Trimming of the three glucose residues decorating nascent N-glycoproteins is a critical step for their entry into the endoplasmic reticulum quality control (ERQC) and recognition by ER chaperones. However, the functional relevance of the second glucose (G2) and the regulatory step upstream of its removal by glucosidase II (GCS2) remain poorly understood. Here, we report that TUSC3, a component of the oligosaccharyltransferase (OST) complex, regulates G2 to G1 trimming on N-glycosylated bone morphogenetic protein 4 (BMP4) and its Drosophila homolog Dpp to promote their ERQC entry. Loss- and gain-of-function genetic experiments and biochemical assays in mammalian cells and flies indicate that TUSC3 serves as a dosage-sensitive gatekeeper that influences the decision between proper folding and secretion versus elimination by ER-associated degradation for the BMP4 molecules, thereby tuning BMP signaling. Together, these data reveal an unrecognized role for an OST component in early glycoprotein maturation, relevant to a major developmental signaling pathway.

CAR-T cell therapy has revolutionized the treatment of hematologic malignancies. Still, durable activity in tumors remains limited by antigen heterogeneity and escape, immunosuppressive tumor microenvironment, and restricted persistence. Genome engineering with CRISPR/Cas systems offers a powerful route to reprogram CAR-T cells; however, translation increasingly depends on how editing payloads are delivered. Viral vectors remain a benchmark for efficient gene transfer, cargo constraints, insertional risk, immunogenicity, and manufacturing complexity motivate the development of safer, more scalable non-viral platforms. In this review, we provide an overview of CAR designs, clinical use, and current ex vivo manufacturing workflow; compare viral and non-viral delivery routes while distinguishing established ex vivo editing from emerging in vivo T-cell programming; and outline genome-engineering strategies organized by therapeutic goals. We highlight feasibility trade-offs and discuss how nanoparticles could enable transient, non-viral delivery of genome editors, while noting that robust T-cell targeting and standardized potency/safety assays remain key bottlenecks.

Background Patients affected by Rett syndrome (RTT) and MECP2 duplication syndrome (MDS) experience disabling muscle weakness and gastrointestinal dysmotility of unclear origin. Whether these defects arise cell-autonomously, rather than secondarily to neural dysfunction, and which developmental windows are most vulnerable to MeCP2 disfunction remains unresolved. MeCP2 is a dosage-sensitive transcriptional regulator, whose functions are tightly linked to chromatin states. Because short-chain fatty acids (SCFAs) are known to inhibit histone deacetylases (HDACs), a tractable in vivo model is needed to test the effect of HDAC modulation on muscle defects. Methods We misexpressed human MECP2 in the Drosophila melanogaster mesoderm that gives rise to skeletal and visceral muscles. We analyzed quantitatively their morphology and function. To assess the effects of SCFA supplementation, we also supplemented diets with sodium butyrate (NaB), Lalbaay®, a NaB-containing supplement, acetate (AcOH), and valproate (VPA). Findings MECP2 misexpression caused pre-eclosion lethality, thinning of larval skeletal fibers with nuclear mispositioning and altered mitochondria. Functionally, it reduced locomotion, decreased food transit and gut peristalsis. Phenotypes were strongest when expression began during development. NaB and VPA supplementation rescue most of these phenotypes, consistent with their histone-deacetylase (HDAC) activity. Defects were not observed upon comparable misexpression of an RTT-associated MeCP2 loss-of-function variant, indicating that they might be relevant to pathogenesis of MECP2-related disorders. Interpretation Our genetic in vivo analysis models peripheral effects of MeCP2 dysregulation and their amelioration, supporting the possibility of HDAC-targeted strategies for MECP2-related muscle and gastrointestinal dysfunction. ### Competing Interest Statement The authors have declared no competing interest.

Visceral myopathy is a rare, life-threatening disease linked to identified genetic mutations in 60% of cases. Mostly due to the dearth of knowledge regarding its pathogenesis, effective treatments are lacking. The disease is most commonly diagnosed in children with recurrent or persistent disabling episodes of functional intestinal obstruction, which can be life threatening, often requiring long-term parenteral or specialized enteral nutritional support. Although these interventions are undisputedly life-saving as they allow affected individuals to avoid malnutrition and related complications, they also seriously compromise their quality of life and can carry the risk of sepsis and thrombosis. Animal models for visceral myopathy, which could be crucial for advancing the scientific knowledge of this condition, are scarce. Clearly, a collaborative network is needed to develop research plans to clarify genotype-phenotype correlations and unravel molecular mechanisms to provide targeted therapeutic strategies. This paper represents a summary report of the first 'European Forum on Visceral Myopathy'. This forum was attended by an international interdisciplinary working group that met to better understand visceral myopathy and foster interaction among scientists actively involved in the field and clinicians who specialize in care of people with visceral myopathy.

AbstractIntestinal barrier dysfunction leads to inflammation and associated metabolic changes. However, the relative impact of infectious versus non-infectious mechanisms on animal health in the context of barrier dysfunction is not well understood. Here, we establish that loss ofDrosophila N-glycanase 1 (Pngl) leads to gut barrier defects, which cause starvation and increased JNK activity. These defects result in Foxo overactivation, which induces a hyperactive innate immune response and lipid catabolism, thereby contributing to lethality associated with loss ofPngl. Notably, germ-free rearing ofPnglmutants did not rescue lethality. In contrast, raisingPnglmutants on isocaloric, fat-rich diets improved animal survival in a dosage-dependent manner. Our data indicate that Pngl functions inDrosophilalarvae to establish the gut barrier, and that the immune and metabolic consequences of loss ofPnglare primarily mediated through non-infectious mechanisms.

