水島 恒裕

RNF213 was originally identified as a susceptibility gene for Moyamoya disease (MMD), and its variants are also associated with other vascular occlusive disorders, including pulmonary arterial hypertension and large-artery atherosclerosis. RNF213 encodes a large protein with AAA+ ATPase and E3 ubiquitin ligase activities and has been implicated in vasculopathy as well as cellular responses to microbial infection and lipid stress. However, the precise biological functions of RNF213 and the pathogenic mechanisms underlying disease-associated mutations remain poorly understood. Recent studies have suggested lipid-induced stress may play a key role in MMD pathogenesis. Therefore, we performed temporal transcriptomic analyses to identify RNF213-regulated signaling pathways in response to palmitate, the most common saturated fatty acid. Our results demonstrate that RNF213 is required for activation of apoptosis, the unfolded protein response, and NFκB signaling, as well as for the regulation of autophagy and oxidative stress responses following palmitate exposure. These functions were dependent on both the AAA+ ATPase and RZ-finger domains of RNF213 while MMD-associated RNF213 mutations enhanced palmitate-induced signaling responses. Together, these findings suggest that gain-of-function RNF213 mutations, combined with dysregulated lipid metabolism, contribute to the pathogenesis of RNF213-associated vasculopathies.

Citrate synthase (CS) is a pivotal enzyme in carbohydrate and energy metabolism, with distinct isoforms present in various eukaryotic compartments, including mitochondria and glyoxysomes in plants. While CSs exhibit diverse oligomeric states, detailed structural information on higher plant non-mitochondrial Type II CSs has been limited. We herein determined the crystal structures of CS 3 from Arabidopsis thaliana (AtCSY3) in complex with oxaloacetate (OAA) and acetyl-coenzyme A (CoA)-OAA at resolutions of 2.0 and 1.7 Å, respectively. These structures revealed that AtCSY3 can form a homo-tetrameric assembly that is distinct from the hexameric Escherichia coli CS and the octameric Ananas comosus CS. The tetrameric arrangement observed in the crystal structure is mediated by hydrogen-bonding and hydrophobic interactions between subunits. Gel filtration chromatography further suggests the presence of a tetrameric species in solution under the purification conditions. Ligand density was observed near the interface between the two dimers in the tetrameric structure; however, no experimental evidence is currently available to determine whether ligand binding affects the oligomeric state or enzymatic activity of AtCSY3. These structures illustrate the structural diversity of CS oligomerization and provide a structural basis for studies of plant glyoxysomal CSs.

Abstract Dysregulation of DNA double-strand break (DSB) repair leads to adaptive immunodeficiency, whereas the remaining lymphocytes are aberrantly activated and provoke inflammations. However, no model mice were available to consistently manifest inflammation under defective DSB repair. We generated mutant mice carrying a missense mutation p.W447C in the gene encoding DNA ligase IV (LIG4), critical for DSB repair.Lig4^W447C/W447Cmice showed growth retardation and severe intestinal inflammations under adaptive immunodeficiency. The inflammations were featured by marked infiltration of T helper type 1 (Th1) cells and macrophages and was dependent on lymphocytes. WhenIfngwas deleted, Th2 and Th17 instead of Th1 cells drove the inflammations.Lig4^W447C/W447Cmice showed expansion of oligoclonal T cells with T cell receptor α repertoire skewed towards more proximal 3’ V and 5’ J gene segments. Thus, our novel hypomorphicLig4mutant mice show that defective DSB repair leads to Th1-dependent intestinal inflammations under severe adaptive immunodeficiency.

The cytosolic peptide:N-glycanase (PNGase) is involved in the quality control of N-glycoproteins via the endoplasmic reticulum-associated degradation (ERAD) pathway. Mutations in the gene encoding cytosolic PNGase (NGLY1 in humans) cause NGLY1 deficiency. Recent findings indicate that the F-box protein FBS2 of the SCFFBS2 ubiquitin ligase complex can be a promising drug target for NGLY1 deficiency. Here, we determined the crystal structure of bovine FBS2 complexed with the adaptor protein SKP1 and a sugar ligand, Man3GlcNAc2, which corresponds to the core pentasaccharide of N-glycan. Our crystallographic data together with NMR data revealed the structural basis of disparate sugar-binding specificities in homologous FBS proteins and identified a potential druggable pocket for in silico docking studies. Our results provide a potential basis for the development of selective inhibitors against FBS2 in NGLY1 deficiency.