Synergistic toxicity with copper contributes to NAT2-associated isoniazid toxicity.

Anti-tuberculosis (AT) medications, including isoniazid (INH), can cause drug-induced liver injury (DILI), but the underlying mechanism remains unclear. In this study, we aimed to identify genetic factors that may increase the susceptibility of individuals to AT-DILI and to examine genetic interactions that may lead to isoniazid (INH)-induced hepatotoxicity. We performed a targeted sequencing analysis of 380 pharmacogenes in a discovery cohort of 112 patients (35 AT-DILI patients and 77 controls) receiving AT treatment for active tuberculosis. Pharmacogenome-wide association analysis was also conducted using 1048 population controls (Korea1K). NAT2 and ATP7B genotypes were analyzed in a replication cohort of 165 patients (37 AT-DILI patients and 128 controls) to validate the effects of both risk genotypes. NAT2 ultraslow acetylators (UAs) were found to have a greater risk of AT-DILI than other genotypes (odds ratio [OR] 5.6 [95% confidence interval; 2.5-13.2], P = 7.2 × 10-6). The presence of ATP7B gene 832R/R homozygosity (rs1061472) was found to co-occur with NAT2 UA in AT-DILI patients (P = 0.017) and to amplify the risk in NAT2 UA (OR 32.5 [4.5-1423], P = 7.5 × 10-6). In vitro experiments using human liver-derived cell lines (HepG2 and SNU387 cells) revealed toxic synergism between INH and Cu, which were strongly augmented in cells with defective NAT2 and ATP7B activity, leading to increased mitochondrial reactive oxygen species generation, mitochondrial dysfunction, DNA damage, and apoptosis. These findings link the co-occurrence of ATP7B and NAT2 genotypes to the risk of INH-induced hepatotoxicity, providing novel mechanistic insight into individual AT-DILI susceptibility. Yoon et al. showed that individuals who carry NAT2 UAs and ATP7B 832R/R genotypes are at increased risk of developing isoniazid hepatotoxicity, primarily due to the increased synergistic toxicity between isoniazid and copper, which exacerbates mitochondrial dysfunction-related apoptosis.

Novel small molecule-mediated restoration of the surface expression and anion exchange activity of mutated pendrin causing Pendred syndrome and DFNB4.

Variants in SLC26A4 (pendrin) are the most common reasons for genetic hearing loss and vestibular dysfunction in East Asians. In patients with Pendred syndrome and DFNB4 (autosomal recessive type of genetic hearing loss 4), caused by variants in SLC26A4, the hearing function is residual at birth and deteriorates over several years, with no curative treatment for these disorders. In the present study, we revealed that a novel small molecule restores the expression and function of mutant pendrin. High-throughput screening of 54,000 small molecules was performed. We observed that pendrin corrector (PC2-1) increased the surface expression and anion exchange activity of p.H723R pendrin (H723R-PDS), the most prevalent genetic variant that causes Pendred syndrome and DFNB4. Furthermore, in endogenous H723R-PDS-expressing human nasal epithelial cells, PC2-1 significantly increased the surface expression of pendrin. PC2-1 exhibited high membrane permeability in vitro and high micromolar concentrations in the cochlear perilymph in vivo. In addition, neither inhibition of Kv11.1 activity in the human ether-a-go-go-related gene assay nor cell toxicity in the cell proliferation assay was observed at a high PC2-1 concentration (30 µM). These preclinical data support the hypothesis of the druggability of mutant pendrin using the novel corrector molecule PC2-1. In conclusion, PC2-1 may be a new therapeutic molecule for ameliorating hearing loss and treating vestibular disorders in patients with Pendred syndrome or DFNB4.

Multicenter Surveillance of Cystic Fibrosis in Korean Children.