Intestinal barrier dysfunction leads to inflammation and associated metabolic changes. However, the relative impact of gut bacteria versus non-bacterial insults on animal health in the context of barrier dysfunction is not well understood. Here, we establish that loss of Drosophila N -glycanase 1 (Pngl) in a specific intestinal cell type leads to gut barrier defects, causing starvation and JNK overactivation. These abnormalities, along with loss of Pngl in enterocytes and fat body, result in Foxo overactivation, leading to hyperactive innate immune response and lipid catabolism and thereby contributing to lethality. Germ-free rearing of Pngl mutants rescued their developmental delay but not lethality. However, raising Pngl mutants on isocaloric, fat-rich diets partially rescued lethality. Our data indicate that Pngl functions in Drosophila larvae to establish the gut barrier, and that the lethality caused by loss of Pngl is primarily mediated through non-bacterial induction of immune and metabolic abnormalities.

The short-chain fatty acid butyrate, produced by the gut microbiota, acts as a potent histone deacetylase (HDAC) inhibitor. We assessed possible ameliorative effects of butyrate, relative to other HDAC inhibitors, in in vitro and in vivo models of Rubinstein–Taybi syndrome (RSTS), a severe neurodevelopmental disorder caused by variants in the genes encoding the histone acetyltransferases CBP and p300. In RSTS cell lines, butyrate led to the patient-specific rescue of acetylation defects at subtoxic concentrations. Remarkably, we observed that the commensal gut microbiota composition in a cohort of RSTS patients is significantly depleted in butyrate-producing bacteria compared to healthy siblings. We demonstrate that the effects of butyrate and the differences in microbiota composition are conserved in a Drosophila melanogaster mutant for CBP, enabling future dissection of the gut–host interactions in an in vivo RSTS model. This study sheds light on microbiota composition in a chromatinopathy, paving the way for novel therapeutic interventions.

Mutations in humanN-glycanase 1 (NGLY1) cause the first known congenital disorder of deglycosylation (CDDG). Patients with this rare disease, which is also known asNGLY1deficiency, exhibit global developmental delay and other phenotypes including neuropathy, movement disorder, and constipation. NGLY1 is known to regulate proteasomal and mitophagy gene expression through activation of a transcription factor called "nuclear factor erythroid 2-like 1" (NFE2L1). Loss of NGLY1 has also been shown to impair energy metabolism, but the molecular basis for this phenotype and itsin vivoconsequences are not well understood. Using a combination of genetic studies, imaging, and biochemical assays, here we report that loss of NGLY1 in the visceral muscle of theDrosophilalarval intestine results in a severe reduction in the level of AMP-activated protein kinase α (AMPKα), leading to energy metabolism defects, impaired gut peristalsis, failure to empty the gut, and animal lethality.Ngly1–/–mouse embryonic fibroblasts andNGLY1deficiency patient fibroblasts also show reducedAMPKαlevels. Moreover, pharmacological activation of AMPK signaling significantly suppressed the energy metabolism defects in these cells. Importantly, the reduced AMPKα level and impaired energy metabolism observed inNGLY1deficiency models are not caused by the loss of NFE2L1 activity. Taken together, these observations identify reduced AMPK signaling as a conserved mediator of energy metabolism defects inNGLY1deficiency and suggest AMPK signaling as a therapeutic target in this disease.

Article Figures and data Abstract Introduction Results Discussion Materials and methods Appendix 1 Data availability References Decision letter Author response Article and author information Metrics Abstract During endoplasmic reticulum-associated degradation (ERAD), the cytoplasmic enzyme N-glycanase 1 (NGLY1) is proposed to remove N-glycans from misfolded N-glycoproteins after their retrotranslocation from the ER to the cytosol. We previously reported that NGLY1 regulates Drosophila BMP signaling in a tissue-specific manner (Galeone et al., 2017). Here, we establish the Drosophila Dpp and its mouse ortholog BMP4 as biologically relevant targets of NGLY1 and find, unexpectedly, that NGLY1-mediated deglycosylation of misfolded BMP4 is required for its retrotranslocation. Accumulation of misfolded BMP4 in the ER results in ER stress and prompts the ER recruitment of NGLY1. The ER-associated NGLY1 then deglycosylates misfolded BMP4 molecules to promote their retrotranslocation and proteasomal degradation, thereby allowing properly-folded BMP4 molecules to proceed through the secretory pathway and activate signaling in other cells. Our study redefines the role of NGLY1 during ERAD and suggests that impaired BMP4 signaling might underlie some of the NGLY1 deficiency patient phenotypes. Introduction N-Glycanase 1 (NGLY1; also known as peptide:N-glycanase or PNGase) is a cytoplasmic enzyme capable of removing N-glycans from glycoproteins (Suzuki et al., 2002). Mutations in human NGLY1 cause an autosomal recessive, multi-system developmental disorder called NGLY1 deficiency (OMIM # 610661) (Need et al., 2012; Enns et al., 2014). NGLY1 and its homologs recognize and cleave N-glycans from their target proteins, changing the asparagine (N) to