水島 恒裕

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.

Deficiencies in mitochondrial protein import are associated with a number of diseases. However, although nonimported mitochondrial proteins are at great risk of aggregation, it remains largely unclear how their accumulation causes cell dysfunction. Here, we show that nonimported citrate synthase is targeted for proteasomal degradation by the ubiquitin ligase SCFUcc1. Unexpectedly, our structural and genetic analyses revealed that nonimported citrate synthase appears to form an enzymatically active conformation in the cytosol. Its excess accumulation caused ectopic citrate synthesis, which, in turn, led to an imbalance in carbon flux of sugar, a reduction of the pool of amino acids and nucleotides, and a growth defect. Under these conditions, translation repression is induced and acts as a protective mechanism that mitigates the growth defect. We propose that the consequence of mitochondrial import failure is not limited to proteotoxic insults, but that the accumulation of a nonimported metabolic enzyme elicits ectopic metabolic stress.