Highlights d p62 directly interacts with the FIP200 C-terminal domain d Structural studies reveal a claw shape of the conserved FIP200 C-terminal domain d p62-ubiquitin condensates recruit FIP200 via the Claw to promote their degradation d LC3B outcompetes FIP200 from p62, suggesting an inbuild directionality in the system

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SUMMARY The lysosomal membrane is the locus for sensing cellular nutrient levels, which are transduced to mTORC1 via the Rag GTPases and the Ragulator complex. The crystal structure of the five-subunit human Ragulator at 1.4 Å resolution was determined. Lamtor1 wraps around the other four subunits to stabilize the assembly. The Lamtor2:Lamtor3 dimer stacks upon Lamtor4:Lamtor5 to create a platform for Rag binding. Hydrogen-deuterium exchange was used to map the Rag binding site to the outer face of the Lamtor2:Lamtor3 dimer and to the N-terminal intrinsically disordered region of Lamtor1. EM was used to reconstruct the assembly of the full-length RagAGTP:RagCGDP dimer bound to Ragulator at 16 Å resolution, revealing that the G-domains of the Rags project away from the Ragulator core. The combined structural model shows how Ragulator functions as a platform for the presentation of active Rags for mTORC1 recruitment, and might suggest an unconventional mechanism for Rag GEF activity.

Mutation of C9ORF72 is the most prevalent defect in amyotrophic lateral sclerosis (ALS) and frontal temporal degeneration (FTD). Together with hexanucleotide repeat expansion, haploinsufficiency of C9ORF72 contributes to neuronal dysfunction. We determined the structure of the SMCR8-C9orf72-WDR41 complex by cryo-EM. C9orf72 and SMCR8 are both longin-DENN domain proteins, while WDR41 is a beta-propeller protein that binds to SMCR8 such that the whole structure resembles an eye slip hook. Contacts between WDR41 and SMCR8 DENN drive lysosomal localization in amino acid starvation. The structure suggested that SMCR8-C9orf72 was a small GTPase activating protein (GAP). We found that SMCR8-C9orf72-WDR41 is a GAP for Arf family small GTPases, and refer to it as the Lysosomal SMCR8-C9orf72 Arf GAP ("L-SCARF") complex. These data rationalize the function of C9orf72 both in normal physiology and in ALS/FTD.

SummaryModerate alcohol consumption has been shown to reduce the risk of ischemic heart disease potentially through its effect on specific endothelial-derived compounds. We tested the hypothesis that ethanol increases the expression of endothelial nitric oxide synthase (eNOS) and nitric oxide (NO) production in bovine aortic endothelial cells (BAEC). Primary cultures of BAEC grown to confluence under standard conditions were treated 3-6 h with 0.1% ethanol in the presence of indomethacin. Ethanol induced a significant increase in both basal and stimulated NO production as determined by chemiluminescence method. This effect was accompanied by a rapid increase of eNOS protein and mRNA expression levels. eNOS mRNA increased two-fold within 3 h and gradually declined, but the increased levels of mRNA persisted for >24 h. A similar increase of eNOS expression was observed in human umbilical endothelial cells exposed to ethanol. These results demonstrate that ethanol augments both basal and stimulated NO production and that this effect is associated with increased eNOS protein and mRNA expression levels. The data are consistent with the hypothesis that the reduced incidence of ischemic heart disease associated with alcohol may be related, at least in part, to the modulation of vascular endothelial cell production of NO

Peptidoglycan (PG) is a highly cross-linked, protective mesh-like sacculus that surrounds the bacterial cytoplasmic membrane. Expansion of PG is tightly coupled to growth of a bacterial cell and requires hydrolases to cleave the cross-links for insertion of nascent PG material. In Escherichia coli, a proteolytic system comprising the periplasmic PDZ-protease Prc and the lipoprotein adaptor NlpI contributes to PG enlargement by regulating cellular levels of MepS, a cross-link-specific hydrolase. Here, we demonstrate how NlpI binds Prc to facilitate the degradation of its substrate MepS by structural and mutational analyses. An NlpI homodimer binds two molecules of Prc and forms three-sided MepS-docking cradles using its tetratricopeptide repeats. Prc forms a monomeric bowl-shaped structure with a lid-like PDZ domain connected by a substrate-sensing hinge that recognizes the bound C terminus of the substrate. In summary, our study reveals mechanistic details of protein degradation by the PDZ-protease Prc bound to its cognate adaptor protein.

