Biallelic Variants in UBA5 Link Dysfunctional UFM1 Ubiquitin-like Modifier Pathway to Severe Infantile-Onset Encephalopathy.

The ubiquitin fold modifier 1 (UFM1) cascade is a recently identified evolutionarily conserved ubiquitin-like modification system whose function and link to human disease have remained largely uncharacterized. By using exome sequencing in Finnish individuals with severe epileptic syndromes, we identified pathogenic compound heterozygous variants in UBA5, encoding an activating enzyme for UFM1, in two unrelated families. Two additional individuals with biallelic UBA5 variants were identified from the UK-based Deciphering Developmental Disorders study and one from the Northern Finland Intellectual Disability cohort. The affected individuals (n = 9) presented in early infancy with severe irritability, followed by dystonia and stagnation of development. Furthermore, the majority of individuals display postnatal microcephaly and epilepsy and develop spasticity. The affected individuals were compound heterozygous for a missense substitution, c.1111G>A (p.Ala371Thr; allele frequency of 0.28% in Europeans), and a nonsense variant or c.164G>A that encodes an amino acid substitution p.Arg55His, but also affects splicing by facilitating exon 2 skipping, thus also being in effect a loss-of-function allele. Using an in vitro thioester formation assay and cellular analyses, we show that the p.Ala371Thr variant is hypomorphic with attenuated ability to transfer the activated UFM1 to UFC1. Finally, we show that the CNS-specific knockout of Ufm1 in mice causes neonatal death accompanied by microcephaly and apoptosis in specific neurons, further suggesting that the UFM1 system is essential for CNS development and function. Taken together, our data imply that the combination of a hypomorphic p.Ala371Thr variant in trans with a loss-of-function allele in UBA5 underlies a severe infantile-onset encephalopathy.

Vesicular and non-vesicular transport feed distinct glycosylation pathways in the Golgi.

Newly synthesized proteins and lipids are transported across the Golgi complex via different mechanisms whose respective roles are not completely clear. We previously identified a non-vesicular intra-Golgi transport pathway for glucosylceramide (GlcCer)--the common precursor of the different series of glycosphingolipids-that is operated by the cytosolic GlcCer-transfer protein FAPP2 (also known as PLEKHA8) (ref. 1). However, the molecular determinants of the FAPP2-mediated transfer of GlcCer from the cis-Golgi to the trans-Golgi network, as well as the physiological relevance of maintaining two parallel transport pathways of GlcCer--vesicular and non-vesicular--through the Golgi, remain poorly defined. Here, using mouse and cell models, we clarify the molecular mechanisms underlying the intra-Golgi vectorial transfer of GlcCer by FAPP2 and show that GlcCer is channelled by vesicular and non-vesicular transport to two topologically distinct glycosylation tracks in the Golgi cisternae and the trans-Golgi network, respectively. Our results indicate that the transport modality across the Golgi complex is a key determinant for the glycosylation pattern of a cargo and establish a new paradigm for the branching of the glycosphingolipid synthetic pathway.

Sqstm1-GFP knock-in mice reveal dynamic actions of Sqstm1 during autophagy and under stress conditions in living cells.

Sqstm1 serves as a signaling hub and receptor for selective autophagy. Consequently, dysregulation of Sqstm1 causes imbalances in signaling pathways and disrupts proteostasis, thereby contributing to the development of human diseases. Environmental stresses influence the level of Sqstm1 by altering its expression and/or autophagic degradation, and also changes the localization of Sqstm1, making it difficult to elucidate the actions and roles of this protein. In this study, we developed knock-in mice expressing Sqstm1 fused to GFP (Sqstm1-GFP(KI/+)). Using these Sqstm1-GFP(KI/+) mice, we revealed for the first time the dynamics of endogenous Sqstm1 in living cells. Sqstm1-GFP was translocated to a restricted area of LC3-positive structures, which primarily correspond to the inside of autophagosomes, and then degraded. Moreover, exposure to arsenite induced expression of Sqstm1-GFP, followed by accumulation of the fusion protein in large aggregates that were degraded by autophagy. Furthermore, suppression of autophagy in Sqstm1-GFP(KI/+) mouse livers caused accumulation of Sqstm1-GFP and formation of GFP-positive aggregate structures, leading to severe hepatic failure. These results indicate that Sqstm1-GFP(KI/+) mice are a useful tool for analyzing Sqstm1 in living cells and intact animals.

