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Alagille syndrome is a rare genetic disease that often presents with severe cholestasis and pruritus. There are no approved drugs for management. Maralixibat, an apical, sodium-dependent, bile acid transport inhibitor, prevents enterohepatic bile acid recirculation. We evaluated the safety and efficacy of maralixibat for children with cholestasis in Alagille syndrome. ICONIC was a placebo-controlled, randomised withdrawal period (RWD), phase 2b study with open-label extension in children (aged 1–18 years) with Alagille syndrome (NCT02160782). Eligible participants had more than three times the normal serum bile acid (sBA) levels and intractable pruritus. After 18 weeks of maralixibat 380 μg/kg once per day, participants were randomly assigned (1:1) to continue maralixibat or receive placebo for 4 weeks. Subsequently, all participants received open-label maralixibat until week 48. During the long-term extension (204 weeks reported), doses were increased up to 380 μg/kg twice per day. The primary endpoint was the mean sBA change during the RWD in participants with at least 50% sBA reduction by week 18. Cholestastic pruritus was assessed using observer-rated, patient-rated, and clinician-rated 0–4 scales. The safety population was defined as all participants who had received at least one dose of maralixibat. This trial was registered with ClinicalTrials.gov, NCT02160782, and is closed to recruitment. Between Oct 28, 2014, and Aug 14, 2015, 31 participants (mean age 5·4 years [SD 4·25]) were enrolled and 28 analysed at week 48. Of the 29 participants who entered the randomised drug withdrawal period, ten (34%) were female and 19 (66%) were male. In the RWD, participants switched to placebo had significant increases in sBA (94 μmol/L, 95% CI 23 to 164) and pruritus (1·7 points, 95% CI 1·2 to 2·2), whereas participants who continued maralixibat maintained treatment effect. This study met the primary endpoint (least square mean difference –117 μmol/L, 95% CI –232 to –2). From baseline to week 48, sBA (–96 μmol/L, –162 to –31) and pruritus (–1·6 pts, –2·1 to –1·1) improved. In participants who continued to week 204 (n=15) all improvements were maintained. Maralixibat was generally safe and well tolerated throughout. The most frequent adverse events were gastrointestinal related. Most adverse events were self-limiting in nature and mild-to-moderate in severity. In children with Alagille syndrome, maralixibat is, to our knowledge, the first agent to show durable and clinically meaningful improvements in cholestasis. Maralixibat might represent a new treatment paradigm for chronic cholestasis in Alagille syndrome. Mirum Pharmaceuticals.

The adaptor ASC is required for caspase-1 activation via the NLRP3 and AIM2 inflammasomes. Mitsuyama and colleagues show that signaling dependent on the kinases Syk and Jnk controls ASC speck formation through ASC phosphorylation. The inflammasome adaptor ASC contributes to innate immunity through the activation of caspase-1. Here we found that signaling pathways dependent on the kinases Syk and Jnk were required for the activation of caspase-1 via the ASC-dependent inflammasomes NLRP3 and AIM2. Inhibition of Syk or Jnk abolished the formation of ASC specks without affecting the interaction of ASC with NLRP3. ASC was phosphorylated during inflammasome activation in a Syk- and Jnk-dependent manner, which suggested that Syk and Jnk are upstream of ASC phosphorylation. Moreover, phosphorylation of Tyr144 in mouse ASC was critical for speck formation and caspase-1 activation. Our results suggest that phosphorylation of ASC controls inflammasome activity through the formation of ASC specks.

Sepsis, severe sepsis and septic shock are the main cause of mortality in non-cardiac intensive care units. Immunometabolism has been linked to sepsis; however, the precise mechanism by which metabolic reprogramming regulates the inflammatory response is unclear. Here we show that aerobic glycolysis contributes to sepsis by modulating inflammasome activation in macrophages. PKM2-mediated glycolysis promotes inflammasome activation by modulating EIF2AK2 phosphorylation in macrophages. Pharmacological and genetic inhibition of PKM2 or EIF2AK2 attenuates NLRP3 and AIM2 inflammasomes activation, and consequently suppresses the release of IL-1β, IL-18 and HMGB1 by macrophages. Pharmacological inhibition of the PKM2–EIF2AK2 pathway protects mice from lethal endotoxemia and polymicrobial sepsis. Moreover, conditional knockout of PKM2 in myeloid cells protects mice from septic death induced by NLRP3 and AIM2 inflammasome activation. These findings define an important role of PKM2 in immunometabolism and guide future development of therapeutic strategies to treat sepsis. Inflammation involves a Warburg effect that switches cellular metabolism to glycolysis. Here the authors show this switch drives IL-1β, IL-18 and HMGB1 release from macrophages by activating the NLRP3 and AIM2 inflammasomes via protein kinase R phosphorylation, a pathway that can be inhibited to prevent sepsis in mice.

