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The classic mode of STING activation is through binding the cyclic dinucleotide 2'3'-cyclic GMP-AMP (cGAMP), produced by the DNA sensor cyclic GMP-AMP synthase (cGAS), which is important for the innate immune response to microbial infection and autoimmune disease. Modes of STING activation that are independent of cGAS are much less well understood. Here, through a spatiotemporally resolved proximity labelling screen followed by quantitative proteomics, we identify the lysosomal membrane protein Niemann-Pick type C1 (NPC1) as a cofactor in the trafficking of STING. NPC1 interacts with STING and recruits it to the lysosome for degradation in both human and mouse cells. Notably, we find that knockout of Npc1 'primes' STING signalling by physically linking or 'tethering' STING to SREBP2 trafficking. Loss of NPC1 protein also 'boosts' STING signalling by blocking lysosomal degradation. Both priming and boosting of STING signalling are required for severe neurological disease in the Npc1.sup.-/- mouse. Genetic deletion of Sting1 (the gene that encodes STING) or Irf3, but not that of Cgas, significantly reduced the activation of microglia and relieved the loss of Purkinje neurons in the cerebellum of Npc1.sup.-/- mice, leading to improved motor function. Our study identifies a cGAS- and cGAMP-independent mode of STING activation that affects neuropathology and provides a therapeutic target for the treatment of Niemann-Pick disease type C. The lysosomal protein Niemann-Pick type C1 (NPC1) is identified as a cofactor in the trafficking of STING, and loss of NPC1 is shown to both 'prime' and 'boost' STING signalling leading to neurological disease., Author(s): Ting-Ting Chu [sup.1] , Xintao Tu [sup.1] , Kun Yang [sup.1] , Jianjun Wu [sup.1] , Joyce J. Repa [sup.2] [sup.3] , Nan Yan [sup.1] [sup.4] Author Affiliations: (1) [...]

Dozens of genes contribute to the wide variation in human pigmentation. Many of these genes encode proteins that localize to the melanosome--the organelle, related to the lysosome, that synthesizes pigment--but have unclear functions.sup.1,2. Here we describe MelanoIP, a method for rapidly isolating melanosomes and profiling their labile metabolite contents. We use this method to study MFSD12, a transmembrane protein of unknown molecular function that, when suppressed, causes darker pigmentation in mice and humans.sup.3,4. We find that MFSD12 is required to maintain normal levels of cystine--the oxidized dimer of cysteine--in melanosomes, and to produce cysteinyldopas, the precursors of pheomelanin synthesis made in melanosomes via cysteine oxidation.sup.5,6. Tracing and biochemical analyses show that MFSD12 is necessary for the import of cysteine into melanosomes and, in non-pigmented cells, lysosomes. Indeed, loss of MFSD12 reduced the accumulation of cystine in lysosomes of fibroblasts from patients with cystinosis, a lysosomal-storage disease caused by inactivation of the lysosomal cystine exporter cystinosin.sup.7-9. Thus, MFSD12 is an essential component of the cysteine importer for melanosomes and lysosomes. A rapid labelling and immunopurification-based method is used to isolate melanosomes and profile their labile metabolites, revealing that MFSD12 has a key role in cysteine import into melanosomes and lysosomes., Author(s): Charles H. Adelmann [sup.1] [sup.2] [sup.3] [sup.4] [sup.5] , Anna K. Traunbauer [sup.1] [sup.2] [sup.3] [sup.4] [sup.5] , Brandon Chen [sup.1] [sup.2] [sup.3] [sup.4] [sup.5] , Kendall J. Condon [...]

Piezo1 forms mechanically activated nonselective cation channels that contribute to endothelial response to fluid flow. Here we reveal an important role in the control of capillary density. Conditional endothelial cell-specific deletion of Piezo1 in adult mice depressed physical performance. Muscle microvascular endothelial cell apoptosis and capillary rarefaction were evident and sufficient to account for the effect on performance. There was selective upregulation of thrombospondin-2 (TSP2), an inducer of endothelial cell apoptosis, with no effect on TSP1, a related important player in muscle physiology. TSP2 was poorly expressed in muscle endothelial cells but robustly expressed in muscle pericytes, in which nitric oxide (NO) repressed the Tsp2 gene without an effect on Tsp1. In endothelial cells, Piezo1 was required for normal expression of endothelial NO synthase. The data suggest an endothelial cell-pericyte partnership of muscle in which endothelial Piezo1 senses blood flow to sustain capillary density and thereby maintain physical capability., Introduction Physical fitness is important for survival. Lack of physical activity results in detraining, lower performance, and ultimately, incapacity. At least 2500 years ago it was first documented that exercise [...]

