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Mutations in the human peptide:N-glycanase gene (NGLY1), which encodes a cytosolic de–N-glycosylating enzyme, cause a congenital autosomal recessive disorder. In rodents, the loss of Ngly1 results in severe developmental delay or lethality, but the underlying mechanism remains unknown. In this study, we found that deletion of Fbxo6 (also known as Fbs2), which encodes a ubiquitin ligase subunit that recognizes glycoproteins, rescued the lethality-related defects in Ngly1-KO mice. In NGLY1-KO cells, FBS2 overexpression resulted in the substantial inhibition of proteasome activity, causing cytotoxicity. Nuclear factor, erythroid 2–like 1 (NFE2L1, also known as NRF1), an endoplasmic reticulum–associated transcriptional factor involved in expression of proteasome subunits, was also abnormally ubiquitinated by SCF(FBS2) in NGLY1-KO cells, resulting in its retention in the cytosol. However, the cytotoxicity caused by FBS2 was restored by the overexpression of “glycan-less” NRF1 mutants, regardless of their transcriptional activity, or by the deletion of NRF1 in NGLY1-KO cells. We conclude that the proteasome dysfunction caused by the accumulation of N-glycoproteins, primarily NRF1, ubiquitinated by SCF(FBS2) accounts for the pathogenesis resulting from NGLY1 deficiency.

Significance Cytosolic peptide: N -glycanase (NGLY1) is a widely conserved enzyme involved in de– N -glycosylation of N -glycosylated proteins. Mutations in the human NGLY1 gene cause global developmental delay and multisystemic symptoms, but the molecular mechanism underlying pathogenesis remains poorly understood. FBXO6/FBS2, a subunit of the SCF (SKP1–CUL1–F-box protein) ubiquitin ligase complex, recognizes N -glycans of cytosolic glycoproteins in the endoplasmic reticulum–associated degradation (ERAD) pathway. This paper reports that high levels of ERAD glycoprotein substrates abnormally ubiquitinated by SCF FBS2 in the absence of NGLY1 impair the proteasome, contributing to the pathogenesis of NGLY1 deficiency. Importantly, knockout of Fbxo6/Fbs2 rescued the lethality of NGLY1 deficiency in mice, suggesting a strategy for developing therapeutics for this intractable disease.

Ubiquitination functions as a signal to recruit autophagic machinery to damaged organelles and induce their clearance. Here, we report the characterization of FBXO27, a glycoprotein-specific F-box protein that is part of the SCF (SKP1/CUL1/F-box protein) ubiquitin ligase complex, and demonstrate that SCFFBXO27 ubiquitinates glycoproteins in damaged lysosomes to regulate autophagic machinery recruitment. Unlike F-box proteins in other SCF complexes, FBXO27 is subject to N-myristoylation, which localizes it to membranes, allowing it to accumulate rapidly around damaged lysosomes. We also screened for proteins that are ubiquitinated upon lysosomal damage, and identified two SNARE proteins, VAMP3 and VAMP7, and five lysosomal proteins, LAMP1, LAMP2, GNS, PSAP, and TMEM192. Ubiquitination of all glycoproteins identified in this screen increased upon FBXO27 overexpression. We found that the lysosomal protein LAMP2, which is ubiquitinated preferentially on lysosomal damage, enhances autophagic machinery recruitment to damaged lysosomes. Thus, we propose that SCFFBXO27 ubiquitinates glycoproteins exposed upon lysosomal damage to induce lysophagy.

Although lysosomes play a crucial role in autophagy, damaged lysosomes are eliminated by autophagy. The molecular mechanisms that recognize lysosomal damage in cells remain poorly understood, but ubiquitination is a known prerequisite for directing autophagic machinery to damaged lysosomes. FBXO27, a substrate-recognition subunit of the SCF (SKP1/CUL1/F-box protein) ubiquitin ligase complex, localizes to the cytosolic surface of endomembranes and binds glycoproteins. This paper reports that SCFFBXO27 ubiquitinates exposed glycoproteins normally sequestered on the lumenal surface of lysosomes following lysosomal damage, resulting in accelerated recruitment of autophagic machinery.

The identification of substrates for ubiquitin ligases has remained challenging, because most substrates are either immediately degraded by the proteasome or processed by deubiquitinating enzymes (DUBs) to remove polyubiquitin. Although a methodology that enables detection of ubiquitinated proteins using ubiquitin Lys-e-Gly-Gly (diGly) remnant antibodies and MS has been developed, it is still insufficient for identification and characterization of the ubiquitin-modified proteome in cells overexpressing a particular ubiquitin ligase. Here, we show that exogenously expressed trypsin-resistant tandem ubiquitin-binding entity(ies) (TR-TUBE) protect polyubiquitin chains on substrates from DUBs and circumvent proteasome-mediated degradation in cells. TR-TUBE effectively associated with substrates ubiquitinated by an exogenously overexpressed ubiquitin ligase, allowing detection of the specific activity of the ubiquitin ligase and isolation of its substrates. Although the diGly antibody enabled effective identification of ubiquitinated proteins in cells, overexpression of an ubiquitin ligase and treatment with a proteasome inhibitor did not increase the level of diGly peptides specific for the ligase relative to the background level of diGly peptides, probably due to deubiquitination. By contrast, in TR-TUBE–expressing cells, the level of substrate-derived diGly peptides produced by the overexpressed ubiquitin ligase was significantly elevated. We developed a method for identifying the substrates of specific ubiquitin ligases using two enrichment strategies, TR-TUBE and diGly remnant antibodies, coupled with MS. Using this method, we identified target substrates of FBXO21, an uncharacterized F-box protein.