Chronic immune thrombocytopenic purpura (ITP) is characterised by accelerated platelet destruction and decreased platelet production. Short-term administration of the thrombopoiesis-stimulating protein, romiplostim, has been shown to increase platelet counts in most patients with chronic ITP. We assessed the long-term administration of romiplostim in splenectomised and non-splenectomised patients with ITP. In two parallel trials, 63 splenectomised and 62 non-splenectomised patients with ITP and a mean of three platelet counts 30×109/L or less were randomly assigned 2:1 to subcutaneous injections of romiplostim (n=42 in splenectomised study and n=41 in non-splenectomised study) or placebo (n=21 in both studies) every week for 24 weeks. Doses of study drug were adjusted to maintain platelet counts of 50×109/L to 200×109/L. The primary objectives were to assess the efficacy of romiplostim as measured by a durable platelet response (platelet count ≥50×109/L during 6 or more of the last 8 weeks of treatment) and treatment safety. Analysis was per protocol. These studies are registered with ClinicalTrials.gov, numbers NCT00102323 and NCT00102336. A durable platelet response was achieved by 16 of 42 splenectomised patients given romplostim versus none of 21 given placebo (difference in proportion of patients responding 38% [95% CI 23·4–52·8], p=0·0013), and by 25 of 41 non-splenectomised patients given romplostim versus one of 21 given placebo (56% [38·7–73·7], p<0·0001). The overall platelet response rate (either durable or transient platelet response) was noted in 88% (36/41) of non-splenectomised and 79% (33/42) of splenectomised patients given romiplostim compared with 14% (three of 21) of non-splenectomised and no splenectomised patients given placebo (p<0·0001). Patients given romiplostim achieved platelet counts of 50×109/L or more on a mean of 13·8 (SE 0·9) weeks (mean 12·3 [1·2] weeks in splenectomised group vs 15·2 [1·2] weeks in non-splenectomised group) compared with 0·8 (0·4) weeks for those given placebo (0·2 [0·1] weeks vs 1·3 [0·8] weeks). 87% (20/23) of patients given romiplostim (12/12 splenectomised and eight of 11 non-splenectomised patients) reduced or discontinued concurrent therapy compared with 38% (six of 16) of those given placebo (one of six splenectomised and five of ten non-splenectomised patients). Adverse events were much the same in patients given romiplostim and placebo. No antibodies against romiplostim or thrombopoietin were detected. Romiplostim was well tolerated, and increased and maintained platelet counts in splenectomised and non-splenectomised patients with ITP. Many patients were able to reduce or discontinue other ITP medications. Stimulation of platelet production by romiplostim may provide a new therapeutic option for patients with ITP.

NEK7, a member of the NIMA-related kinase family, is identified as a regulator of NLRP3 inflammasome oligomerization and activation; NEK7 functions downstream of potassium efflux in a manner that is independent of its kinase activity. NEK7 mediates NLRP3 inflammasome activation The NLRP3 inflammasome, a critical component of the innate immune system, has been linked to multiple acquired and inherited diseases. However, the molecular mechanism that leads to NLRP3 oligomerization and activation remains elusive. Here Gabriel Núñez and colleagues identify a member of the family of NIM related kinases (NEK7) as a regulator of NLRP3 inflammasome oligomerization and activation. NEK7 functions downstream of potassium efflux in a manner that is independent of its kinase activity. Inflammasomes are intracellular protein complexes that drive the activation of inflammatory caspases 1 . So far, four inflammasomes involving NLRP1, NLRP3, NLRC4 and AIM2 have been described that recruit the common adaptor protein ASC to activate caspase-1, leading to the secretion of mature IL-1β and IL-18 proteins 2 , 3 . The NLRP3 inflammasome has been implicated in the pathogenesis of several acquired inflammatory diseases 4 , 5 as well as cryopyrin-associated periodic fever syndromes (CAPS) caused by inherited NLRP3 mutations 6 , 7 . Potassium efflux is a common step that is essential for NLRP3 inflammasome activation induced by many stimuli 8 , 9 . Despite extensive investigation, the molecular mechanism leading to NLRP3 activation in response to potassium efflux remains unknown. Here we report the identification of NEK7, a member of the family of mammalian NIMA-related kinases (NEK proteins) 10 , as an NLRP3-binding protein that acts downstream of potassium efflux to regulate NLRP3 oligomerization and activation. In the absence of NEK7, caspase-1 activation and IL-1β release were abrogated in response to signals that activate NLRP3, but not NLRC4 or AIM2 inflammasomes. NLRP3-activating stimuli promoted the NLRP3–NEK7 interaction in a process that was dependent on potassium efflux. NLRP3 associated with the catalytic domain of NEK7, but the catalytic activity of NEK7 was shown to be dispensable for activation of the NLRP3 inflammasome. Activated macrophages formed a high-molecular-mass NLRP3–NEK7 complex, which, along with ASC oligomerization and ASC speck formation, was abrogated in the absence of NEK7. NEK7 was required for macrophages containing the CAPS-associated NLRP3(R258W) activating mutation to activate caspase-1. Mouse chimaeras reconstituted with wild-type, Nek7 −/− or Nlrp3 −/− haematopoietic cells showed that NEK7 was required for NLRP3 inflammasome activation in vivo . These studies demonstrate that NEK7 is an essential protein that acts downstream of potassium efflux to mediate NLRP3 inflammasome assembly and activation.