aspartic acid (D) upon removing the sugar chain (Hirayama et al., 2015). N-Glycans are added to proteins in the ER and undergo trimming and maturation in the ER and in the Golgi apparatus, where the properly folded proteins are sorted toward various cellular destinations (Taniguchi and Aebi, 2015). However, proteins that fail to fold properly are recognized, retrotranslocated from the ER into the cytosol and undergo proteasomal degradation through a process called ER-associated degradation (ERAD) (Smith et al., 2011; Brodsky, 2012). The yeast NGLY1 homolog (PNG1) is part of the ERAD mechanism (Hirayama et al., 2015), and NGLY1 is proposed to contribute to ERAD by removing N-glycans from misfolded proteins after their retrotranslocation, thereby promoting their degradation (Suzuki et al., 2016). A fraction of NGLY1 molecules are associated with the ER (Suzuki et al., 1997a; Katiyar et al., 2004), likely through protein-protein interaction with valosin containing protein (VCP; also called p97) (Ye et al., 2001; Li et al., 2006). However, the functional significance of NGLY1’s ER recruitment is not known, and the temporal relationship between retrotranslocation and deglycosylation has not been determined. During Drosophila embryonic development, signaling by a bone morphogenetic protein (BMP) called Decapentaplegic (Dpp) is responsible for the specification of two regions in the middle part of the intestine (midgut), the gastric caeca region and the acid zone (Panganiban et al., 1990; Newfeld et al., 1996; Dubreuil, 2004). Dpp is first expressed in narrow bands in parasegments 3 (PS3) and PS7 of the embryonic visceral mesoderm (VM). Dpp then uses a paracrine/autocrine loop to sustain high levels of its own expression in the VM. As Dpp level increases in the PS3 and PS7 regions of the VM, it activates BMP signaling in the neighboring endoderm and induces the formation of gastric caeca and acid zone regions of the midgut (Panganiban et al., 1990; Hursh et al., 1993; Bienz, 1997; Galeone et al., 2017). We have previously shown that the Drosophila homolog of NGLY1 (PNGase-like or Pngl) is required in the VM to promote Dpp autoactivation in this tissue and consequently BMP signaling in the midgut endoderm (Galeone et al., 2017). However, the direct target of Pngl in the BMP pathway and the mechanism for the regulation of BMP signaling by Pngl are not known. Moreover, given the tissue-specific BMP defects observed in Drosophila Pngl mutants (Galeone et al., 2017), it remained to be seen whether NGLY1 regulates BMP signaling in mammals as well. Here, we provide mechanistic evidence of the regulation of BMP pathway by Pngl/NGLY1 in flies and mammals. Our data indicate that Pngl/NGLY1 promotes Dpp/BMP4 signaling by removing N-glycans from misfolded Dpp/BMP4. Analysis of Ngly1–/– mouse embryos shows developmental abnormalities accompanied by a severe decrease in the expression of the BMP effector pSMAD1/5 in the heart and brain. Unexpectedly, our data suggest that BMP4 deglycosylation is specifically mediated by NGLY1 molecules recruited to the ER membrane, not the free cytosolic pool of NGLY1. Moreover, loss of NGLY1 or impaired recruitment of NGLY1 to the ER results in the accumulation of misfolded BMP4 in the ER not in the cytosol, strongly suggesting that deglycosylation of BMP4 by NGLY1 occurs before BMP4 is fully retrotranslocated from the ER. Our studies identify a new biologically relevant target of deglycosylation by NGLY1 and challenge the current assumption about the order of events during glycoprotein ERAD. Results Drosophila Dpp is a direct target of Pngl/NGLY1 in vivo BMP ligands have a number of N-glycosylation sites, many of which are experimentally verified (Gelbart, 1989; Groppe et al., 1998; Tauscher et al., 2016). Therefore, one potential mechanism to explain the impaired Dpp signaling in Pngl mutants is that removal of N-glycans from Dpp promotes Dpp signaling in the VM. To test this hypothesis, we first overexpressed a GFP-tagged, functional version of Dpp (Teleman and Cohen, 2000) in the mesoderm using the GAL4/UAS system (Brand and Perrimon, 1993) and asked whether RNAi-mediated Pngl knock-down affects the migration of Dpp-GFP in western blots. In control embryos, immunoblotting with an anti-GFP antibody recognized a number of bands, corresponding in size to full-length Dpp-GFP and its cleavage products (Figure 1A). Upon Pngl knock-down, one of the Dpp-GFP bands shifted upwards, consistent with the presence of N-glycans that would normally be removed by Pngl. Incubation of the protein extract from these animals with PNGase F, an enzyme that removes N-glycans from N-glycoproteins (Freeze and Kranz, 2010), restored Dpp-GFP migration in mesodermal Pngl knock-down larvae (Figure 1A). None of the 13 asparagine residues present in GFP conform to the N-glycosylation minimal consensus sequence to which the majority of N-glycans are attached (N-X-S/T, where S is serine, T is threonine and X can be any amino acid other than proline) (Taniguchi and Aebi, 2015). These data provide strong evidence that Pngl deglycosylates a significant fraction of Dpp in Drosophila embryos. Figure 1 with 1 supplement see all Download asset Open asset Deglycosylation of Dpp by Pngl is essential for BMP signaling during Drosophila midgut development. (A) Western blot with α-GFP on protein lysates from embryos of indicated genotypes. The shift