The serotonin transporter (SERT) regulates neurotransmitter homeostasis through the sodium- and chloride-dependent recycling of serotonin into presynaptic neurons.sup.1-3. Major depression and anxiety disorders are treated using selective serotonin reuptake inhibitors--small molecules that competitively block substrate binding and thereby prolong neurotransmitter action.sup.2,4. The dopamine and noradrenaline transporters, together with SERT, are members of the neurotransmitter sodium symporter (NSS) family. The transport activities of NSSs can be inhibited or modulated by cocaine and amphetamines.sup.2,3, and genetic variants of NSSs are associated with several neuropsychiatric disorders including attention deficit hyperactivity disorder, autism and bipolar disorder.sup.2,5. Studies of bacterial NSS homologues--including LeuT--have shown how their transmembrane helices (TMs) undergo conformational changes during the transport cycle, exposing a central binding site to either side of the membrane.sup.1,6-12. However, the conformational changes associated with transport in NSSs remain unknown. To elucidate structure-based mechanisms for transport in SERT we investigated its complexes with ibogaine, a hallucinogenic natural product with psychoactive and anti-addictive properties.sup.13,14. Notably, ibogaine is a non-competitive inhibitor of transport but displays competitive binding towards selective serotonin reuptake inhibitors.sup.15,16. Here we report cryo-electron microscopy structures of SERT-ibogaine complexes captured in outward-open, occluded and inward-open conformations. Ibogaine binds to the central binding site, and closure of the extracellular gate largely involves movements of TMs 1b and 6a. Opening of the intracellular gate involves a hinge-like movement of TM1a and the partial unwinding of TM5, which together create a permeation pathway that enables substrate and ion diffusion to the cytoplasm. These structures define the structural rearrangements that occur from the outward-open to inward-open conformations, and provide insight into the mechanism of neurotransmitter transport and ibogaine inhibition. Cryo-electron microscopy reveals three conformations of the serotonin transporter in complex with ibogaine, detailing the structural rearrangements that occur between the different stages of its transport cycle., Author(s): Jonathan A. Coleman [sup.1] , Dongxue Yang [sup.1] , Zhiyu Zhao [sup.2] , Po-Chao Wen [sup.2] , Craig Yoshioka [sup.3] , Emad Tajkhorshid [sup.2] , Eric Gouaux [sup.1] [sup.4] [...]

The present study was designed to examine the possible neuroprotective and antiepileptic effects of metformin (Metf) in a rat model of pentylenetetrazole (PTZ)-induced epilepsy and its possible underlying mechanisms. Forty male albino rats were assigned to 4 groups of equal size: (1) normal control (NC) group, (2) Metf group: daily treatment with Metf (200 mg/kg, i.p.) for 2 weeks, (3) PTZ group: treatment with PTZ (50 mg/kg, i.p.) every other day for 2 weeks, and (4) Metf + PTZ group: daily treatment with PTZ and metformin (200 mg/kg, i.p.) for 2 weeks. Administration of PTZ caused a significant increase in seizure score and duration, induced a state of oxidative stress (high malondialdehyde, low reduced glutathione and catalase activity), and led to the upregulation of [beta]-catenin, caspase-3, and its cleavage products, Hsp70 and [alpha]-synuclein, in hippocampal regions as well as a significant reduction in seizure latency. While Metf treatment significantly ameliorated PTZ-induced seizures, attenuated oxidative stress, and upregulated [alpha]-synuclein and [beta]-catenin expression, it also inhibited caspase-3 activation and the release of the cleavage product and caused more upregulation in Hsp70 expression in hippocampal regions (p < 0.05). In conclusion, the antiepileptic and neuroprotective effects of Metf in PTZ-induced epilepsy might be due to the inhibition of apoptosis, attenuation of oxidative stress and [alpha]-synuclein expression, and upregulation of Hsp70.Key words: metformin, pentylenetetrazole, epilepsy, Hsp70, [alpha]-synuclein, caspase-3, [beta]-catenin.Cette etude avait pour but d'examiner les effets neuroprotecteurs et antiepileptiques eventuels de la metformine (Metf), ainsi que des modes d'action sous-jacents eventuels dans un modele d'epilepsie provoquee par le pentylenetetrazole (PTZ) chez le rat. Nous avons reparti 40 rats albinos males dans 4 groupes de tailles egales : (1) temoins normaux, (2) Metf (200 mg/kg i.p.) quotidiennement pendant 2 semaines, (3) PTZ (50 mg/kg, i.p.) un jour sur deux pendant 2 semaines et (4) Metf + PTZ suivant les memes posologies pendant 2 semaines. L'administration de PTZ a entraine une augmentation notable de l'indice de convulsions et de leur duree avec la production d'un etat de stress oxydatif (hausse des taux de malondialdehyde et baisse des taux de glutathion et de catalase), et menait a une regulation a la hausse de la [beta]-catenine, de la caspase-3 et de ses produits de clivage, des proteines de choc thermique Hsp70 (pour << Heat shock proteins >>) et de l'[alpha]-synucleine dans les regions de l'hippocampe, ainsi qu'une nette diminution de la latence des convulsions. Par ailleurs, l'administration de Metf permettait d'attenuer nettement les convulsions provoquees par le PTZ, ainsi que le stress oxydatif et entrainait une regulation a la hausse de l'expression de l'[alpha]-synucleine et de la [beta]-catenine, avec aussi une inhibition de l'activation de la caspase-3 et la liberation de produits de clivage entrainant une augmentation de la regulation a la hausse de l'expression des Hsp70 dans les regions de l'hippocampe (p < 0,05). En conclusion, les proprietes antiepileptiques et neuroprotectrices de la Metf dans l'epilepsie provoquee par le PTZ pourraient etre le fait de l'inhibition de l'apoptose, l'attenuation du stress oxydatif et de l'expression de l'[alpha]-synucleine avec une regulation a la hausse des Hsp70. [Traduit par la Redaction]Mots-cles : metformine, pentylenetetrazole, epilepsie, Hsp70, [alpha]-synucleine, caspase-3, [beta]-catenine., IntroductionEpilepsy is a common neurological disease that affects about 0.5%-2% of the world's population (Naseer et al. 2009). According to previous clinical studies, nearly 30% of patients suffer from resistant [...]