Proteasome dysfunction activates autophagy and the Keap1-Nrf2 pathway.

The ubiquitin-proteasome system and autophagy are crucially important for proteostasis in cells. These pathways are interdependent, and dysfunction in either pathway causes accumulation of ubiquitin-positive aggregates, a hallmark of human pathological conditions. To elucidate in vivo compensatory action(s) against proteasomal dysfunction, we developed mice with reduced proteasome activity in their livers. The mutant mice exhibited severe liver damage, accompanied by formation of aggregates positive for ubiquitin and p62/Sqstm1, an adaptor protein for both selective autophagy and the anti-oxidative Keap1-Nrf2 pathway. These aggregates were selectively entrapped by autophagosomes, and pathological features of livers with impaired proteasome activity were exacerbated by simultaneous suppression of autophagy. In contrast, concomitant loss of p62/Sqstm1 had no apparent effect on the liver pathology though p62/Sqstm1 was indispensable for the aggregates formation. Furthermore, defective proteasome function led to transcriptional activation of the Nrf2, which served as a physiological adaptation. Our in vivo data suggest that cells contain networks of cellular defense mechanisms against defective proteostasis.

Endosomal protein expression of γ1-adaptin is associated with tumor growth activity and relapse-free survival in breast cancer.

BACKGROUND: γ1-Adaptin is a subunit of adaptor protein complex-1 (AP-1), which regulates intracellular transport between the trans-Golgi network (TGN) and endosomes. Since expression levels of AP-1 subunits have been reported to be associated with cell proliferation and cancer malignancy, we investigated the relationships between the immunohistochemical expression of γ1-adaptin and both clinicopathological factors and relapse-free survival (RFS) in breast cancer tissue. MATERIALS AND METHODS: SK-BR-3 cell line depleted of γ1-adaptin was used for cell proliferation, migration, and invasion assay. Intracellular localization of γ1-adaptin was examined with immunohistochemistry (IHC) using an antibody against γ1-adaptin, and with double immunohistofluorescence (IHF) microscopy using markers for the TGN and endosome. γ1-Adaptin intensities in IHC samples from 199 primary breast cancer patients were quantified and assessed in relation to clinicopathological factors and RFS. RESULTS: Cell growth, migration, and invasion of SK-BR-3 cells were significantly suppressed by the depletion of γ1-adaptin. Although the staining patterns in the cancer tissues varied among cases by IHC, double IHF demonstrated that γ1-adaptin was mainly localized in EEA1-positive endosomes, but not in the TGN. γ1-Adaptin intensity was significantly higher in the tumor regions than in non-tumor regions. It was also higher in patients with Ki-67 (high), ER (-), PgR (-), and HER2 (+). Among subtypes of breast cancer, γ1-adaptin intensity was higher in HER2 than in luminal A or luminal B. The results of the survival analysis indicated that high γ1-adaptin intensity was significantly associated with worse RFS, and this association was also observed in group with ER (+), PgR (+), HER2 (-), Ki-67 (high), or luminal B. In addition, the Cox proportional hazards model showed that high γ1-adaptin intensity was an independent prognostic factor. CONCLUSION: These results suggest that the endosomal expression of γ1-adaptin is positively correlated with breast cancer malignancy and could be a novel prognostic marker.

STING signalling is terminated through ESCRT-dependent microautophagy of vesicles originating from recycling endosomes.