in band size upon treatment with PNGase F (black arrowhead) shows that full-length Dpp-GFP retains N-glycans upon RNAi-mediated Pngl knock-down in the embryonic mesoderm. The mature Dpp-GFP is indicated by the red arrowhead. (B) (i) Schematic representation of DppHA, which contains an HA tag in the active domain (red box). (ii) Schematic representation of dppHA knock-in allele and its mutant version dppHA-3NQ, in which three of Dpp’s four N-glycosylation sites are ablated by N-to-Q mutations. (C) Eclosion tests of Pngl–/– flies harboring a copy of dppHA (1.3% of the expected Mendelian ratio, n = 153 total progeny scored) or dppHA-3NQ (20.8% of the expected Mendelian ratio, n = 158 total progeny scored). One copy of dppHA-3NQ partially rescues the lethality of Pngl mutant flies. (D) Immunofluorescence staining of parasegment 3 (PS3) to PS7 region of embryonic midguts (marked by the dashed yellow lines) of animals with the indicated genotypes. Dpp is expressed in PS3 and PS7 of the embryonic visceral mesoderm (VM) and induces BMP signaling (evidenced by pMad expression) in PS3 and PS7, both in the VM (paracrine/autocrine) and in the endoderm. pMad staining (red) indicates that one copy of dppHA-3NQ, but not dppHA, restores the Dpp signaling during Pngl–/– midgut development. n = 5 for each genotype. Scale bar, 50 μm. Removal of Dpp N-glycans by Pngl/NGLY1 is essential for BMP signaling in Drosophila embryonic midgut Given the above observations, we sought to examine whether removing N-glycans from Dpp by Pngl affects BMP signaling in the embryonic midgut. To this end, we took advantage of two transposable elements flanking the second (last) coding exon of dpp and generated a knock-in, HA-tagged allele of dpp in which the three N-glycosylation sites located in this exon are replaced by N-to-Q mutations (Figure 1B, dppHA-3NQ). If Pngl promotes BMP signaling in the VM by directly removing N-glycans from Dpp (as opposed to affecting other potential targets and indirectly regulating the function of Dpp), DppHA-3NQ should not depend on Pngl for signaling in the VM. To test this hypothesis, we set crosses leading to the generation of Pngl–/– animals in which one of the two endogenous copies of dpp is replaced with dppHA or dppHA-3NQ. In agreement with our previous report on Pngl–/– animals (Galeone et al., 2017), only a small fraction (1.3%) of dppHA/+ Pngl–/– animals reached adulthood (Figure 1C). However, ~21% of dppHA-3NQ/+ Pngl–/– animals reached adulthood (Figure 1C). We have previously reported that loss of BMP signaling in the midgut is only responsible for up to 30% of the lethality in Pngl–/– animals (Galeone et al., 2017). Therefore, the BMP-dependent component of Pngl–/– lethality is largely rescued by genetic removal of these three N-glycans from Dpp. In control (Pngl+/–) embryos, both dppHA and dppHA-3NQ drive high levels of Dpp expression in PS3 and PS7 (Figure 1D), similar to endogenous Dpp expression pattern (Galeone et al., 2017). To assess the level of BMP signaling, we performed antibody staining for phosphorylated Mothers against dpp (pMad), which is the transducer of BMP signaling in flies (Newfeld et al., 1996). The pMad expression domain is broader in PS7 of dppHA-3NQ/+ Pngl+/– embryos compared to dppHA/+ Pngl+/– embryos (Figure 1D), suggesting that the 3NQ mutations might enhance BMP signaling in PS7. Similar to Pngl–/– embryos (Galeone et al., 2017), dppHA/+ Pngl–/– embryos show a severe decrease in pMad expression accompanied by weak and narrow Dpp expression domains in PS3 and PS7 (Figure 1D). Strikingly, one copy of dppHA-3NQ is sufficient to rescue Dpp and pMad expression in PS3 and PS7 of Pngl–/– embryos (Figure 1D). These observations indicate that Pngl regulates BMP signaling in the embryonic VM by removing these three N-glycans or a subset of them from Dpp, and that the N-to-D conversion of N-glycosylated amino acids upon Pngl-mediated deglycosylation is not essential for BMP signaling during Drosophila midgut development. We also examined the effect of homozygosity for dppHA and dppHA-3NQ alleles on BMP signaling in embryos and on animal survival. Staining dppHA-3NQ/HA-3NQ and control dppHA/HA embryos for HA revealed a similar expression pattern for DppHA and DppHA-3NQ but was somewhat broader for DppHA-3NQ (Figure 1—figure supplement 1). While the overall pMad expression pattern was similar in these two genotypes, pMad staining in the PS3 and PS7 areas of the developing midgut was expanded in dppHA-3NQ/HA-3NQ animals (Figure 1—figure supplement 1). These data indicate that mutating these three Dpp N-glycosylation sites does not impair Dpp signaling during embryogenesis, and suggest that the mutations might even enhance Dpp’s range of signaling in the midgut region. The control dppHA/HA animals reached adulthood at the expected Mendelian ratio (n = 225). However, dppHA-3NQ/HA-3NQ animals did not reach adulthood (n = 97) and died by the second instar stage. While we do not know the reason for the lethality of dppHA-3NQ/HA-3NQ animals, the HA and pMad staining data suggest that the lethality is not due to the loss of BMP signaling in the embryos. Ngly1-mutant mouse embryos show a severe decrease in BMP signaling in some contexts NGLY1 deficiency patients exhibit global developmental delay, seizures, involuntary movements, chronic constipation, osteopenia, small hands and feet, lack of tears and other