RESUMEN
The Cytomatrix Assembled at the active Zone (CAZ) of a presynaptic terminal displays electron-dense appearance and defines the center of the synaptic vesicle release. The protein constituents of CAZ are multiple-domain scaffolds that interact extensively with each other and also with an ensemble of synaptic vesicle proteins to ensure docking, fusion, and recycling. Reflecting the central roles of the active zone in synaptic transmission, CAZ proteins are highly conserved throughout evolution. As the nervous system increases complexity and diversity in types of neurons and synapses, CAZ proteins expand in the number of gene and protein isoforms and interacting partners. This chapter summarizes the discovery of the core CAZ proteins and current knowledge of their functions.
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Sinapsis , Vesículas Sinápticas , Humanos , Neuronas , Transmisión SinápticaRESUMEN
In neuronal synapses, autophagosome biogenesis is coupled with the activity-dependent synaptic vesicle cycle via ATG-9. How vesicles containing ATG-9 are sorted at the presynapse is unknown. We performed forward genetic screens at single synapses of C. elegans neurons for mutants that disrupt ATG-9 presynaptic localization, and identified the long isoform of the active zone protein CLA-1 (Clarinet; CLA-1 L). We find that disrupting CLA-1 L results in abnormal accumulation of ATG-9-containing vesicles enriched with clathrin. The adaptor protein complexes and proteins at the periactive zone genetically interact with CLA-1 L in ATG-9 sorting. Moreover, the phenotype of the ATG-9 protein in cla-1(L) mutants was not observed for integral synaptic vesicle proteins, suggesting distinct mechanisms that regulate sorting of ATG-9-containing vesicles and synaptic vesicles. Our findings reveal novel roles for active zone proteins in the sorting of ATG-9 and in presynaptic macroautophagy/autophagy.
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Autofagia , Caenorhabditis elegans , Animales , Caenorhabditis elegans/metabolismo , Proteínas Portadoras/metabolismo , Terminales Presinápticos/metabolismo , Sinapsis/metabolismo , Vesículas Sinápticas/metabolismoRESUMEN
Glucagon-like peptide-1 receptor agonist (GLP-1RA) has been clinically proven to protect endothelial function. Previously, we demonstrated that endothelial NO synthase (eNOS) was activated by high-density lipoprotein (HDL) via its scavenger receptor of the B class/human homologue of SR-BI, CD36 and LIMPII analogous-1(hSR-BI/CLA-1). Here, we investigated the effect of GLP-1RA and exendin-4 on the expression of hSR-BI/CLA-1 in HUVECs. Our results confirmed that GLP-1R was expressed in HUVECs by PCR and exendin-4 significantly enhanced HDL-induced eNOS activation. Next, exendin-4 increased the expression of hSR-BI/CLA-1 and a blockade of GLP-1R cancelled this effect. Further, the hSR-BI/CLA-1 transcriptional activity was enhanced by exendin-4, which was diminished by the inhibition of AMPK or dominant-negative AMPK-α-subunit. Moreover, AMPK was phosphorylated by the activation of GLP-1R. Next, ChIP assay demonstrated that exendin-4 increased the FoxO1-binding in the hSR-BI/CLA-1 promoter by upregulation of FoxO1. Mutation of FoxO1-binding or silencing of FoxO1 cancelled the effect of exendin-4 on hSR-BI/CLA-1 expression. Exendin-4 reduced FoxO1 phosphorylation and induced its nuclear accumulation, while this effect was altered by the blocking of GLP-1R or inhibition of AMPK pathway. In summary, our results proved that exendin-4 increased hSR-BI/CLA-1 expression via the AMPK/FoxO1 pathway to activate eNOS, providing a basic mechanism underlining the protective effect of GLP-1RA on endothelial function.