Stimulator of interferon genes (STING) is essential for the type I interferon response against a variety of DNA pathogens. Upon emergence of cytosolic DNA, STING translocates from the endoplasmic reticulum to the Golgi where STING activates the downstream kinase TBK1, then to lysosome through recycling endosomes (REs) for its degradation. Although the molecular machinery of STING activation is extensively studied and defined, the one underlying STING degradation and inactivation has not yet been fully elucidated. Here we show that STING is degraded by the endosomal sorting complexes required for transport (ESCRT)-driven microautophagy. Airyscan super-resolution microscopy and correlative light/electron microscopy suggest that STING-positive vesicles of an RE origin are directly encapsulated into Lamp1-positive compartments. Screening of mammalian Vps genes, the yeast homologues of which regulate Golgi-to-vacuole transport, shows that ESCRT proteins are essential for the STING encapsulation into Lamp1-positive compartments. Knockdown of Tsg101 and Vps4, components of ESCRT, results in the accumulation of STING vesicles in the cytosol, leading to the sustained type I interferon response. Knockdown of Tsg101 in human primary T cells leads to an increase the expression of interferon-stimulated genes. STING undergoes K63-linked ubiquitination at lysine 288 during its transit through the Golgi/REs, and this ubiquitination is required for STING degradation. Our results reveal a molecular mechanism that prevents hyperactivation of innate immune signalling, which operates at REs.

The UFM1 system regulates ER-phagy through the ufmylation of CYB5R3.

Protein modification by ubiquitin-like proteins (UBLs) amplifies limited genome information and regulates diverse cellular processes, including translation, autophagy and antiviral pathways. Ubiquitin-fold modifier 1 (UFM1) is a UBL covalently conjugated with intracellular proteins through ufmylation, a reaction analogous to ubiquitylation. Ufmylation is involved in processes such as endoplasmic reticulum (ER)-associated protein degradation, ribosome-associated protein quality control at the ER and ER-phagy. However, it remains unclear how ufmylation regulates such distinct ER-related functions. Here we identify a UFM1 substrate, NADH-cytochrome b5 reductase 3 (CYB5R3), that localizes on the ER membrane. Ufmylation of CYB5R3 depends on the E3 components UFL1 and UFBP1 on the ER, and converts CYB5R3 into its inactive form. Ufmylated CYB5R3 is recognized by UFBP1 through the UFM1-interacting motif, which plays an important role in the further uyfmylation of CYB5R3. Ufmylated CYB5R3 is degraded in lysosomes, which depends on the autophagy-related protein Atg7- and the autophagy-adaptor protein CDK5RAP3. Mutations of CYB5R3 and genes involved in the UFM1 system cause hereditary developmental disorders, and ufmylation-defective Cyb5r3 knock-in mice exhibit microcephaly. Our results indicate that CYB5R3 ufmylation induces ER-phagy, which is indispensable for brain development.

Clathrin adapters AP-1 and GGA2 support expression of epidermal growth factor receptor for cell growth.

The role of Golgi/endosome-localized clathrin adapters in the maintenance of steady-state cell surface epidermal growth factor receptor (EGFR) is not well known. Here, we show that EGFR associates preferentially with both AP-1 and GGA2 in vitro. AP-1 depletion caused a reduction in the EGFR protein by promoting its lysosomal degradation. Triple immunofluorescence microscopy and proximity ligation assays demonstrated that the interaction of EGFR with AP-1 or GGA2 occurred more frequently in Rab11-positive recycling endosomes than in Rab5-positive early endosomes. Biochemical recycling assay revealed that the depletion of AP-1 or GGA2 significantly suppressed EGFR recycling to the plasma membrane regardless of the EGF stimulation. Depletion of AP-1 or GGA2 also reduced cell contents of other tyrosine kinases, MET and ErbB4, and therefore, suppressed the growth of H1975 cancer cells in culture and xenograft model. Moreover, AP-1 was expressed in endosomes at higher levels in some cancer tissues. Collectively, these results suggest that AP-1 and GGA2 function in recycling endosomes to retrieve endocytosed EGFR, thereby sustaining its cell surface expression and, consequently, cancer cell growth.

Homeostatic regulation of STING by retrograde membrane traffic to the ER.

Coat protein complex I (COP-I) mediates the retrograde transport from the Golgi apparatus to the endoplasmic reticulum (ER). Mutation of the COPA gene, encoding one of the COP-I subunits (α-COP), causes an immune dysregulatory disease known as COPA syndrome. The molecular mechanism by which the impaired retrograde transport results in autoinflammation remains poorly understood. Here we report that STING, an innate immunity protein, is a cargo of the retrograde membrane transport. In the presence of the disease-causative α-COP variants, STING cannot be retrieved back to the ER from the Golgi. The forced Golgi residency of STING results in the cGAS-independent and palmitoylation-dependent activation of the STING downstream signaling pathway. Surf4, a protein that circulates between the ER/ ER-Golgi intermediate compartment/ Golgi, binds STING and α-COP, and mediates the retrograde transport of STING to the ER. The STING/Surf4/α-COP complex is disrupted in the presence of the disease-causative α-COP variant. We also find that the STING ligand cGAMP impairs the formation of the STING/Surf4/α-COP complex. Our results suggest a homeostatic regulation of STING at the resting state by retrograde membrane traffic and provide insights into the pathogenesis of COPA syndrome.

