RESUMO
The activity-dependent plasticity of synapses is believed to be the cellular basis of learning. These synaptic changes are mediated through the coordination of local biochemical reactions in synapses and changes in gene transcription in the nucleus to modulate neuronal circuits and behavior. The protein kinase C (PKC) family of isozymes has long been established as critical for synaptic plasticity. However, because of a lack of suitable isozyme-specific tools, the role of the novel subfamily of PKC isozymes is largely unknown. Here, through the development of fluorescence lifetime imaging-fluorescence resonance energy transfer activity sensors, we investigate novel PKC isozymes in synaptic plasticity in CA1 pyramidal neurons of mice of either sex. We find that PKCδ is activated downstream of TrkB and DAG production, and that the spatiotemporal nature of its activation depends on the plasticity stimulation. In response to single-spine plasticity, PKCδ is activated primarily in the stimulated spine and is required for local expression of plasticity. However, in response to multispine stimulation, a long-lasting and spreading activation of PKCδ scales with the number of spines stimulated and, by regulating cAMP response-element binding protein activity, couples spine plasticity to transcription in the nucleus. Thus, PKCδ plays a dual functional role in facilitating synaptic plasticity.SIGNIFICANCE STATEMENT Synaptic plasticity, or the ability to change the strength of the connections between neurons, underlies learning and memory and is critical for brain health. The protein kinase C (PKC) family is central to this process. However, understanding how these kinases work to mediate plasticity has been limited by a lack of tools to visualize and perturb their activity. Here, we introduce and use new tools to reveal a dual role for PKCδ in facilitating local synaptic plasticity and stabilizing this plasticity through spine-to-nucleus signaling to regulate transcription. This work provides new tools to overcome limitations in studying isozyme-specific PKC function and provides insight into molecular mechanisms of synaptic plasticity.
Assuntos
Isoenzimas , Transdução de Sinais , Animais , Camundongos , Transdução de Sinais/fisiologia , Sinapses/fisiologia , Plasticidade Neuronal/fisiologia , Proteína Quinase C/metabolismoRESUMO
The development of faster and less expensive methods to discover bioactive small molecules remains a high priority in chemical biology. This article discusses one alternative to traditional high-throughput screening: the synthesis and screening of one bead one compound (OBOC) libraries. Protocols are provided to create and screen libraries of peptoid displayed on TentaGel beads, which is a cheap and relatively straightforward process for the identification of selective protein ligands. However, peptoids bind to proteins with modest affinity in most cases. Therefore, we also describe protocols to create libraries of stiffer oligomers called PICCOs (peptoid-inspired, conformationally constrained oligomers) that have proven to be a superior source of high affinity ligands.
Assuntos
Técnicas de Química Combinatória/métodos , Peptoides/síntese química , Peptoides/farmacologia , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Descoberta de Drogas/métodos , Ensaios de Triagem em Larga Escala/métodos , Humanos , Ligantes , Microesferas , Modelos Moleculares , Peptoides/química , Proteínas/metabolismo , Bibliotecas de Moléculas Pequenas/química , Técnicas de Síntese em Fase Sólida/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodosRESUMO
The circulating antibody repertoire encodes a patient's health status and pathogen exposure history, but identifying antibodies with diagnostic potential usually requires knowledge of the antigen(s). We previously circumvented this problem by screening libraries of bead-displayed small molecules against case and control serum samples to discover "epitope surrogates" (ligands of IgGs enriched in the case sample). Here, we describe an improved version of this technology that employs DNA-encoded libraries and high-throughput FACS-based screening to discover epitope surrogates that differentiate noninfectious/latent (LTB) patients from infectious/active TB (ATB) patients, which is imperative for proper treatment selection and antibiotic stewardship. Normal control/LTB (10 patients each, NCL) and ATB (10 patients) serum pools were screened against a library (5 × 106 beads, 448â¯000 unique compounds) using fluorescent antihuman IgG to label hit compound beads for FACS. Deep sequencing decoded all hit structures and each hit's occurrence frequencies. ATB hits were pruned of NCL hits and prioritized for resynthesis based on occurrence and homology. Several structurally homologous families were identified and 16/21 resynthesized representative hits validated as selective ligands of ATB serum IgGs (p < 0.005). The native secreted TB protein Ag85B (though not the E. coli recombinant form) competed with one of the validated ligands for binding to antibodies, suggesting that it mimics a native Ag85B epitope. The use of DNA-encoded libraries and FACS-based screening in epitope surrogate discovery reveals thousands of potential hit structures. Distilling this list down to several consensus chemical structures yielded a diagnostic panel for ATB composed of thermally stable and economically produced small molecule ligands in place of protein antigens.