Nearly all mitochondrial proteins are encoded by the nuclear genome and imported into mitochondria after synthesis on cytosolic ribosomes. These precursor proteins are translocated into mitochondria by the TOM complex, a protein-conducting channel in the mitochondrial outer membrane. We have determined high-resolution cryo-EM structures of the core TOM complex from Saccharomyces cerevisiae in dimeric and tetrameric forms. Dimeric TOM consists of two copies each of five proteins arranged in two-fold symmetry: pore-forming β-barrel protein Tom40 and four auxiliary α-helical transmembrane proteins. The pore of each Tom40 has an overall negatively charged inner surface attributed to multiple functionally important acidic patches. The tetrameric complex is essentially a dimer of dimeric TOM, which may be capable of forming higher-order oligomers. Our study reveals the detailed molecular organization of the TOM complex and provides new insights about the mechanism of protein translocation into mitochondria.

A protein complex composed of KPTN, ITFG2, C12orf66 and SZT2, named KICSTOR, is necessary for lysosomal localization of GATOR1, interaction of GATOR1 with the Rag GTPases and GATOR2, and nutrient-dependent mTORC1 modulation. KICSTOR is a negative regulator of mTORC1 signalling The mechanistic target of rapamycin complex 1 (mTORC1) is a central regulator of cell growth and organismal homeostasis and is deregulated in many human diseases, including epilepsy and cancer. In response to nutrients, mTORC1 is recruited to the lysosome by the Rag family of GTPases, whose activity is regulated by the GATOR complex. Here David Sabatini and colleagues identify a four-membered protein complex that they term KICSTOR. It localizes to lysosomes and interacts with GATOR to negatively regulate the pathway through which mTORC1 senses nutrients. In mice lacking one of the KICSTOR subunits, SZT2, mTORC1 signalling is hyperactivated in several tissues. A related paper in this week's issue of Nature from Ming Li and colleagues identifies the protein SZT2 as a negative regulator of mTORC1 signalling. Together, the two papers offer insight into mTORC1 regulation at the lysosome and could have implications for diseases associated with hyperactive mTORC1 signalling. The mechanistic target of rapamycin complex 1 (mTORC1) is a central regulator of cell growth that responds to diverse environmental signals and is deregulated in many human diseases, including cancer and epilepsy 1 , 2 , 3 . Amino acids are a key input to this system, and act through the Rag GTPases to promote the translocation of mTORC1 to the lysosomal surface, its site of activation 4 . Multiple protein complexes regulate the Rag GTPases in response to amino acids, including GATOR1, a GTPase activating protein for RAGA, and GATOR2, a positive regulator of unknown molecular function. Here we identify a protein complex (KICSTOR) that is composed of four proteins, KPTN, ITFG2, C12orf66 and SZT2, and that is required for amino acid or glucose deprivation to inhibit mTORC1 in cultured human cells. In mice that lack SZT2, mTORC1 signalling is increased in several tissues, including in neurons in the brain. KICSTOR localizes to lysosomes; binds and recruits GATOR1, but not GATOR2, to the lysosomal surface; and is necessary for the interaction of GATOR1 with its substrates, the Rag GTPases, and with GATOR2. Notably, several KICSTOR components are mutated in neurological diseases associated with mutations that lead to hyperactive mTORC1 signalling 5 , 6 , 7 , 8 , 9 , 10 . Thus, KICSTOR is a lysosome-associated negative regulator of mTORC1 signalling, which, like GATOR1, is mutated in human disease 11 , 12 .