Emerging roles of Golgi/endosome-localizing monomeric clathrin adaptors GGAs.

GGAs (Golgi-localized, γ-adaptin ear-containing, ADP ribosylation factor [Arf]-binding proteins) are a family of ubiquitously expressed, Arf-dependent monomeric clathrin adaptor proteins, and are conserved from yeast to humans. Mammals have three GGAs (GGA1-3) that work not only at the trans-Golgi network, but also in endosomes to sort transmembrane cargo proteins such as mannose 6-phosphate receptors, sortilin, ß-site amyloid precursor protein cleaving enzyme 1, and epidermal growth factor receptor. The cytoplasmic regions of these cargoes possess motifs of acidic amino acid cluster-dileucine and/or ubiquitination sites, which can be recognized by GGAs. Despite seminal investigations of the three molecules, their in vivo roles and functional redundancies, as well as relationships with a heterotetrameric adaptor protein, AP-1, which is functionally similar to GGAs were still poorly understood. Studies over the past two decades, however, discovered several new GGA cargoes, their interaction modes, and accessory proteins. These findings collectively suggest distinct and more fundamental roles of each GGA in regulating neuronal survival, lipid metabolism, and cell proliferation. This review aims to provide an update to the GGA research focusing on how GGAs became considered not only as players in the context of the TGN-endosome transport, but also as key regulators for physiologically and pathologically important phenomena.

Author Correction: GGA2 interacts with EGFR cytoplasmic domain to stabilize the receptor expression and promote cell growth.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Neuron-specific recombination by Cre recombinase inserted into the murine tau locus.

To determine the neuronal function of genes in vivo, the neuron-specific deletion of a target gene in animals is required. Tau, a microtubule-associated protein, is expressed abundantly in neurons but scarcely in glias and other tissues. Therefore, to generate mice that express Cre recombinase in neurons, we inserted Cre recombinase into the tau locus. By crossing these tau-Cre mice with ROSA26 lacZ reporter mice, we observed Cre recombinase activity in the neurons from most of the central nervous system, but not in glias nor in non-neuronal tissues. This neuronal-specific activity appeared during embryogenesis. We further crossed tau-Cre mice with rab8 'floxed' mice, and showed that the recombination was nearly complete in the brain, but incomplete or non-detectable in other tissues. Thus, tau-Cre knockin mouse is a useful tool for studying the neuronal function of a gene in vivo.

Neuron-specific and inducible recombination by Cre recombinase in the mouse.

To investigate the neuronal function of genes in vivo, a neuron-specific and inducible gene targeting system is desirable. In this study, we generated a knockin mouse line that expresses a fusion protein consisting of the Cre recombinase and the progesterone receptor (CrePR) in neurons. The neuron-specific expression of CrePR was attained by inserting CrePR gene into the tau locus, because tau is expressed strongly in neurons but scarcely in glias and other tissues. By crossing this knockin mouse line (tau(CrePR)) with ROSA26 lacZ reporter mouse line (R26R), we observed that the antiprogesterone RU486 could induce recombinase activity of the CrePR specifically in neurons. Thus, tau (CrePR) knockin line is a useful tool for studying neuronal gene functions.

GGA2 interacts with EGFR cytoplasmic domain to stabilize the receptor expression and promote cell growth.