symptoms (Need et al., 2012; Enns et al., 2014; Lam et al., 2017). A loss-of-function mutation in the mouse Ngly1 (Ngly1tm1.1Tasuz) results in late embryonic lethality accompanied by ventricular septal defects (VSD) (Fujihira et al., 2017). To examine whether mammalian BMP signaling is affected by the loss of Ngly1, we performed pSMAD1/5 staining in E15.5 mouse embryos homozygous for a newly generated Ngly1 null mutant (Ngly1em4Lutzy; Figure 2—figure supplement 1) and sibling controls. We focused our analysis on the heart (because of the above-mentioned report [Fujihira et al., 2017]) and on the CNS (because of human patient phenotypes [Need et al., 2012; Enns et al., 2014; Lam et al., 2017]). All five Ngly1–/– mutant embryos examined in our studies had VSDs (not shown). Ngly1+/+ (control) embryos showed elaborate myocardial trabeculae expressing high level of pSMAD1/5 (Figure 2A, arrowheads). We found an overall decrease in the heart size and a remarkable reduction of myocardial trabeculae in mutant embryos (Figure 2B). The remaining trabeculae showed a much weaker pSMAD1/5 staining compared to controls (Figure 2B, arrowhead). In the CNS, we found significant abnormalities in the 4th ventricle choroid plexus. In Ngly1+/+ E15.5 embryos, the choroid plexus consists of slender structures that express high levels of pSMAD1/5 and reach both sides of the ventricle (Figure 2C and E). However, Ngly1–/– embryos have a rudimentary, stump-like choroid plexus with a dramatic decrease in pSMAD1/5 expression (Figure 2D and F, arrowheads). pSMAD1/5 staining was also observed in control E15.5 cerebella (Figure 2E, arrowheads) but was significantly reduced in mutant cerebella (Figure 2F), although the morphological difference between control and mutant cerebella was less stark than that between control and mutant choroid plexus (Figure 2C and D, asterisks). BMP signaling, mediated by several ligands including BMP4, is essential for normal cardiovascular and choroid plexus development (Hébert et al., 2002; Hébert et al., 2003; Morrell et al., 2016), and BMP4 is sufficient to induce choroid plexus epithelial fate in neuroepithelial progenitors (Lehtinen et al., 2013). Therefore, these data indicate that similar to fly Pngl, mouse NGLY1 is required for BMP signaling in specific contexts and suggest a possible causative role between decreased BMP signaling and the observed phenotypes. Figure 2 with 1 supplement see all Download asset Open asset Ngly1 is required for BMP signaling in embryonic heart and cerebellum. (A,B) Immunofluorescence staining in sagittal sections of the embryonic heart of control (Ngly1+/+) and Ngly1–/– animals at E15.5. Nuclei (DAPI) are in blue and pSMAD1/5 in red. n = 5 for each genotype. pSMAD1/5 staining is severely reduced in mutant embryos (compare grey scale images), suggesting that loss of Ngly1 leads to reduced BMP signaling in the heart. Arrowheads mark the myocardial trabeculae. (C–F) H&E (C,D) and immunofluorescence (E,F) staining in sagittal sections of embryonic cerebellum of control (Ngly1+/+) and Ngly1–/– animals at E15.5. n = 5 for each genotype. Loss of Ngly1 leads to significant morphological abnormalities in the 4th ventricle choroid plexus (close-up images from blue boxes) and severe reduction in pSMAD1/5 staining (in grey scale). Scale bars are 100 µm in A,B,E,F and 200 µm in c,d (both low magnification and close-up images). These data suggest that Ngly1 has a role in BMP signaling in developing choroid plexus. NGLY1 promotes BMP4 signal-sending in mouse embryonic fibroblasts To directly test the role of NGLY1 in mammalian BMP signaling, we performed staining and immunoblotting with an anti-pSMAD1/5 antibody in Ngly1–/– and control mouse embryonic fibroblasts (MEFs) (Huang et al., 2015). A low level of pSMAD1/5 expression was detected in wild-type MEFs, which did not show a statistically significant decrease in Ngly1–/– MEFs (Figure 3A,C and D). Transfection of control MEFs with a construct expressing double-tagged BMP4 (HA-tag in the prodomain and Myc-tag in the active domain) resulted in a 12-fold increase in BMP signaling (Figure 3B–D). However, BMP signaling in Ngly1–/– MEFs remained at the baseline level upon Bmp4HA-Myc transfection (Figure 3B–D). Of note, treating control and Ngly1–/– cells with recombinant BMP4 resulted in similar levels of pSMAD1/5 expression (Figure 3C,D), indicating that NGLY1 is not essential for receiving the BMP4 signal. Together, these data indicate impaired BMP4 signal-sending in Ngly1–/– MEFs, similar to the impaired Dpp signal-sending observed in Pngl–/– fly VM (Galeone et al., 2017). Figure 3 with 1 supplement see all Download asset Open asset Loss of Ngly1 leads to impaired BMP4 signal-sending in mouse embryonic fibroblasts. (A) Representative immunofluorescence staining of DAPI (blue) and pSMAD1/5 (red) in untransfected WT and Ngly1 mutant (–/–) MEFs used as a baseline for pSMAD1/5 expression (grey scale). (B) WT and Ngly1 mutant MEFs co-transfected with expression vectors for BMP4HA-Myc and GFP. GFP expression is used as control for transfection efficiency. pSMAD1/5 expression (grey scale) is used as readout of BMP signaling. Scale bars, 50 μm. n = 6 biologically independent samples. Loss of Ngly1 severely reduces pSMAD1/5 levels. (C) Representative western blot of pSMAD1/5 from WT and Ngly1 mutant MEF cell lysates. The first two lanes are without transfection (control); the middle two lanes are co-transfected with expression vectors for BMP4HA-Myc and GFP (for transfection efficiency); the last two lanes are treated with recombinant BMP4 in the culture media. (D) Quantification of the data shown in C. For each blot, the pSMAD1/5 expression was normalized to tubulin and the value of Ngly1+/+ cells without transfection was set as 1. n = 3 biologically independent samples. Two-way ANOVA with Tukey's multiple comparisons test was used for statistical analysis. Mean ± s.d. are shown. ****p<0.0001, NS, not significant. These data exhibit that BMP4 signal-sending, but not signal-receiving, is impaired in MEFs upon loss of Ngly1, similarly to the impaired Dpp signal-sending in Drosophila embryonic midguts (Galeone et al., 2017). Figure 3—source data 1 Raw data and statistical analysis for panel D. https://cdn.elifesciences.org/articles/55596/elife-55596-fig3-data1-v2.xlsx Download elife-55596-fig3-data1-v2.xlsx To determine whether inhibition of NGLY1 leads to impairment of endogenous BMP4 signaling in a mouse cell line, we used the preadipocyte 3T3-L1 cells line, which expresses Bmp4 and depends on endogenous BMP4 signaling for pSMAD1/5 expression and adipocyte differentiation (Suenaga et al., 2010; Suenaga et al., 2013). As shown in Figure 3—figure supplement 1, treating 3T3-L1 cells with low and high concentrations of the NGLY1 inhibitor Z-VAD-fluoromethylketone (fmk) (Misaghi et al., 2004) resulted in a significant decrease in pSMAD1/5 levels in these cells, indicating reduced BMP signaling. Z-VAD-fmk is also a potent pan-caspase inhibitor, especially at high concentrations (Misaghi et al., 2004). To rule out the possibility that Z-VAD-fmk reduced BMP signaling in 3T3-L1 cells through its caspase inhibitory effect, we treated these cells with another broad spectrum caspase inhibitor, Q-VD-OPh, that does not inhibit NGLY1 (Caserta et al., 2003; Misaghi et al., 2004; Tomlin et al., 2017). As shown in Figure 3—figure supplement 1, 3T3-L1 cells treated with Q-VD-OPh did not exhibit any reduction in the level of pSMAD1/5. These data are in agreement with our Drosophila and mouse staining results and our MEF overexpression studies, and suggest that NGLY1 plays a key role in endogenous BMP4 signaling in some contexts. Loss of NGLY1 results in accumulation of BMP4 in the ER and upregulation of the ER stress markers Our observations in MEFs and mouse embryos, combined with data establishing Dpp as a target of Pngl, prompted us to examine whether loss of Ngly1 affects the glycosylation status and trafficking of BMP4. Double staining for HA and the ER marker KDEL showed that BMP4HA-Myc only partially localizes to the ER in control MEFs (Figure 4A), likely reflecting the exocytic trafficking of BMP4 from ER to Golgi and beyond. In contrast, BMP4HA-Myc almost fully colocalized with KDEL in Ngly1–/– MEFs (Figure 4A). This suggests that in the absence of NGLY1, BMP4 is trapped in the ER. The anti-KDEL antibody used in our studies is raised against the KDEL-containing C-terminal part of the GRP78/BiP and can recognize this protein in immunoblots (Cai et al., 1998; Barra et al., 2017). GRP78/Bip (official name: heat shock protein family A member 5, HSPA5) is a major ER luminal chaperone whose expression can be used to monitor ER stress (Lee, 2005). As shown in Figure 4B and C, the level of GRP78/BiP was significantly increased in control MEFs transfected with Bmp4HA-Myc, suggesting some BMP4 misfolding in these cells. Untransfected Ngly1–/– MEFs also showed a similar increase in GRP78/BiP expression (Figure 4B and C), suggesting some degree of ER stress in these cells (Kozutsumi et al., 1988; Gülow et al., 2002). Upon Bmp4HA-Myc transfection, Ngly1–/– cells showed further increase in GRP78/BiP level (Figure 4B and C), suggesting that expression of BMP4HA-Myc leads to additional ER stress in Ngly1–/– cells. Figure 4 with 1 supplement see all Download asset Open asset In Ngly1 mutant MEFs, BMP4 retains N-glycans and is trapped in the ER. (A) Confocal sections of WT and Ngly1 mutant MEFs co-transfected with Bmp4HA-Myc and GFP (for transfection efficiency) and stained for HA (to mark BMP4HA-Myc, green) and the ER marker KDEL (red). HA-tagged BMP4 almost fully colocalizes with KDEL/GRP78 in Ngly1 mutant cells, suggesting impaired exocytic trafficking. Scale bars, 10 μm. n = 8 biologically independent samples. (B,C) Western blot with α-KDEL antibody, which marks GRP78 (BiP), and α-pIRE1α, α-IRE1α, and α-OS9 antibodies on protein lysates from WT and Ngly1 mutant MEFs with or without Bmp4HA-Myc transfection. ER stress is induced in Ngly1–/– MEFs and is significantly enhanced upon Bmp4 transfection, likely due to the accumulation of misfolded BMP4. A representative image of three independent blots is shown. For each blot, the protein expression was normalized to tubulin and the value of Ngly1+/+ cells without transfection was set as 1. Two-way ANOVA with Tukey's multiple comparisons test was used for statistical analysis. Mean ± s.d. is shown. *p<0.05; **p<0.01; ***p<0.001; NS, not significant. (D) Western blot for HA and Myc on cell lysates and media from WT and Ngly1 mutant MEFs transfected with expression vectors for BMP4HA-Myc and GFP or GFP only (last lane). Protein extracts were treated with PNGase and Endo H for glycosylation profiling. Note the accumulation of a slow-migrating HA+ band in