We examined mechanotranscriptional regulation of the contractile gene, ␣-smooth muscle actin (SMA), in osteoblastic cells. Tensile forces were applied through collagen-coated magnetite beads to ROS17/2.8 cells. These cells were desmin؊, vimentin؉ and expressed low levels of SMA. After force application (480 piconewton/ cell), SMA protein and mRNA were increased but ␤-actin was unchanged. Beads coated with bovine serum albumin or poly-L-lysine produced no change of SMA. In cells transiently transfected with plasmids containing the SMA promoter fused to ␤-galactosidase or green fluorescent protein coding sequences, SMA promoter activity was increased by ϳ60% after 4 h of force, whereas control (Rous sarcoma virus) promoter activity was unaffected. Transfections with ␤-galactosidase or green fluorescent protein reporter constructs showed that forceloaded cells exhibited higher ␤-galactosidase activity than cells without force. Cytochalasin D and latrunculin B inhibited force-induced increases of SMA promoter activity. Deletion analyses showed that SMA promoter activity was increased ϳ70% after force with a minimal construct containing 155 bp upstream of the translation start site. The force effect on the SMA promoter was abrogated in cells transfected with CArG-B box mutants. Gel mobility shift analyses of nuclear extracts showed strong binding to the CArG-B motif after force. We conclude that the CArG-B box is a force-responsive element in the SMA promoter.

1 The antiarrhythmic potential and electromechanical effects of liriodenine, an aporphine alkaloid isolated from the plant, Fissistigma glaucescens, were examined. 2 In the Langendorff perfused (with constant pressure) rat heart, at a concentration of 0.3 to 3 gM, liriodenine was able to convert a polymorphic ventricular tachyrhythmia induced by the ischaemiareperfusion (EC50 = 0.3 gM).3 In isolated atrial and ventricular muscle, liriodenine increased the contractile force and slowed the spontaneous beating of the right atrium.4 The liriodenine-induced positive inotropy was markedly attenuated by a transient outward K+ channel blocker, 4-aminopyridine (4-AP) but was not significantly affected by prazosin, propranolol, verapamil or carbachol. 5 In rat isolated ventricular myocytes, liriodenine prolonged action potential duration and decreased the maximal upstroke velocity of phase 0 depolarization (7,,ma) and resting membrane potential in a concentration-dependent manner. The action potential amplitude was not significantly changed. 6 Whole-cell voltage clamp study revealed that liriodenine blocked the Na+ channel (INa) concentration-dependently (IC50 = 0.7 gM) and caused a leftward shift of its steady-state inactivation curve. However, its recovery rate from the inactivated state was not affected. The L-type Ca2" currents (ICa) were also decreased, but to a lesser degree (ICo = 2.5 gM, maximal inhibition = 35%). 7 Liriodenine inhibited the 4-AP-sensitive transient outward current (I,.) (IC50 = 2.8 gM) and moderately accelerated its rate of decay. The block of Ito was not associated with changes in the voltage-dependence of the steady-state inactivation curve or in the process of recovery from inactivation of the current. Liriodenine also reduced the amplitude of a slowly inactivating, steady-state outward current (ISS) (IC50 = 1.9 ,M). These effects were consistent with its prolonging effect on action potential duration. The inwardly rectifying background K+ current (IK1), was also decreased but to a less degree. 8 Compared to quinidine, liriodenine exerted a stronger degree of block on INa, comparable degree of block on IKi, and lesser extent of block on ICa and In,. 9 It is concluded that, through inhibition of Na+ and the Ito channel, liriodenine can suppress ventricular arrhythmias induced by myocardial ischaemia reperfusion. The positive inotropic effect can be explained by inhibition of the Ito channel and the subsequent prolongation of action potential duration. These results provide a satisfactory therapeutic potential for the treatment of cardiac arrhythmias.