Epidermal growth factor receptor (EGFR) signaling and its downregulation upon ligand binding have been extensively documented. However, the mechanisms by which cells maintain steady-state EGFR expression remain poorly understood. Here, we report a novel role of Golgi-localized, γ-adaptin ear-containing, ADP ribosylation factor-binding protein 2 (GGA2) in the control of EGFR turnover. Whereas GGA1- or GGA3-depletion increased EGFR expression, GGA2-depletion by RNAi greatly reduced steady-state expression of EGFR, reflecting enhanced lysosomal degradation of EGFR. Subsequent pull-down assays showed interactions of VHS-GAT domains from three GGAs with the cytoplasmic juxtamembrane region (jxt) of EGFR, which was dependent on N108 in the VHS domain. Proximity ligation assay also revealed the steady-state interaction between GGA2 and EGFR in situ. Moreover, reduced expression of EGFR in GGA2-depleted cells was reversed by additional depletion of GGA1 or GGA3, suggesting that GGA1 and GGA3 promote EGFR degradation. In addition, GGA2-depleted cells had reduced EGF signaling and cell proliferation in cell culture and xenograft experiments. Finally, GGA2 was upregulated in 30.8% of human hepatocellular carcinomas and 23.3% of colorectal cancers. Together, these results indicate that GGA2 supports cell growth by interacting with EGFR for sustaining the receptor expression.

Intracellular localization of GGA accessory protein p56 in cell lines and central nervous system neurons.

Adaptor protein complex-1 (AP-1) and Golgi associated, γ-adaptin ear containing, Arf binding proteins (GGAs) are clathrin adaptors that regulate membrane trafficking between the trans-Golgi network (TGN) and endosomes. p56 is a clathrin adaptor accessory protein that may modulate the function of GGAs in mammalian cell lines. However, the precise relationship between p56 and the three GGAs (GGA1-3), as well as the physiological role of p56 in tissue cells, remain unknown. To this end, we generated an antibody against p56 and determined its cellular localization. In ARPE-19 cells and mouse embryonic fibroblasts, p56 was found to be localized as fine puncta in the TGN. Interestingly, the depletion of each clathrin adaptor by RNAi revealed that this localization was dependent on the expression of GGA1, but not that of GGA2, GGA3, or AP-1. Using immunohistofluorescence microscopy in the mouse central nervous system (CNS), p56 was clearly detected as scattered cytoplasmic puncta in spinal motor neurons, cerebellar Purkinje cells, and pyramidal neurons of the hippocampus and cerebral cortex. Moreover, double labeling with organelle markers revealed that the majority of these puncta were closely associated with the TGN; however, a small fraction was associated with endosomes or lysosomes in spinal motor neurons. Collectively, these results indicate a functional association of p56 with GGA1, suggesting an important role of p56 in larger CNS neurons.

14-3-3 proteins and protein phosphatases are not reduced in tau-deficient mice.

Tau is an axonal microtubule-associated protein, whose dysfunction causes neurodegenerative diseases such as Alzheimer's disease and other tauopathies. Earlier studies have shown the interactions of tau with glycogen synthase kinase-3beta, 14-3-3zeta, protein phosphatase 1 and protein phosphatase 2A. In this study, we compared the amounts of these tau-interacting proteins in brain microtubule-enriched fractions from wild-type and tau-deficient mice. Contrary to our expectation, we detected no difference in the amount of these proteins between wild-type and tau-deficient mice. Our findings indicate that only a small portion of tau-interacting proteins are bound to tau in vivo, and suggest the existence of other scaffolding proteins. We propose that tau-deficient mice are an ideal system for confirming the function of tau-interacting proteins.

Differing susceptibility to autophagic degradation of two LC3-binding proteins: SQSTM1/p62 and TBC1D25/OATL1.

MAP1LC3/LC3 (a mammalian ortholog family of yeast Atg8) is a ubiquitin-like protein that is essential for autophagosome formation. LC3 is conjugated to phosphatidylethanolamine on phagophores and ends up distributed both inside and outside the autophagosome membrane. One of the well-known functions of LC3 is as a binding partner for receptor proteins, which target polyubiquitinated organelles and proteins to the phagophore through direct interaction with LC3 in selective autophagy, and their LC3-binding ability is essential for degradation of the polyubiquitinated substances. Although a number of LC3-binding proteins have been identified, it is unknown whether they are substrates of autophagy or how their interaction with LC3 is regulated. We previously showed that one LC3-binding protein, TBC1D25/OATL1, plays an inhibitory role in the maturation step of autophagosomes and that this function depends on its binding to LC3. Interestingly, TBC1D25 seems not to be a substrate of autophagy, despite being present on the phagophore. In this study we investigated the molecular basis for the escape of TBC1D25 from autophagic degradation by performing a chimeric analysis between TBC1D25 and SQSTM1/p62 (sequestosome 1), and the results showed that mutant TBC1D25 with an intact LC3-binding site can become an autophagic substrate when TBC1D25 is forcibly oligomerized. In addition, an ultrastructural analysis showed that TBC1D25 is mainly localized outside autophagosomes, whereas an oligomerized TBC1D25 mutant rather uniformly resides both inside and outside the autophagosomes. Our findings indicate that oligomerization is a key factor in the degradation of LC3-binding proteins and suggest that lack of oligomerization ability of TBC1D25 results in its asymmetric localization at the outer autophagosome membrane.