The cullin–RING ubiquitin E3 ligase (CRL) family comprises over 200 members in humans. The COP9 signalosome complex (CSN) regulates CRLs by removing their ubiquitin-like activator NEDD8. The CUL4A–RBX1–DDB1–DDB2 complex (CRL4A DDB2 ) monitors the genome for ultraviolet-light-induced DNA damage. CRL4A DBB2 is inactive in the absence of damaged DNA and requires CSN to regulate the repair process. The structural basis of CSN binding to CRL4A DDB2 and the principles of CSN activation are poorly understood. Here we present cryo-electron microscopy structures for CSN in complex with neddylated CRL4A ligases to 6.4 Å resolution. The CSN conformers defined by cryo-electron microscopy and a novel apo-CSN crystal structure indicate an induced-fit mechanism that drives CSN activation by neddylated CRLs. We find that CSN and a substrate cannot bind simultaneously to CRL4A, favouring a deneddylated, inactive state for substrate-free CRL4 complexes. These architectural and regulatory principles appear conserved across CRL families, allowing global regulation by CSN. Much of the intracellular protein degradation in eukaryotes is controlled by cullin–RING ubiquitin ligases (CRLs), a vast class of enzymes which are regulated by the COP9 signalosome (CSN); structural characterization of CSN–N8CRL4A complexes by cryo-electron microscopy reveals an induced-fit mechanism of CSN activation triggered only by catalytically activated CRLs without bound substrate, explaining how CSN acts as a global regulator of CRLs. Control of intracellular protein degradation Much of the intracellular protein degradation in eukaryotes is controlled by cullin–RING ubiquitin ligases (CRLs). The structure of these enzymes and their substrates vary greatly, yet all are regulated by a single complex — the COP9 signalosome (CSN). What enables CSN to be a master regulator of diverse CRLs? Nicolas Thomä and colleagues present biochemical data and cryo-electron microscopy of CSN–CRL4 complexes revealing an induced-fit mechanism that activates CSN only in the presence of a catalytically activated CRL not bound to a substrate. The authors identify both unique and less-specific CSN–CRL contacts.

Iron–sulfur (Fe/S) clusters are essential protein cofactors crucial for many cellular functions including DNA maintenance, protein translation, and energy conversion. De novo Fe/S cluster synthesis occurs on the mitochondrial scaffold protein ISCU and requires cysteine desulfurase NFS1, ferredoxin, frataxin, and the small factors ISD11 and ACP (acyl carrier protein). Both the mechanism of Fe/S cluster synthesis and function of ISD11-ACP are poorly understood. Here, we present crystal structures of three different NFS1-ISD11-ACP complexes with and without ISCU, and we use SAXS analyses to define the 3D architecture of the complete mitochondrial Fe/S cluster biosynthetic complex. Our structural and biochemical studies provide mechanistic insights into Fe/S cluster synthesis at the catalytic center defined by the active-site Cys of NFS1 and conserved Cys, Asp, and His residues of ISCU. We assign specific regulatory rather than catalytic roles to ISD11-ACP that link Fe/S cluster synthesis with mitochondrial lipid synthesis and cellular energy status. Fe/S clusters are synthesized by the mitochondrial iron-sulfur cluster assembly (ISC) machinery. Here the authors combine crystallography and small angle X-ray scattering measurements to structurally characterize the core ISC complex and give functional insights into eukaryotic Fe/S cluster synthesis.

Mutation of C9orf72 is the most prevalent defect associated with amyotrophic lateral sclerosis and frontotemporal degeneration 1 . Together with hexanucleotide-repeat expansion 2 , 3 , haploinsufficiency of C9orf72 contributes to neuronal dysfunction 4 – 6 . Here we determine the structure of the C9orf72–SMCR8–WDR41 complex by cryo-electron microscopy. C9orf72 and SMCR8 both contain longin and DENN (differentially expressed in normal and neoplastic cells) domains 7 , and WDR41 is a β-propeller protein that binds to SMCR8 such that the whole structure resembles an eye slip hook. Contacts between WDR41 and the DENN domain of SMCR8 drive the lysosomal localization of the complex in conditions of amino acid starvation. The structure suggested that C9orf72–SMCR8 is a GTPase-activating protein (GAP), and we found that C9orf72–SMCR8–WDR41 acts as a GAP for the ARF family of small GTPases. These data shed light on the function of C9orf72 in normal physiology, and in amyotrophic lateral sclerosis and frontotemporal degeneration. The cryo-electron microscopy structure of C9orf72–SMCR8–WDR41 suggests that this complex is a GTPase-activating protein for ARF-family small GTPases, which sheds light on the role of C9orf72 mutations in neuronal dysfunction.