Ngly1–/– MEFs which returns to normal size upon treatment with both enzymes, indicating the retention of N-glycans on BMP4 upon loss of Ngly1. Note also the severe decrease in the level of active BMP4 (Myc blot) secreted by Ngly1–/– MEFs into the culture media. (E) Western blot for HA, pSMAD1/5 and Myc on cell lysates and media from WT MEFs transfected with an expression vector for BMP4HA-Myc-4NQ compared with WT and Ngly1–/– MEFs expressing wild-type BMP4HA-Myc. n = 3 independent biological samples. These data indicate that N-glycans on BMP4 are not necessary for its secretion (Myc) and signaling (pSMAD1/5) and that NGLY1 promotes BMP4 signaling by removing one or more N-glycans from BMP4, similar to their Drosophila counterparts. Figure 4—source data 1 Raw data and statistical analysis for panel C. https://cdn.elifesciences.org/articles/55596/elife-55596-fig4-data1-v2.xlsx Download elife-55596-fig4-data1-v2.xlsx To provide further evidence for ER stress in Ngly1–/– cells especially upon BMP4 overexpression, we examined the levels of two additional ER stress markers: phosphorylated IRE1α (pIRE1α), which is an indicator of unfolded protein response activation (Zhang and Kaufman, 2004; Korennykh et al., 2009), and OS9, an ER lectin which is upregulated upon ER stress, selectively binds misfolded glycoproteins, and facilitates their transport to the retrotranslocation machinery (Kim et al., 2005; Alcock and Swanton, 2009; Satoh et al., 2010). The levels of pIRE1α and OS9 were moderately increased in Ngly1–/– cells, further suggesting that loss of Ngly1 leads to ER stress, potentially due to misfolded glycoprotein accumulation (Figure 4B and C). Moreover, upon Bmp4HA-Myc transfection, the levels of pIRE1α and OS9 in Ngly1–/– cells increased to 2.5 to 3-fold of pIRE1α and OS9 in untransfected Ngly1–/– cells. Together with GRP78 accumulation, these data demonstrate that Bmp4 transfection leads to significant ER stress in Ngly1–/– MEFs, potentially due to the accumulation of misfolded BMP4 in the ER. Removal of BMP4 N-glycans by NGLY1 promotes BMP4 signal-sending in MEFs The full-length BMP proteins dimerize in the ER, but the cleavages that release the dimerized active domain of BMP ligands from the prodomains are thought to occur in the Golgi apparatus and/or further along the secretory pathway (Figure 4—figure supplement 1; Nelsen and Christian, 2009). As expected from reduced signaling activity by BMP4HA-Myc in the absence of NGLY1, conditioned media from Ngly1–/– MEFs showed a severe decrease in the level of BMP4Myc active domain secreted by Ngly1–/– cells compared to control MEFs (Figure 4D). Ngly1–/– cells accumulated a band corresponding to the full-length BMP4HA-Myc that migrated slower than the full-length BMP4HA-Myc from control cells in SDS-PAGE gels, compatible with retention of N-glycans upon loss of NGLY1 (Figure 4D). Upon digestion of the cell lysates with PNGase F and Endo H enzymes (Freeze and Kranz, 2010), the BMP4HA-Myc band migrated similarly in Ngly1+/+ and Ngly1–/– cells, indicating that BMP4 retains ER-type (high mannose) N-glycans in Ngly1–/– cells (Figure 4D). Moreover, a mutant version of BMP4HA-Myc harboring N-to-Q mutations in all four N-glycosylation sites (BMP4HA-Myc-4NQ) was secreted efficiently by MEFs and was able to induce robust BMP signaling (Figure 4E). These observations indicate that in Ngly1–/– MEFs, BMP4HA-Myc accumulates in the ER and retains ER-type N-glycans. The data further support the notion that like Dpp, removal of BMP4 N-glycan(s) by NGLY1 is required for BMP4 signaling. BMP4/Dpp deglycosylation is specifically mediated by NGLY1 molecules recruited to the ER The current model for the role of NGLY1 in ERAD posits that NGLY1 deglycosylates misfolded proteins in the cytosol after their retrotranslocation from the ER to facilitate proteasomal degradation (Suzuki et al., 2016) and predicts that loss of NGLY1 should result in the accumulation of its targets in the cytosol. Given the accumulation of glycosylated BMP4 in the ER of Ngly1–/– MEFs, we sought to reevaluate this model. NGLY1 is likely recruited from the cytosol to the ER membrane via its association with valosin containing protein (VCP; also called p97) (Li et al., 2006), an ATPase which is recruited to the ER upon the accumulation of misfolded proteins in the ER (Kondratyev et al., 2007) and plays a critical role in retrotranslocation of misfolded proteins from the ER to cytosol (Ye et al., 2001). Staining of Ngly1–/– MEFs transfected with an expression vector for a V5-tagged version of wild-type human NGLY1 (NGLY1V5-WT) with anti-V5 and anti-KDEL antibodies did not show a clear association between NGLY1 and ER (Figure 5A). However, co-transfection of Bmp4HA-Myc with NGLY1V5-WT resulted in a significant overlap between V5 and KDEL signals (Figure 5A and B), suggesting that NGLY1 is recruited to the ER upon ER stress, similar to VCP (Kondratyev et al., 2007). Next, we generated two mutant versions of human NGLY1 (N41P and G79A/F80A) previously shown to abolish NGLY1-VCP interaction (Li et al., 2006). As shown in Figure 5A, ER recruitment of NGLY1 was abolished in both mutant versions. These observations support the notion that upon ER stress, NGLY1 is recruited to the ER, potentially through its interaction with VCP. Figure 5 Download asset Open asset Mutations in VCP-binding sites abolish the recruitment of NGLY1 to ER. (A) Confocal