Proteasome assembly defect due to a proteasome subunit beta type 8 (PSMB8) mutation causes the autoinflammatory disorder, Nakajo-Nishimura syndrome

Abstract

Nakajo-Nishimura syndrome (NNS) is a disorder that segregates in an autosomal recessive fashion. Symptoms include periodic fever, skin rash, partial lipomuscular atrophy, and joint contracture. Here, we report a mutation in the human proteasome subunit beta type 8 gene (PSMB8) that encodes the immunoproteasome subunit β5i in patients with NNS. This G201V mutation disrupts the β-sheet structure, protrudes from the loop that interfaces with the β4 subunit, and is in close proximity to the catalytic threonine residue. The β5i mutant is not efficiently incorporated during immunoproteasome biogenesis, resulting in reduced proteasome activity and accumulation of ubiquitinated and oxidized proteins within cells expressing immunoproteasomes. As a result, the level of interleukin (IL)-6 and IFN-γ inducible protein (IP)-10 in patient sera is markedly increased. Nuclear phosphorylated p38 and the secretion of IL-6 are increased in patient cells both in vitro and in vivo, which may account for the inflammatory response and periodic fever observed in these patients. These results show that a mutation within a proteasome subunit is the direct cause of a human disease and suggest that decreased proteasome activity can cause inflammation.

Fig. 1.

SNP microarray-based homozygosity mapping and mutation search. (A) Homozygosity mapping for NNS patients and nonaffected siblings. ROH regions were detected using a hidden Markov model-based algorithm. The sole candidate region identified within 6p21.31–32 is shown. Green vertical lines indicate heterozygous SNPs and the background gray area indicates a region without heterozygous SNP calls. To be conservative, we did not regard isolated single heterozygous calls as delimiting ROH regions. The physical positions are shown in NCBI build 36.1. Patient numbers correspond to Figs. S1A and S1C and Table S1. No history of consanguineous marriage was apparent for patients 3 and 5, according to the family history interview. (B) Chromatograms for a control, a patient's father, and a patient. A mutation in PSMB8 exon 5 identified in NNS patients by sequencing is highlighted in yellow. (C) Amino acid comparisons with other species. The glycine at the mutation site (red box) is highly conserved among vertebrates.

Fig. 2.

G201V mutation in β5i reduces proteasome activity in immunoproteasome-expressing cells. (A) Close-up view of the mutation site (G201V) within β5i. Structural models of G201V β5i (orange) and wild-type β5i (green) were created from the β5-subunit structure [Protein Data Bank (PDB) ID code 1IRU]. The secondary structure elements for β5i are labeled. Val201 and Thr73 are shown in the stick model. Thr73 is a catalytic residue of β5i. (B) A ribbon diagram of the β4–β5i complex. The arrow shows the difference in the β-sheet between β5i (green) and β5i^G201V (orange). Arrowheads show the protruding S8–H3 loop of β5i^G201V. (C) Peptidase activity of LCLs. Extracts were fractionated by glycerol gradient centrifugation (8–32% glycerol from fraction 1–32). Arrowheads indicate the peak positions of the 20S and 26S proteasomes (open arrows, single-capped 26S; closed arrows, double-capped 26S). (D) Western blot analysis of fractionated total LCL extracts. Western blot analysis of proteasome subunits from fractions 1–32 fractionated in C. The sedimenting positions of the immature 20S, 20S, and 26S proteasomes are indicated by arrowheads. The mature and incompletely cleaved β5i^G201V subunits are indicated by arrows. The mature β5i subunit is cleaved within a C-terminal polypeptide between Gly72 and Thr73. The insufficiently cleaved β5i subunit is probably cleaved at a site toward the N terminus site, yielding a fragment with a higher molecular weight. The same amount of protein was subjected to glycerol gradient ultracentrifugation. The level of proteasome is reduced in NNS patients. Control, LCL extract from healthy control; NNS, LCL extract from patient with NNS.

Fig. 3.

Decrease of proteolytic activity and accumulation of polyubiquitinated and oxidized proteins in NNS cells. (A) In vitro proteolytic activity of the mutant proteasome. Degradation of recombinant ³⁵S-labeled ODC was expressed as % total ODC as described previously (11). Error bars indicated the SD of the mean (n = 3). *P < 0.05, **P < 0.01. (B and C) Accumulation of ubiquitinated proteins in LCLs (B) and fibroblasts (C). Western blot analysis of ubiquitinated proteins using an antiubiquitin antibody (Left), an anti-K48 polyubiquitinated protein antibody (Middle), and an anti-K63 polyubiquitinated protein antibody (Right). Tubulin was used as a loading control (Lower). NHDF, adult normal human dermal fibroblasts. (D) Levels of oxidized proteins determined by Oxyblot. NHDF and NNS fibroblasts were stimulated with or without 100 units of IFN-γ for 24 h. Tubulin was used as a loading control. (E) Immunofluorescence staining of CD68 and ubiquitinated proteins. Staining for CD68 (green) and ubiquitinated proteins (red) in skin sections from an NNS patient and a fasciitis patient. NNS ubiquitin signals showed a 4.7-fold increase with ImageJ (http://rsb.info.nih.gov/ij/) compared with fasciitis signals. (Scale bar, 10 μm.)

Fig. 4.

Analyses of the level of IL-6 in NNS and the signal transduction system related to cytokine production. (A) IL-6 concentrations in sera from healthy controls, patients with NNS, and patients with rheumatoid arthritis. IL-6 levels in sera were determined by ELISA. (B) IL-6 production by cultured fibroblasts. The concentrations of IL-6 in conditioned media were determined by ELISA (in triplicate). (C) Western blot analysis for NF-κB and MAPK. Whole cell extracts and nuclear extracts were immunoblotted using antibodies against IκBα, p-IκBα, p65, p-ERK, p-JNK, and p-p38. (D) Western blot analysis of p-p38 in peripheral blood lymphocytes. Nuclear extracts from the peripheral blood lymphocytes of a healthy control, a heterozygous family member, and a NNS patient were blotted and visualized with anti–p-p38. Error bars indicate SD of the mean. *P < 0.05, **P < 0.01, ***P < 0.001 [Mann-Whitney u test (A) and two-tailed Welch's t test (B)]. Signal intensities were quantified using ImageJ and expressed as fold changes relative to a healthy control normalized to histone H3 (D).

Fig. 5.

Schematic model showing induction of inflammation in NNS patients with the PSMB8 mutation. Our data are based on the scheme proposed by Bulua et al. (40). (A) In a normal cell, ubiquitinated or oxidized proteins generated by various stressors, including cytokines, are cleared by proteasomes. (B) The ubiquitinated and oxidized proteins accumulate in a cell with the PSMB8 mutation (NNS cell). ROS and/or oxidized proteins may cause phosphorylation of p-38 to predominate over the nonphosphorylated form by inhibiting MAPK phosphatase or by activating MAPK.