Activation of STING requires palmitoylation at the Golgi.

Stimulator of interferon genes (STING) is essential for the type I interferon response against DNA pathogens. In response to the presence of DNA and/or cyclic dinucleotides, STING translocates from the endoplasmic reticulum to perinuclear compartments. However, the role of this subcellular translocation remains poorly defined. Here we show that palmitoylation of STING at the Golgi is essential for activation of STING. Treatment with palmitoylation inhibitor 2-bromopalmitate (2-BP) suppresses palmitoylation of STING and abolishes the type I interferon response. Mutation of two membrane-proximal Cys residues (Cys88/91) suppresses palmitoylation, and this STING mutant cannot induce STING-dependent host defense genes. STING variants that constitutively induce the type I interferon response were found in patients with autoimmune diseases. The response elicited by these STING variants is effectively inhibited by 2-BP or an introduction of Cys88/91Ser mutation. Our results may lead to new treatments for cytosolic DNA-triggered autoinflammatory diseases.

p62/Sqstm1 promotes malignancy of HCV-positive hepatocellular carcinoma through Nrf2-dependent metabolic reprogramming.

p62/Sqstm1 is a multifunctional protein involved in cell survival, growth and death, that is degraded by autophagy. Amplification of the p62/Sqstm1 gene, and aberrant accumulation and phosphorylation of p62/Sqstm1, have been implicated in tumour development. Herein, we reveal the molecular mechanism of p62/Sqstm1-dependent malignant progression, and suggest that molecular targeting of p62/Sqstm1 represents a potential chemotherapeutic approach against hepatocellular carcinoma (HCC). Phosphorylation of p62/Sqstm1 at Ser349 directs glucose to the glucuronate pathway, and glutamine towards glutathione synthesis through activation of the transcription factor Nrf2. These changes provide HCC cells with tolerance to anti-cancer drugs and proliferation potency. Phosphorylated p62/Sqstm1 accumulates in tumour regions positive for hepatitis C virus (HCV). An inhibitor of phosphorylated p62-dependent Nrf2 activation suppresses the proliferation and anticancer agent tolerance of HCC. Our data indicate that this Nrf2 inhibitor could be used to make cancer cells less resistant to anticancer drugs, especially in HCV-positive HCC patients.

A cluster of thin tubular structures mediates transformation of the endoplasmic reticulum to autophagic isolation membrane.

Recent findings have suggested that the autophagic isolation membrane (IM) might originate from a domain of the endoplasmic reticulum (ER) called the omegasome. However, the morphological relationships between ER, omegasome, and IM remain unclear. In the present study, we found that hybrid structures composed of a double FYVE domain-containing protein 1 (DFCP1)-positive omegasome and the IM accumulated in Atg3-deficient mouse embryonic fibroblasts (MEFs). Moreover, correlative light and electron microscopy and immunoelectron microscopy revealed that green fluorescent protein (GFP)-tagged DFCP1 was localized on tubular or vesicular elements adjacent to the IM rims. Through detailed morphological analyses, including optimization of a fixation method and electron tomography, we observed a cluster of thin tubular structures between the IM edges and ER, part of which were continuous with IM and/or ER. The formation of these thin tubular clusters was observed in several cell lines and MEFs deficient for Atg5, Atg7, or Atg16L1 but not in FIP200-deficient cells, suggesting that they were relevant to the earlier events in autophagosome formation. Taken together, our findings indicate that these tubular profiles represent a part of the omegasome that links the ER with the IM.