand these complex processes model substrates have been developed to recapitulate the spatial presentation of ligands as gradients and single cell patterns. However, until now, the interplay between ligand affinity, ligand density and ligand area at the nanoscale on cell behavior has received little attention due to the lack of synergistic surface chemistry, microscopy, cell biology, and nanopatterning technologies. In this report, we develop biospecific nanopatterned peptide array substrates to examine how the nano-environment controls cell behavior utilizing parallel dip-pen nanolithography.
Assuntos
Divisão Celular , Polaridade Celular , Matriz Extracelular/química , Oligopeptídeos/química , Células 3T3 , Animais , CamundongosRESUMO
We report a switchable redox click and cleave reaction strategy for conjugating and releasing a range of molecules on demand. This chemoselective redox-responsive ligation (CRRL) and release strategy is based on a redox switchable oxime linkage that is controlled by mild chemical or electrochemical redox signals and can be performed at physiological conditions without the use of a catalyst. Both conjugation and release reactions are kinetically well behaved and quantitative. The CRRL strategy is synthetically modular and easily monitored and characterized by routine analytical techniques. We demonstrate how the CRRL strategy can be used for the dynamic generation of cyclic peptides and the ligation of two different peptides that are stable but can be selectively cleaved upon changes in the redox environment. We also demonstrate a new redox based delivery of cargoes to live cells strategy via the CRRL methodology by synthesizing a FRET redox-responsive probe that is selectively activated within a cellular environment. We believe the ease of the CRRL strategy should find wide use in a range of applications in biology, tissue engineering, nanoscience, synthetic chemistry, and material science and will expand the suite of current conjugation and release strategies.
Assuntos
Peptídeos Cíclicos/metabolismo , Animais , Adesão Celular/efeitos dos fármacos , Transferência Ressonante de Energia de Fluorescência , Camundongos , Oxirredução , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/química , Células Swiss 3T3RESUMO
How cells interact with the extracellular matrix (ECM) is important for a number of fundamental -processes in cell biology. However, the ECM is highly complex and in order to simplify the matrix for cell biological studies, it has been modeled with self-assembled monolayers (SAMs) of alkanethiolates on gold substrates. In this chapter, we outline procedures to create dynamic surfaces by functionalizing SAMs. SAMs based on quinone, oxyamine, and alcohol-terminated thiols were used to immobilize cell adhesive peptides with spatial control. Cells were seeded to these surfaces to provide cell co-culture -patterns suitable for biological studies.
Assuntos
Movimento Celular , Técnicas de Cocultura/métodos , Células 3T3 , Álcoois/química , Aldeídos/química , Animais , Benzoquinonas/química , Adesão Celular , Eletroquímica , Proteínas Imobilizadas/química , Indicadores e Reagentes/química , Camundongos , Oximas/química , Peptídeos/química , Impressão , Propriedades de SuperfícieRESUMO
To generate model substrates for cell adhesion, we have developed two different biocompatible strategies based on self-assembled monolayers (SAMs) of alkanethiolates on gold terminated with latent ketones and aldehydes. Under spatial control, the hydroquinone and alcohol-terminated SAMs can be oxidized to allow for oxyamine ligand patterning on the surface with microfluidic cassettes. These immobilization strategies were characterized by electrochemistry, fluorescence, and utilizing a cell adhesive peptide, cell patterns were generated.
Assuntos
Adesão Celular , Eletroquímica/métodos , Microfluídica/métodos , Adesividade , Álcoois/química , Animais , Fibroblastos/citologia , Hidroquinonas/química , Ligantes , Camundongos , Microfluídica/instrumentação , Oxirredução , Células Swiss 3T3RESUMO
We report the fabrication of hybrid nanohole array surfaces to study the role of the surface nanoevironment on cell adhesion and cell migration. We use polystyrene beads and reactive ion etching to control the size and the spacing between nanoholes on a tailored self-assembled monolayer inert gold surface. The arrays were characterized by scanning electron microscopy and brightfield microscopy. For cell adhesion studies, cells were seeded to these substrates to study the effect of ligand spacing on cell spreading, stress fiber formation, and focal adhesion structure and size. Finally, comparative cell migration rates were examined on the various nanohole array surfaces using time-lapse microscopy.
Assuntos
Movimento Celular , Nanoestruturas/química , Nanotecnologia , Células 3T3 , Animais , Adesão Celular , Fibroblastos/citologia , Vidro/química , Ouro/química , Ligantes , Camundongos , Microscopia , Microscopia Eletrônica de Varredura , Fibras de Estresse/metabolismo , Propriedades de Superfície , Fatores de TempoRESUMO
In this work, we develop a new, rapid and inexpensive method to generate spatially controlled aldehyde and carboxylic acid surface groups by microfluidic oxidation of 11-hydroxyundecylphosphonic acid self-assembled monolayers (SAMs) on indium tin oxide (ITO) surfaces. SAMs are activated and patterned using a reversibly sealable, elastomeric polydimethylsiloxane cassette, fabricated with preformed micropatterns by soft lithography. By flowing the mild oxidant pyridinium chlorochromate through the microchannels, only selected areas of the SAM are chemically altered. This microfluidic oxidation strategy allows for ligand immobilization by two chemistries originating from a single SAM composition. ITO is robust, conductive, and transparent, making it an ideal platform for studying interfacial interactions. We display spatial control over the immobilization of a variety of ligands on ITO and characterize the resulting oxime and amide linkages by electrochemistry, X-ray photoelectron spectroscopy, contact angle, fluorescence microscopy, and atomic force microscopy. This general method may be used with many other materials to rapidly generate patterned and tailored surfaces for studies ranging from molecular electronics to biospecific cell-based assays and biomolecular microarrays.