PURPOSE: Cystic fibrosis (CF), caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, is rare among non-Caucasians. We aimed to identify the clinical features and CFTR mutations in Korean children. METHODS: We included 18 pediatric patients with CF diagnosed using sweat chloride test or genetic analysis for 30 years. HEK293 cells were transfected with wild-type CFTR, ΔF508-CFTR, and L441P-CFTR mutant plasmids for 24 hours and treated with CFTR correctors (VX809 and VX661). RESULTS: The median age at diagnosis was 9.2 years. Eleven patients had growth retardation, and 6 had a respiratory failure at diagnosis. Genetic analysis was used for all patients, while sweat testing was for 8 patients. At diagnosis, the median z scores of forced expiratory volume in one second (FEV1), FEV1/forced vital capacity, and forced expiratory flow at 25%-75% of forced vital capacity were -3.61 (-5.78, 1.78), -3.38 (-4.40, -0.60), and -4.45 (-5.78, 0.54), respectively. Two patients were treated with dornase alfa and only one with CFTR modulator. Patients were followed up for 3.7 years as a median. Four patients died at 10.6 years, with 4.2 years of post-diagnosis survival. The most common mutation was exon 16-17b deletion (19.4%). Among 11 single nucleotide variants, c.1322T>C (p.Leu441Pro, L441P) was detected in 4 patients. In the functional assay, L441P-CFTR correction was well restored by CFTR correctors compared with ΔF508. CONCLUSIONS: CF is extremely rare in Korean children and is caused by different mutations from those commonly observed in Caucasians. Early diagnosis and treatment availability may improve outcomes. CFTR modulators may be effective for Asian patients with rare CFTR mutations, c.1322T>C (p.Leu441Pro).

Autophagy-Related Pathways in Vesicular Unconventional Protein Secretion.

Cellular proteins directed to the plasma membrane or released into the extracellular space can undergo a number of different pathways. Whereas the molecular mechanisms that underlie conventional ER-to-Golgi trafficking are well established, those associated with the unconventional protein secretion (UPS) pathways remain largely elusive. A pathway with an emerging role in UPS is autophagy. Although originally known as a degradative process for maintaining intracellular homeostasis, recent studies suggest that autophagy has diverse biological roles besides its disposal function and that it is mechanistically involved in the UPS of various secretory cargos including both leaderless soluble and Golgi-bypassing transmembrane proteins. Here, we summarize current knowledge of the autophagy-related UPS pathways, describing and comparing diverse features in the autophagy-related UPS cargos and autophagy machineries utilized in UPS. Additionally, we also suggest potential directions that further research in this field can take.

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).

TMED3 Complex Mediates ER Stress-Associated Secretion of CFTR, Pendrin, and SARS-CoV-2 Spike.

Under ER stress conditions, the ER form of transmembrane proteins can reach the plasma membrane via a Golgi-independent unconventional protein secretion (UPS) pathway. However, the targeting mechanisms of membrane proteins for UPS are unknown. Here, this study reports that TMED proteins play a critical role in the ER stress-associated UPS of transmembrane proteins. The gene silencing results reveal that TMED2, TMED3, TMED9 and TMED10 are involved in the UPS of transmembrane proteins, such as CFTR, pendrin and SARS-CoV-2 Spike. Subsequent mechanistic analyses indicate that TMED3 recognizes the ER core-glycosylated protein cargos and that the heteromeric TMED2/3/9/10 complex mediates their UPS. Co-expression of all four TMEDs improves, while each single expression reduces, the UPS and ion transport function of trafficking-deficient ΔF508-CFTR and p.H723R-pendrin, which cause cystic fibrosis and Pendred syndrome, respectively. In contrast, TMED2/3/9/10 silencing reduces SARS-CoV-2 viral release. These results provide evidence for a common role of TMED3 and related TMEDs in the ER stress-associated, Golgi-independent secretion of transmembrane proteins.

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.

Specific autophagy and ESCRT components participate in the unconventional secretion of CFTR.

