Amelioration of SARS-CoV-2 infection by ANO6 phospholipid scramblase inhibition.

As an enveloped virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) delivers its viral genome into host cells via fusion of the viral and cell membranes. Here, we show that ANO6/TMEM16F-mediated cell surface exposure of phosphatidylserine is critical for SARS-CoV-2 entry and that ANO6-selective inhibitors are effective against SARS-CoV-2 infections. Application of the SARS-CoV-2 Spike pseudotyped virus (SARS2-PsV) evokes a cytosolic Ca2+ elevation and ANO6-dependent phosphatidylserine externalization in ACE2/TMPRSS2-positive mammalian cells. A high-throughput screening of drug-like chemical libraries identifies three different structural classes of chemicals showing ANO6 inhibitory effects. Among them, A6-001 displays the highest potency and ANO6 selectivity and it inhibits the single-round infection of SARS2-PsV in ACE2/TMPRSS2-positive HEK 293T cells. More importantly, A6-001 strongly inhibits authentic SARS-CoV-2-induced phosphatidylserine scrambling and SARS-CoV-2 viral replications in Vero, Calu-3, and primarily cultured human nasal epithelial cells. These results provide mechanistic insights into the viral entry process and offer a potential target for pharmacological intervention to protect against coronavirus disease 2019 (COVID-19).

Secretory autophagy machinery and vesicular trafficking are involved in HMGB1 secretion.

Nuclear protein HMGB1 is secreted in response to various stimuli and functions as a danger-associated molecular pattern. Extracellular HMGB1 induces inflammation, cytokine production, and immune cell recruitment via activation of various receptors. As HMGB1 does not contain an endoplasmic reticulum-targeting signal peptide, HMGB1 is secreted via the endoplasmic reticulum-Golgi independently via an unconventional secretion pathway. However, the mechanism underlying HMGB1 secretion remains largely unknown. Here, we investigated the role of secretory autophagy machinery and vesicular trafficking in HMGB1 secretion. We observed that HSP90AA1 (heat shock protein 90 alpha family class A member 1), a stress-inducible protein, regulates the translocation of HMGB1 from the nucleus to the cytoplasm and its secretion through direct interaction. Additionally, geldanamycin, an HSP90AA1 inhibitor, reduced HMGB1 secretion. GORASP2/GRASP55 (golgi reassembly stacking protein 2), ARF1Q71L (ADP ribosylation factor 1), and SAR1AT39N (secretion associated Ras related GTPase 1A), which promoted unconventional protein secretion, increased HMGB1 secretion. HMGB1 secretion was inhibited by an early autophagy inhibitor and diminished in ATG5-deficient cells even when GORASP2 was overexpressed. In contrast, a late autophagy inhibitor increased HMGB1 secretion under the same conditions. The multivesicular body formation inhibitor GW4869 dramatically decreased HMGB1 secretion under HMGB1 secretion-inducing conditions. Thus, we demonstrated that secretory autophagy and multivesicular body formation mediate HMGB1 secretion.

Grasp55-/- mice display impaired fat absorption and resistance to high-fat diet-induced obesity.

The Golgi apparatus plays a central role in the intracellular transport of macromolecules. However, molecular mechanisms of Golgi-mediated lipid transport remain poorly understood. Here, we show that genetic inactivation of the Golgi-resident protein GRASP55 in mice reduces whole-body fat mass via impaired intestinal fat absorption and evokes resistance to high-fat diet induced body weight gain. Mechanistic analyses reveal that GRASP55 participates in the Golgi-mediated lipid droplet (LD) targeting of some LD-associated lipases, such as ATGL and MGL, which is required for sustained lipid supply for chylomicron assembly and secretion. Consequently, GRASP55 deficiency leads to reduced chylomicron secretion and abnormally large LD formation in intestinal epithelial cells upon exogenous lipid challenge. Notably, deletion of dGrasp in Drosophila causes similar defects of lipid accumulation in the midgut. These results highlight the importance of the Golgi complex in cellular lipid regulation, which is evolutionary conserved, and uncover potential therapeutic targets for obesity-associated diseases.

IRE1α kinase-mediated unconventional protein secretion rescues misfolded CFTR and pendrin.