RESUMEN
Vascular complications are the main cause of morbidity and mortality in diabetic patients, and advanced glycation end products (AGEs) play a critical role in promoting diabetic vascular dysfunction. The human homolog of scavenger receptor class B type I (SR-BI), CD36, and LIMPII analog-1 (hSR-BI/CLA-1) facilitates the cellular uptake of cholesterol from HDL. In endothelial cells, HDL activates endothelial nitric oxide synthase (eNOS) via hSR-BI/CLA-1. In this study, we elucidated the effects of AGEs on hSR-BI/CLA-1 expression in human umbilical vein endothelial cells (HUVECs). HSR-BI/CLA-1 expression was examined by real-time PCR, western blot analysis, and reporter gene assay in HUVECs incubated with AGEs. eNOS activity was assessed by detecting the phosphorylation (Ser 1179) of eNOS. Our results showed that AGEs decreased the endogenous expression of hSR-BI/CLA-1. AGEs also inhibited the activity of the hSR-BI/CLA-1 promoter and its mRNA expression via receptor RAGE. We identified the binding site for Smad1 on the hSR-BI/CLA-1 promoter: Smad1 bound to its promoter. AGE treatment stimulated the transcriptional activity of Smad1, and mutation of the Smad1 binding site inhibited the effect of AGEs on the hSR-BI/CLA-1 promoter. HDL-treatment enhanced the phosphorylation of eNOS at Ser 1179, but pretreatment with AGEs inhibited the phosphorylation of eNOS Ser 1179. These results suggested that AGEs downregulate the expression of the endothelial hSR-BI/CLA-1 via the Smad1 pathway, which may be a therapeutic target for diabetic endothelial dysfunction.
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Antígenos CD36/genética , Regulación de la Expresión Génica , Productos Finales de Glicación Avanzada/farmacología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Proteína Smad1/metabolismo , Secuencia de Bases , Antígenos CD36/metabolismo , ADN/genética , Activación Enzimática/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Humanos , Óxido Nítrico Sintasa de Tipo III/metabolismo , Fosforilación/efectos de los fármacos , Regiones Promotoras Genéticas , Receptor para Productos Finales de Glicación Avanzada/metabolismo , Transcripción Genética/efectos de los fármacosRESUMEN
Halogenated compounds are widely discovered in nature, and many of them exhibit biological activities, such as an important chlorinated natural product salinosporamide A serving as a potential anticancer agent. Compared with bromination, iodination and fluorination, chlorination is the mainly important modification. To shed light on the mechanism of SAM-dependent chlorinases, a recombinant chlorinase ClA1 was expressed in Escherichia coli and further purified for crystallization and X-ray diffraction experiments. The flake crystals of ClA1 were able to diffract to a resolution of 1.85 Å. The crystals belonged to space group R3, with unit-cell parameters α = ß = 90.0°, γ = 120.0°. By determining the structure of ClA1, it is revealed that the side chain of Arg242 in ClA1 may have contacts with the L-Met. However, in SalL the equivalent Arg243's side chain is far from L-Met. Considering the ClA1 and SalL are from different environments and their enzyme kinetics are quite different, it is suggested that the side chain conformation differences of the conserved arginine are possibly related with the enzyme activity differences of the two chlorinases.
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Proteínas Bacterianas/química , Streptomyces/química , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Cristalografía por Rayos X , Halogenación , Conformación Proteica , S-Adenosilmetionina/metabolismo , Alineación de Secuencia , Microbiología del Suelo , Streptomyces/metabolismo , Especificidad por SustratoRESUMEN
Phosphatidylcholine is a constituent of Chlamydia trachomatis membranes that must be acquired from its mammalian host to support bacterial proliferation. The CLA1 (SR-B1) receptor is a bi-directional phosphatidylcholine/cholesterol transporter that is recruited to the inclusion of Chlamydia-infected cells along with ABCA1. C. trachomatis growth was inhibited in a dose-dependent manner by BLT-1, a selective inhibitor of CLA1 function. Expression of a BLT-1-insensitive CLA1(C384S) mutant ameliorated the effect of the drug on chlamydial growth. CLA1 knockdown using shRNAs corroborated an important role for CLA1 in the growth of C. trachomatis. Trafficking of a fluorescent phosphatidylcholine analogue to Chlamydia was blocked by the inhibition of CLA1 or ABCA1 function, indicating a critical role for these transporters in phosphatidylcholine acquisition by this organism. Our analyses using a dual-labelled fluorescent phosphatidylcholine analogue and mass spectrometry showed that the phosphatidylcholine associated with isolated Chlamydia was unmodified host phosphatidylcholine. These results indicate that C. trachomatis co-opts host phospholipid transporters normally used to assemble lipoproteins to acquire host phosphatidylcholine essential for growth.