The most common mutation in cystic fibrosis patients is a phenylalanine deletion at position 508 (ΔF508) in the CFTR (cystic fibrosis transmembrane conductance regulator) gene. This mutation impairs cell-surface trafficking of CFTR. During cellular stress, core-glycosylated CFTRΔF508 is transported to the cell surface from the endoplasmic reticulum (ER) via an unconventional route that bypasses the Golgi. However, the mechanisms for this unconventional secretory pathway of CFTR are not well delineated. Here, we report that components of the macroautophagy/autophagy and ESCRT (endosomal sorting complex required for transport) pathways are involved in unconventional secretion of CFTR. In mammalian cells, we found that autophagic pathways were modulated by conditions that also stimulate unconventional secretion, namely ER stress and an ER-to-Golgi transport blockade. Additionally, we found that knockdown of early autophagy components, ATG5 and ATG7, and treatment with pharmacological autophagy inhibitors, wortmannin and 3-methyladenine, abolished the unconventional secretion of CFTR that had been stimulated by ER stress and an ER-to-Golgi blockade. Interestingly, immunoelectron microscopy revealed that GORASP2/GRASP55, which mediates unconventional CFTR trafficking, is present in multivesicular bodies (MVB) and autophagosomal structures under ER stress conditions. A custom small-interfering RNA screen of mammalian ESCRT proteins that mediate MVB biogenesis showed that silencing of some ESCRTs, including MVB12B, inhibited unconventional CFTRΔF508 secretion. Furthermore, MVB12B overexpression partially rescued cell-surface expression and Cl- channel function of CFTRΔF508. Taken together, these results suggest that components involved in early autophagosome formation and the ESCRT/MVB pathway play a key role in the stress-induced unconventional secretion of CFTR.

ZMYND10 stabilizes intermediate chain proteins in the cytoplasmic pre-assembly of dynein arms.

Zinc finger MYND-type-containing 10 (ZMYND10), a cytoplasmic protein expressed in ciliated cells, causes primary ciliary dyskinesia (PCD) when mutated; however, its function is poorly understood. Therefore, in this study, we examined the roles of ZMYND10 using Zmynd10-/-mice exhibiting typical PCD phenotypes, including hydrocephalus and laterality defects. In these mutants, morphology, the number of motile cilia, and the 9+2 axoneme structure were normal; however, inner and outer dynein arms (IDA and ODA, respectively) were absent. ZMYND10 interacted with ODA components and proteins, including LRRC6, DYX1C1, and C21ORF59, implicated in the cytoplasmic pre-assembly of DAs, whose levels were significantly reduced in Zmynd10-/-mice. LRRC6 and DNAI1 were more stable when co-expressed with ZYMND10 than when expressed alone. DNAI2, which did not interact with ZMYND10, was not stabilized by co-expression with ZMYND10 alone, but was stabilized by co-expression with DNAI1 and ZMYND10, suggesting that ZMYND10 stabilized DNAI1, which subsequently stabilized DNAI2. Together, these results demonstrated that ZMYND10 regulated the early stage of DA cytoplasmic pre-assembly by stabilizing DNAI1.

Sec16A is critical for both conventional and unconventional secretion of CFTR.

CFTR is a transmembrane protein that reaches the cell surface via the conventional Golgi mediated secretion pathway. Interestingly, ER-to-Golgi blockade or ER stress induces alternative GRASP-mediated, Golgi-bypassing unconventional trafficking of wild-type CFTR and the disease-causing ΔF508-CFTR, which has folding and trafficking defects. Here, we show that Sec16A, the key regulator of conventional ER-to-Golgi transport, plays a critical role in the ER exit of protein cargos during unconventional secretion. In an initial gene silencing screen, Sec16A knockdown abolished the unconventional secretion of wild-type and ΔF508-CFTR induced by ER-to-Golgi blockade, whereas the knockdown of other COPII-related components did not. Notably, during unconventional secretion, Sec16A was redistributed to cell periphery and associated with GRASP55 in mammalian cells. Molecular and morphological analyses revealed that IRE1α-mediated signaling is an upstream regulator of Sec16A during ER-to-Golgi blockade or ER stress associated unconventional secretion. These findings highlight a novel function of Sec16A as an essential mediator of ER stress-associated unconventional secretion.

Functional characterization of ABCB4 mutations found in progressive familial intrahepatic cholestasis type 3.

Multidrug resistance 3 (MDR3), encoded by the ATP-binding cassette, subfamily B, member 4 gene (ABCB4), localizes to the canalicular membrane of hepatocytes and translocates phosphatidylcholine from the inner leaflet to the outer leaflet of the canalicular membrane. Progressive familial intrahepatic cholestasis type 3 (PFIC3) is a rare hepatic disease caused by genetic mutations of ABCB4. In this study, we characterized 8 ABCB4 mutations found in PFIC3 patients, using in vitro molecular assays. First, we examined the transport activity of each mutant by measuring its ATPase activity using paclitaxel or phosphatidylcholine. Then, the pathogenic mechanisms by which these mutations affect MDR3 were examined through immunoblotting, cell surface biotinylation, and immunofluorescence. As a result, three ABCB4 mutants showed significantly reduced transport activity. Among these mutants, one mutation A364V, located in intracellular domains, markedly decreased MDR3 expression on the plasma membrane, while the others did not affect the expression. The expression of MDR3 on the plasma membrane and transport activity of A364V was rescued by a pharmacological chaperone, cyclosporin A. Our study provides the molecular mechanisms of ABCB4 mutations and may contribute to the understanding of PFIC3 pathogenesis and the development of a mutation-specific targeted treatment for PFIC3.