The Skp1-Cul1-F-box protein (SCF) complex catalyzes protein ubiquitination in diverse cellular processes and is one of the best-characterized ubiquitin ligases. F-box proteins determine the substrate specificities of SCF ubiquitin ligases. Among these, Fbs1/FBG1/FBXO2, Fbs2/FBG2/FBXO6, and Fbs3/FBG5/FBXO27 recognize the N-glycans of glycoproteins, whereas FBG3/FBXO44 has no sugar-binding activity, despite the high sequence homology and conservation of the residues necessary for oligosaccharide binding between Fbs1.3 and FBG3. Here we determined the crystal structure of the Skp1.FBG3 complex at a resolution of 2.6 A. The substrate-binding domain of FBG3 is composed of a 10-stranded antiparallel β-sandwich with three helices. Although the overall structure of FBG3 is similar to that of Fbs1, the residues that form the Fbs1 carbohydrate-binding pocket failed to be superposed with the corresponding residues of FBG3. Structure-based mutational analysis shows that distinct hydrogen bond networks of four FBG3 loops, i.e., β2-β3, β5-β6, β7-β8, and β9-β10, prevent the formation of the carbohydrate-binding pocket shown in Fbs1.

We performed a cluster analysis of human and animal pathogenic Microsporum species and their teleomorphic states, Arthroderma species, including A. otae-related species (M. canis, M. audouinii, M. distortum, M. equinum, M. langeronii, and M. ferrugineum) and M. gypseum complex (A. fulvum, A. gypseum, and A. incurvatum) using DNA sequences of nuclear ribosomal internal transcribed spacer 1 (ITS1). The dendrogram showed the members of A. otae-related species to be monophyletic and to construct an extremely closely related cluster with a long horizontal branch. This ITS1-homologous group of A. otae was organized in 6 unique genotypes, while sequences of the members of the ITS1-homologous group of M. gypseum complex are more diverse. This ITS1-based database of Microsporum species and their teleomorphic states will provide a useful and reliable species identification system: it is time-saving (takes two to three days), accurate and applicable even to strains with atypical morphological features or in a non-culturable state.

The SCF ubiquitin ligase complex consists of four components, Skp1, Cul1, ROC1/Rbx1, and a variable subunit F-box protein, which serves as a receptor for target proteins. The F-box proteins consist of an N-terminal ∼40 amino acid F-box domain that binds to Skp1 and the C-terminal substrate-binding domain. We have reported previously that Fbs1 and Fbs2 are N-linked glycoprotein-specific F-box proteins. In addition, other three F-box proteins, Fbg3, Fbg4, and Fbg5, show high homology to Fbs1 and Fbs2, but their functions remain largely unknown. Here we report that Skp1 assists in correct folding of exogenously expressed F-box proteins. Fbs2 as well as Fbg3, Fbg4, and Fbg5 proteins formed SCF complexes but did not bind to N-glycoproteins when exogenously expressed alone. However, co-expression of Fbs2 and Fbg5 with Skp1 facilitated their binding to glycoproteins that reacted with ConA. Furthermore, Skp1 increased the cellular concentrations of F-box proteins by preventing aggregate formation. These observations suggest that Skp1 plays an important role in stabilizing the conformation of these F-box proteins, which increases their expression levels and substrate-binding.

Fbs1 is an F-box protein present abundantly in the nervous system. Similar to the ubiquitously expressed Fbs2, Fbs1 recognizes N-glycans at the innermost position as a signal for unfolded glycoproteins, probably in the endoplasmic reticulum-associated degradation pathway. Here, we show that the in vivo majority of Fbs1 is present as Fbs1-Skp1 heterodimers or Fbs1 monomers but not SCF(Fbs1) complex. The inefficient SCF complex formation of Fbs1 and the restricted presence of SCF(Fbs1) bound on the endoplasmic reticulum membrane were due to the short linker sequence between the F-box domain and the sugar-binding domain. In vitro, Fbs1 prevented the aggregation of the glycoprotein through the N-terminal unique sequence of Fbs1. Our results suggest that Fbs1 assists clearance of aberrant glycoproteins in neuronal cells by suppressing aggregates formation, independent of ubiquitin ligase activity, and thus functions as a unique chaperone for those proteins.

Species identification of genus Malassezia is important in epidemiological and etiological studies, however, is difficult by the conventional system. A specific and rapid identification system based on sequences of internal transcribed spacer 1 of ribosomal DNA has therefore been developed. Using this system, we could identify two or more species mixed in the clinical samples.