The two-membrane envelope is a defining feature of chloroplasts. Chloroplasts evolved from a Gram-negative cyanobacterial endosymbiont. During evolution, genes of the endosymbiont have been transferred to the host nuclear genome. Most chloroplast proteins are synthesized in the cytosol as higher-molecular-mass preproteins with an N-terminal transit peptide. Preproteins are transported into chloroplasts by the TOC and TIC (translocons at the outer- and inner-envelope membranes of chloroplasts, respectively) machineries.sup.1,2, but how TOC and TIC are assembled together is unknown. Here we report the identification of the TIC component TIC236; TIC236 is an integral inner-membrane protein that projects a 230-kDa domain into the intermembrane space, which binds directly to the outer-membrane channel TOC75. The knockout mutation of TIC236 is embryonically lethal. In TIC236-knockdown mutants, a smaller amount of the inner-membrane channel TIC20 was associated with TOC75; the amount of TOC-TIC supercomplexes was also reduced. This resulted in a reduced import rate into the stroma, though outer-membrane protein insertion was unaffected. The size and the essential nature of TIC236 indicate that--unlike in mitochondria, in which the outer- and inner-membrane translocons exist as separate complexes and a supercomplex is only transiently assembled during preprotein translocation.sup.3,4--a long and stable protein bridge in the intermembrane space is required for protein translocation into chloroplasts. Furthermore, TIC236 and TOC75 are homologues of bacterial inner-membrane TamB.sup.5 and outer-membrane BamA, respectively. Our evolutionary analyses show that, similar to TOC75, TIC236 is preserved only in plants and has co-evolved with TOC75 throughout the plant lineage. This suggests that the backbone of the chloroplast protein-import machinery evolved from the bacterial TamB-BamA protein-secretion system.TIC236 is an inner-membrane protein that binds to the outer-membrane channel TOC75 to create a long, stable bridge that enables the transport of proteins into chloroplasts., Author(s): Yih-Lin Chen [sup.1] , Lih-Jen Chen [sup.1] , Chiung-Chih Chu [sup.1] , Po-Kai Huang [sup.1] [sup.2] , Jie-Ru Wen [sup.1] , Hsou-min Li [sup.1] Author Affiliations:(1) Institute of Molecular [...]