The most prevalent pathogenic mutations in the CFTR (ΔF508) and SLC26A4/pendrin (p.H723R), which cause cystic fibrosis and congenital hearing loss, respectively, evoke protein misfolding and subsequent defects in their cell surface trafficking. Here, we report that activation of the IRE1α kinase pathway can rescue the cell surface expression of ΔF508-CFTR and p.H723R-pendrin through a Golgi-independent unconventional protein secretion (UPS) route. In mammalian cells, inhibition of IRE1α kinase, but not inhibition of IRE1α endonuclease and the downstream effector XBP1, inhibited CFTR UPS. Treatment with the IRE1α kinase activator, (E)-2-(2-chlorostyryl)-3,5,6-trimethyl-pyrazine (CSTMP), rescued cell surface expression and functional activity of ΔF508-CFTR and p.H723R-pendrin. Treatment with a nontoxic dose of CSTMP to ΔF508-CFTR mice restored CFTR surface expression and CFTR-mediated anion transport in the mouse colon. These findings suggest that UPS activation via IRE1α kinase is a strategy to treat diseases caused by defective cell surface trafficking of membrane proteins, including ΔF508-CFTR and p.H723R-pendrin.

Molecular Diagnosis of Craniosynostosis Using Targeted Next-Generation Sequencing.

BACKGROUND: Genetic factors play an important role in the pathogenesis of craniosynostosis (CRS). However, the molecular diagnosis of CRS in clinical practice is limited because of its heterogeneous etiology. OBJECTIVE: To investigate the genomic landscape of CRS in a Korean cohort and also to establish a practical diagnostic workflow by applying targeted panel sequencing. METHODS: We designed a customized panel covering 34 CRS-related genes using in-solution hybrid capture method. We enrolled 110 unrelated Korean patients with CRS, including 40 syndromic and 70 nonsyndromic cases. A diagnostic pipeline was established by combining in-depth clinical reviews and multiple bioinformatics tools for analyzing single-nucleotide variants (SNV)s and copy number variants (CNV)s. RESULTS: The diagnostic yield of the targeted panel was 30.0% (33/110). Twenty-five patients (22.7%) had causal genetic variations resulting from SNVs or indels in 9 target genes (TWIST1, FGFR3, TCF12, ERF, FGFR2, ALPL, EFNB1, FBN1, and SKI, in order of frequency). CNV analysis identified 8 (7.3%) additional patients with chromosomal abnormalities involving 1p32.3p31.3, 7p21.1, 10q26, 15q21.3, 16p11.2, and 17p13.3 regions; these cases mostly presented with syndromic clinical features. CONCLUSION: The present study shows the wide genomic landscape of CRS, revealing various genetic factors for CRS pathogenesis. In addition, the results demonstrate that an efficient diagnostic workup using target panel sequencing provides great clinical utility in the molecular diagnosis of CRS.

A coding variant in FTO confers susceptibility to thiopurine-induced leukopenia in East Asian patients with IBD.

OBJECTIVE: Myelosuppression is a life-threatening complication of thiopurine therapy, and the incidence of thiopurine-induced myelosuppression is higher in East Asians than in Europeans. We investigated genetic factors associated with thiopurine-induced leukopenia in patients with IBD. DESIGN: A genome-wide association study (GWAS) was conducted in thiopurine-treated patients with IBD, followed by high-throughput sequencing of genes identified as significant in the GWAS or those involved in thiopurine metabolism (n=331). Significant loci associated with thiopurine-induced leukopenia were validated in two additional replication cohorts (n=437 and n=330). Functional consequences of FTO (fat mass and obesity-associated) variant were examined both in vitro and in vivo. RESULTS: The GWAS identified two loci associated with thiopurine-induced leukopenia (rs16957920, FTO intron; rs2834826, RUNX1 intergenic). High-throughput targeted sequencing indicated that an FTO coding variant (rs79206939, p.A134T) linked to rs16957920 is associated with thiopurine-induced leukopenia. This result was further validated in two replication cohorts (combined p=1.3×10-8, OR=4.3). The frequency of FTO p.A134T is 5.1% in Koreans but less than 0.1% in Western populations. The p.A134T variation reduced FTO activity by 65% in the nucleotide demethylase assay. In vivo experiments revealed that Fto-/- and Fto+/- mice were more susceptible to thiopurine-induced myelosuppression than wild-type mice. CONCLUSIONS: The results suggest that the hypomorphic FTO p.A134T variant is associated with thiopurine-induced leukopenia. These results shed light on the novel physiological role of FTO and provide a potential pharmacogenetic biomarker for thiopurine therapy.