Assuntos
Álcoois/química , Técnicas Analíticas Microfluídicas/métodos , Organofosfonatos/química , Aldeídos/química , Amidas/química , Ácidos Carboxílicos/química , Técnicas Eletroquímicas , Microscopia de Força Atômica , Microscopia de Fluorescência , Oxirredução , Oximas/química , Compostos de Estanho/químicaRESUMO
To generate patterned substrates of self-assembled monolayers (SAMs) for cell adhesion and migration studies, a variety of gold/glass hybrid substrates were fabricated from gold evaporated on glass. A variety of surfaces were generated including gradients of gold height, completely etched gold/glass hybrids, and partially etched gold surfaces for pattern visualization. Etch rates were controlled by the alkanethiol present on the surface. Gradients of gold height were created using an electrochemical etch with control over the position and slope of the gold height gradient. Cells were seeded to these surfaces, and their adhesion to the gold was controlled by the surface chemistry present in the channel regions. In the future, the etched gold surfaces will be used to simulate the varying nanotopology experienced by the migrating cell in vivo.
Assuntos
Movimento Celular , Eletroquímica/métodos , Ouro/química , Técnicas Analíticas Microfluídicas/métodos , Animais , Adesão Celular , Vidro/química , Iodetos/química , Camundongos , Propriedades de SuperfícieRESUMO
In this report, we show the successful transfer of a sophisticated electroactive immobilization and release strategy to an indium tin oxide (ITO) surface to generate (1) optically transparent, robust, and renewable surfaces, (2) inert surfaces that resist nonspecific protein adsorption and cell attachment, and (3) tailored biospecific surfaces for live-cell high-resolution fluorescence microscopy of cell culture. By comparing the surface chemistry properties on both ITO and gold surfaces, we demonstrate the ITO surfaces are superior to gold as a renewable surface, in robustness (durability), and as an optically transparent material for live-cell fluorescence microscopy studies of cell behavior. These advantages will make ITO surfaces a desired platform for numerous biosensor and microarray applications and as model substrates for various cell biological studies.
Assuntos
Eletroquímica/métodos , Óptica e Fotônica , Compostos de Estanho/química , Animais , Biofísica/métodos , Adesão Celular , Físico-Química/métodos , Fibroblastos/metabolismo , Ouro/química , Concentração de Íons de Hidrogênio , Ligantes , Microscopia de Fluorescência/métodos , Modelos Químicos , Ratos , Propriedades de SuperfícieRESUMO
An expedient and inexpensive method to generate patterned aldehydes on self-assembled monolayers (SAMs) of alkanethiolates on gold with control of density for subsequent chemoselective immobilization from commercially available starting materials has been developed. Utilizing microfluidic cassettes, primary alcohol oxidation of tetra(ethylene glycol) undecane thiol and 11-mercapto-1-undecanol SAMs was performed directly on the surface generating patterned aldehyde groups with pyridinium chlorochromate. The precise density of surface aldehydes generated can be controlled and characterized by electrochemistry. For biological applications, fibroblast cells were seeded on patterned surfaces presenting biospecifc cell adhesive (Arg-Glyc-Asp) RGD peptides.
Assuntos
Aldeídos/química , Células 3T3 , Animais , Adesão Celular , Físico-Química/métodos , Eletroquímica/métodos , Etilenoglicol/química , Álcoois Graxos/química , Ligantes , Camundongos , Microfluídica , Microscopia de Fluorescência/métodos , Modelos Químicos , Compostos de Sulfidrila/química , Propriedades de SuperfícieRESUMO
A straightforward, flexible, and inexpensive method to create patterned self-assembled monolayers (SAMs) on gold using microfluidics-microfluidic lithography-has been developed. Using a microfluidic cassette, alkanethiols were rapidly patterned on gold surfaces to generate monolayers and mixed monolayers. The patterning methodology is flexible and, by controlling the solvent conditions and thiol concentration, permeation of alkanethiols into the surrounding PDMS microfluidic cassette can be advantageously used to create different patterned feature sizes and to generate well-defined SAM surface gradients with a single microfluidic chip. To demonstrate the utility of microfluidic lithography, multiple cell experiments were conducted. By patterning cell adhesive regions in an inert background, a combination of selective masking of the surface and centrifugation achieved spatial and temporal control of patterned cells, enabling the design of both dynamic surfaces for directed cell migration and contiguous cocultures. Cellular division and motility resulted in directed, dynamic migration, while the centrifugation-aided seeding of a second cell line produced contiguous cocultures with multiple sites for heterogeneous cell-cell interactions.