T cell-mediated responses are dependent on their secretion of key effector molecules. However, the critical molecular determinants of the secretion of these proteins are largely undefined. Here, we demonstrate that T cell activation increases trafficking via the ER-to-Golgi pathway. To study the functional role of this pathway, we generated mice with a T cell- specific deletion in SEC23B, a core subunit of coat protein complex II (COPII). We found that SEC23B critically regulated the T cell secretome following activation. SEC23B-deficient T cells exhibited a proliferative defect and reduced effector functions in vitro, as well as in experimental models of allogeneic and xenogeneic hematopoietic cell transplantation in vivo. However, T cells derived from 3 patients with congenital dyserythropoietic anemia II (CDAII), which results from Sec23b mutation, did not exhibit a similar phenotype. Mechanistic studies demonstrated that unlike murine KO T cells, T cells from patients with CDAII harbor increased levels of the closely related paralog, SEC23A. In vivo rescue of murine KO by expression of Sec23a from the Sec23b genomic locus restored T cell functions. Together, our data demonstrate a critical role for the COPII pathway, with evidence for functional overlap in vivo between SEC23 paralogs in the regulation of T cell immunity in both mice and humans., Introduction Coat protein complex II (COPII) drives the formation of ER-derived vesicles that transport newly synthesized proteins to the Golgi compartment. Among their critical roles, COPII-coated vesicles enable the extracellular [...]

We aimed to explore whether myocardial intercellular channel protein connexin-43 (Cx43) along with PKC[epsilon] and MMP-2 might be implicated in responses to acute cardiac injury induced by 2 distinct sublethal interventions in Wistar rats. Animals underwent either single chest irradiation at dose of 25 Gy or subcutaneous injection of isoproterenol (ISO, 120 mg/kg) and were compared with untreated controls. Forty-two days post-interventions, the hearts were excised and left ventricles were used for analysis. The findings showed an increase of total as well as phosphorylated forms of myocardial Cx43 regardless of the type of interventions. Enhanced phosphorylation of Cx43 coincided with increased PKC[epsilon] expression in both models. Elevation of Cx43 was associated with its enhanced distribution on lateral surfaces of the cardiomyocytes in response to both interventions, while focal areas of fibrosis without Cx43 were found in post-ISO but not post-irradiated rat hearts. In parallel, MMP-2 activity was decreased in the former while increased in the latter. Cardiac function was maintained and the susceptibility of the hearts to ischemia or malignant arrhythmias was not deteriorated 42 days after interventions when compared with controls. Altogether, the findings indicate that myocardial Cx43 is most likely implicated in potentially salutary responses to acute heart injury. Key words: acute heart injury, connexin-43, PKC[epsilon], MMP-2. Nous visions a evaluer si la connexine 43 (Cx43), une proteine canalaire intercardiomyocytes, ainsi que la PKC[epsilon] et la MMP-2 pourraient jouer un role dans les lesions cardiaques aigues provoquees par deux interventions subletales distinctes chez le rat Wistar. Les animaux ont subi une irradiation unique du thorax a 25 Gy ou l'injection sous-cutanee d'isoproterenol (ISO, a 120 mg/kg), et nous les avons compares a des temoins non traites. Quarante-deux jours apres les interventions, nous avons preleve les creurs et procede a une analyse des ventricules gauches. Les resultats ont montre une augmentation des taux de Cx43 myocardique totaux et des formes phosphorylees, sans egard au type d'intervention. Dans les deux modeles, l'augmentation de la phosphorylation de la Cx43 coincidait avec l'augmentation de l'expression de la PKC[epsilon]. La hausse des taux de Cx43 etait associee a une accentuation de sa distribution sur les surfaces laterales des cardiomyocytes en reaction aux deux interventions, alors que nous avons observe dans les creurs de rat des regions de fibrose sans Cx43 apres l'administration d'ISO, mais pas apres l'irradiation. En parallele, nous avons observe une diminution de l'activite de la MMP-2 dans le premier cas, mais une augmentation dans le second. Comparativement aux temoins, la fonction cardiaque se maintenait et la susceptibilite des creurs a l'ischemie ou aux arythmies malignes ne se deteriorait pas 42 jours apres les interventions. Dans l'ensemble, les resultats montrent que la Cx43 myocardique joue tres probablement un role dans des reactions a l'ischemie aigue cardiaque qui seraient potentiellement salutaires. [Traduit par la Redaction] Mots-cles: lesion cardiaque aigue, connexine 43, PKC[epsilon], MMP-2., Introduction Intercellular coupling at gap junction connexin channels plays a fundamental role in mediating communication for establishment and maintenance of coordinated heart function. Moreover, intercellular communication regulates adaptive functions of [...]

Author(s): Mohammad T. Mazhab-Jafari [1]; Alexis Rohou [2]; Carla Schmidt [3]; Stephanie A. Bueler [1]; Samir Benlekbir [1]; Carol V. Robinson [3]; John L. Rubinstein (corresponding author) [1, 4, 5] [...]