Monomerization and ER Relocalization of GRASP Is a Requisite for Unconventional Secretion of CFTR.

Induction of endoplasmic reticulum (ER)-to-Golgi blockade or ER stress induces Golgi reassembly stacking protein (GRASP)-mediated, Golgi-independent unconventional cell-surface trafficking of the folding-deficient ΔF508-cystic fibrosis transmembrane conductance regulator (CFTR). However, molecular mechanisms underlying this process remain elusive. Here, we show that phosphorylation-dependent dissociation of GRASP homotypic complexes and subsequent relocalization of GRASP to the ER play a critical role in the unconventional secretion of CFTR. Immunolocalization analyses of mammalian cells revealed that the Golgi protein GRASP55 was redistributed to the ER by stimuli that induce unconventional secretion of ΔF508-CFTR, such as induction of ER-to-Golgi blockade by the Arf1 mutant. Notably, the same stimuli also induced phosphorylation of regions near the C-terminus of GRASP55 and dissociation of GRASP homomultimer complexes. Furthermore, phosphorylation-mimicking mutations of GRASP55 induced the monomerization and ER relocalization of GRASP55, and these changes were nullified by phosphorylation-inhibiting mutations. These results provide mechanistic insights into how GRASP accesses the ER-retained ΔF508-CFTR and mediates the ER stress-induced unconventional secretion pathway.

Shank2 mutant mice display a hypersecretory response to cholera toxin.

Shank2 is a PDZ (PSD-95/discs large/ZO-1)-based adaptor that has been suggested to regulate membrane transporting proteins in the brain and epithelial tissues. Here, we report that Shank2 mutant (Shank2(-/-)) mice exhibit aberrant fluid and ion transport in the intestine. Molecular characterization using epithelial tissues from Shank2(+/+) and Shank2(-/-) mice revealed that a long spliceoform of Shank2 (Shank2E) is predominantly expressed in the pancreatic, renal and intestinal epithelia. In functional assays, deletion of Shank2 increased the cystic fibrosis transmembrane conductance regulator (CFTR)-dependent short-circuit currents by 84% (P < 0.05) and 101% (P < 0.05) in the mouse colon and rectum, respectively. Disruption of the CFTR-Shank2-phosphodiesterase 4D protein complex appeared to be mostly responsible for the changes in CFTR activities. Notably, Shank2 deletion profoundly increased cholera toxin-induced fluid accumulation in the mouse intestine (∼90%, P < 0.01). Analyses with chemical inhibitors confirmed that the hyperactivation of CFTR channel function is responsible for the increased response to cholera toxin. These results suggest that Shank2 is a key molecule that participates in epithelial homeostasis, in particular to prevent overt secretory responses caused by epithelial pathogens.

Rescue of ΔF508-CFTR trafficking via a GRASP-dependent unconventional secretion pathway.

The most prevalent disease-causing mutation of CFTR is the deletion of Phe508 (ΔF508), which leads to defects in conventional Golgi-mediated exocytosis and cell surface expression. We report that ΔF508-CFTR surface expression can be rescued in vitro and in vivo by directing it to an unconventional GRASP-dependent secretion pathway. An integrated molecular and physiological analysis indicates that mechanisms associated with ER stress induce cell surface trafficking of the ER core-glycosylated wild-type and ΔF508-CFTR via the GRASP-dependent pathway. Phosphorylation of a specific site of GRASP and the PDZ-based interaction between GRASP and CFTR are critical for this unconventional surface trafficking. Remarkably, transgenic expression of GRASP in ΔF508-CFTR mice restores CFTR function and rescues mouse survival without apparent toxicity. These findings provide insight into how unconventional protein secretion is activated, and offer a potential therapeutic strategy for the treatment of cystic fibrosis and perhaps diseases stemming from other misfolded proteins.