sections of Ngly1–/– MEFs transfected with expression vectors for wild-type or VCP-binding mutant versions of human NGLY1 tagged with V5 and stained with V5 (green) and KDEL (ER marker, red). Note the ER recruitment of wild-type NGLY1 upon Bmp4 transfection, which is likely due to the accumulation of misfolded BMP4 in the ER. Mutant NGLY1 proteins do not show ER recruitment. (B) Analysis of relative fluorescence overlap of V5-tagged NGLY1 (green) and KDEL signals (red) in A. The signal overlap is quantified by Pearson correlation analysis of five images from three independent experiments. Scale bars, 10 μm. Mean ± s.d. is shown. ***p=0.00011; NS, not significant. These results show that mutations in VCP-binding sites lead to a failure of the NGLY1 recruitment to ER. Figure 5—source data 1 Raw data and statistical analysis for panel B. https://cdn.elifesciences.org/articles/55596/elife-55596-f

During endoplasmic reticulum-associated degradation (ERAD), the cytoplasmic enzyme N-glycanase 1 (NGLY1) is proposed to remove N-glycans from misfolded N-glycoproteins after their retrotranslocation from the ER to the cytosol. We previously reported that NGLY1 regulates Drosophila BMP signaling in a tissue-specific manner (Galeone et al., 2017). Here, we establish the Drosophila Dpp and its mouse ortholog BMP4 as biologically relevant targets of NGLY1 and find, unexpectedly, that NGLY1-mediated deglycosylation of misfolded BMP4 is required for its retrotranslocation. Accumulation of misfolded BMP4 in the ER results in ER stress and prompts the ER recruitment of NGLY1. The ER-associated NGLY1 then deglycosylates misfolded BMP4 molecules to promote their retrotranslocation and proteasomal degradation, thereby allowing properly-folded BMP4 molecules to proceed through the secretory pathway and activate signaling in other cells. Our study redefines the role of NGLY1 during ERAD and suggests that impaired BMP4 signaling might underlie some of the NGLY1 deficiency patient phenotypes.

Mutations in the human N-glycanase 1 (NGLY1) cause a rare, multisystem congenital disorder with global developmental delay. However, the mechanisms by which NGLY1 and its homologs regulate embryonic development are not known. Here we show that Drosophila Pngl encodes an N-glycanase and exhibits a high degree of functional conservation with human NGLY1. Loss of Pngl results in developmental midgut defects reminiscent of midgut-specific loss of BMP signaling. Pngl mutant larvae also exhibit a severe midgut clearance defect, which cannot be fully explained by impaired BMP signaling. Genetic experiments indicate that Pngl is primarily required in the mesoderm during Drosophila development. Loss of Pngl results in a severe decrease in the level of Dpp homodimers and abolishes BMP autoregulation in the visceral mesoderm mediated by Dpp and Tkv homodimers. Thus, our studies uncover a novel mechanism for the tissue-specific regulation of an evolutionarily conserved signaling pathway by an N-glycanase enzyme.

Toxicity of silver nanoparticles (AgNPs) is supported by many observations in literature, but no mechanism details have been proved yet. Here we confirm and quantify the toxic potential of fully characterized AgNPs in HeLa and A549 cells. Notably, through a specific fluorescent probe, we demonstrate the intracellular release of Ag(+) ions in living cells after nanoparticle internalization, showing that in-situ particle degradation is promoted by the acidic lysosomal environment. The activation of metallothioneins in response to AgNPs and the possibility to reverse the main toxic pathway by Ag(+) chelating agents demonstrate a cause/effect relationship between ions and cell death. We propose that endocytosed AgNPs are degraded in the lysosomes and the release of Ag(+) ions in the cytosol induces cell damages, while ions released in the cell culture medium play a negligible effect. These findings will be useful to develop safer-by-design nanoparticles and proper regulatory guidelines of AgNPs. From the clinical editor: The authors describe the toxic potential of silver nanoparticles (AgNP) in human cancer cell lines. Cell death following the application of AgNPs is dose-dependent, and it is mostly due to Ag+ ions. Further in vivo studies should be performed to gain a comprehensive picture of AgNP-toxicity in mammals.

We have studied in vitro toxicity of iron oxide nanoparticles (NPs) coated with a thin silica shell (Fe3O4/SiO2 NPs) on A549 and HeLa cells. We compared bare and surface passivated Fe3O4/SiO2 NPs to evaluate the effects of the coating on the particle stability and toxicity. NPs cytotoxicity was investigated by cell viability, membrane integrity, mitochondrial membrane potential (MMP), reactive oxygen species (ROS) assays, and their genotoxicity by comet assay. Our results show that NPs surface passivation reduces the oxidative stress and alteration of iron homeostasis and, consequently, the overall toxicity, despite bare and passivated NPs show similar cell internalization efficiency. We found that the higher toxicity of bare NPs is due to their stronger in-situ degradation, with larger intracellular release of iron ions, as compared to surface passivated NPs. Our results indicate that surface engineering of Fe3O4/SiO2 NPs plays a key role in improving particles stability in biological environments reducing both cytotoxic and genotoxic effects.