Assuntos
Imunoglobulina G/imunologia , Tuberculose Latente/imunologia , Mycobacterium tuberculosis , Oligopeptídeos/imunologia , Tuberculose Pulmonar/imunologia , Aciltransferases/imunologia , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , DNA/genética , Epitopos/imunologia , Escherichia coli , Ensaios de Triagem em Larga Escala , Humanos , Imunoglobulina G/sangue , Tuberculose Latente/sangue , Tuberculose Latente/microbiologia , Ligantes , Oligopeptídeos/síntese química , Biblioteca de Peptídeos , Técnicas de Síntese em Fase Sólida , Relação Estrutura-Atividade , Tuberculose Pulmonar/sangue , Tuberculose Pulmonar/microbiologiaRESUMO
PrP(C), a host protein which in prion-infected animals is converted to PrP(Sc), is linked to the cell membrane by a GPI anchor. Mice expressing PrP(C) without GPI anchor (tgGPIâ» mice), are susceptible to prion infection but accumulate anchorless PrP(Sc) extra-, rather than intracellularly. We investigated whether tgGPIâ» mice could faithfully propagate prion strains despite the deviant structure and location of anchorless PrP(Sc). We found that RML and ME7, but not 22L prions propagated in tgGPIâ» brain developed novel cell tropisms, as determined by the Cell Panel Assay (CPA). Surprisingly, the levels of proteinase K-resistant PrP(Sc) (PrP(res)) in RML- or ME7-infected tgGPIâ» brain were 25-50 times higher than in wild-type brain. When returned to wild-type brain, ME7 prions recovered their original properties, however RML prions had given rise to a novel prion strain, designated SFL, which remained unchanged even after three passages in wild-type mice. Because both RML PrP(Sc) and SFL PrP(Sc) are stably propagated in wild-type mice we propose that the two conformations are separated by a high activation energy barrier which is abrogated in tgGPIâ» mice.
Assuntos
Proteínas PrPC/química , Proteínas PrPSc/química , Scrapie/transmissão , Animais , Western Blotting , Ensaio de Imunoadsorção Enzimática , Glicoproteínas de Membrana/deficiência , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas PrPC/metabolismo , Proteínas PrPSc/metabolismo , Conformação Proteica , Scrapie/metabolismoRESUMO
Prions consist mainly of PrP(Sc), a pathogenic conformer of host-encoded PrP(C). Prion populations with distinct phenotypes but associated with PrP(Sc), having the same amino acid sequence, constitute distinct strains. Strain identity is thought to be encoded by the conformation of PrP(Sc) and to be maintained by seeded conversion. Prion strains can be distinguished by the cell panel assay, which measures their ability to infect distinct cell lines. Brain-derived 22L prions characteristically are able to infect R33 cells (i.e., are "R33 competent"), as well as PK1 cells in the presence of the inhibitor swainsonine (i.e. are "swa resistant"). Here we report that 22L prions retained their characteristic cell tropism and swa resistance when transferred from brain to R33 cells. However, when transferred from the R33 cells to PK1 cells, they gradually became R33 incompetent and swa sensitive, unless the transfer was in the presence of swa, in which case swa resistance and R33 competence were retained. PrP(Sc) associated with swa-resistant/R33-competent and swa-sensitive/R33-incompetent prions had different conformational stabilities. When cloned R33-incompetent/swa-sensitive prions were again propagated in brain, their properties gradually reverted to those of the original brain-derived 22L prions. Our results support the view that 22L prion populations are heterogeneous and that distinct prion variants are selected in different cellular environments.
Assuntos
Encéfalo/metabolismo , Mutação , Proteínas PrPSc/metabolismo , Príons/metabolismo , Animais , Antineoplásicos Fitogênicos/farmacologia , Western Blotting , Encéfalo/patologia , Linhagem Celular Tumoral , Resistência a Medicamentos/genética , Variação Genética , Especificidade de Hospedeiro/genética , Camundongos , Camundongos Endogâmicos C57BL , Proteínas PrPSc/química , Proteínas PrPSc/genética , Príons/efeitos dos fármacos , Príons/genética , Conformação Proteica , Swainsonina/farmacologiaRESUMO
A key adaptation enabling the fetus to survive in a limited energy environment may be the reprogramming of mitochondrial function, which can have deleterious effects. Critical questions are whether mitochondrial dysfunction progressively declines after birth, and if so, what mechanism might underlie this process. To address this, we developed a model of intrauterine growth retardation (IUGR) in the rat that leads to diabetes in adulthood. Reactive oxygen species (ROS) production and oxidative stress gradually increased in IUGR islets. ATP production was impaired and continued to deteriorate with age. The activities of complex I and III of the electron transport chain progressively declined in IUGR islets. Mitochondrial DNA point mutations accumulated with age and were associated with decreased mitochondrial DNA content and reduced expression of mitochondria-encoded genes in IUGR islets. Mitochondrial dysfunction resulted in impaired insulin secretion. These results demonstrate that IUGR induces mitochondrial dysfunction in the fetal beta-cell, leading to increased production of ROS, which in turn damage mitochondrial DNA. A self-reinforcing cycle of progressive deterioration in mitochondrial function leads to a corresponding decline in beta-cell function. Finally, a threshold in mitochondrial dysfunction and ROS production is reached, and diabetes ensues.