Additive loss-of-function proteasome subunit mutations in CANDLE/PRAAS patients promote type I IFN production
Anja Brehm, … , Ivona Aksentijevich, Raphaela Goldbach-Mansky
Anja Brehm, … , Ivona Aksentijevich, Raphaela Goldbach-Mansky
Published October 20, 2015
Citation Information: J Clin Invest. 2015;125(11):4196-4211. https://doi.org/10.1172/JCI81260.
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Research Article Immunology

Additive loss-of-function proteasome subunit mutations in CANDLE/PRAAS patients promote type I IFN production

Abstract

Autosomal recessive mutations in proteasome subunit β 8 (PSMB8), which encodes the inducible proteasome subunit β5i, cause the immune-dysregulatory disease chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature (CANDLE), which is classified as a proteasome-associated autoinflammatory syndrome (PRAAS). Here, we identified 8 mutations in 4 proteasome genes, PSMA3 (encodes α7), PSMB4 (encodes β7), PSMB9 (encodes β1i), and proteasome maturation protein (POMP), that have not been previously associated with disease and 1 mutation in PSMB8 that has not been previously reported. One patient was compound heterozygous for PSMB4 mutations, 6 patients from 4 families were heterozygous for a missense mutation in 1 inducible proteasome subunit and a mutation in a constitutive proteasome subunit, and 1 patient was heterozygous for a POMP mutation, thus establishing a digenic and autosomal dominant inheritance pattern of PRAAS. Function evaluation revealed that these mutations variably affect transcription, protein expression, protein folding, proteasome assembly, and, ultimately, proteasome activity. Moreover, defects in proteasome formation and function were recapitulated by siRNA-mediated knockdown of the respective subunits in primary fibroblasts from healthy individuals. Patient-isolated hematopoietic and nonhematopoietic cells exhibited a strong IFN gene-expression signature, irrespective of genotype. Additionally, chemical proteasome inhibition or progressive depletion of proteasome subunit gene transcription with siRNA induced transcription of type I IFN genes in healthy control cells. Our results provide further insight into CANDLE genetics and link global proteasome dysfunction to increased type I IFN production.

Authors

Anja Brehm, Yin Liu, Afzal Sheikh, Bernadette Marrero, Ebun Omoyinmi, Qing Zhou, Gina Montealegre, Angelique Biancotto, Adam Reinhardt, Adriana Almeida de Jesus, Martin Pelletier, Wanxia L. Tsai, Elaine F. Remmers, Lela Kardava, Suvimol Hill, Hanna Kim, Helen J. Lachmann, Andre Megarbane, Jae Jin Chae, Jilian Brady, Rhina D. Castillo, Diane Brown, Angel Vera Casano, Ling Gao, Dawn Chapelle, Yan Huang, Deborah Stone, Yongqing Chen, Franziska Sotzny, Chyi-Chia Richard Lee, Daniel L. Kastner, Antonio Torrelo, Abraham Zlotogorski, Susan Moir, Massimo Gadina, Phil McCoy, Robert Wesley, Kristina I. Rother, Peter W. Hildebrand, Paul Brogan, Elke Krüger, Ivona Aksentijevich, Raphaela Goldbach-Mansky

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Figure 3

Maturation and incorporation of mutant proteasome subunits into proteasome complex in vitro in HeLa cells.

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Maturation and incorporation of mutant proteasome subunits into proteaso...
WT and mutant versions of the subunits PSMA3/α7, PSMB4/β7, and PSMB8/β5i/LMP7 were ectopically expressed with V5 epitope tags in HeLa cells. (A) Schematic representation of V5-tagged subunits and their maturation behavior. Of note, most of the β subunits (but not α subunits) are expressed as proforms and have to be matured by autocatalytic propeptide cleavage during proteasome assembly. Maturation of active site subunits such as β5i is a 2-step procedure resulting in proform, intermediate proform, and mature form of the subunit. (B) Immunoblot of HeLa cells transfected with V5 epitope–tagged subunits and detected by a V5-specific antibody. MOCK, empty vector backbone. Asterisks indicate unspecific background staining. β–Tubulin served as loading control. Expression of α7-R233del-V5 or β5i-C135X-V5 revealed almost no detectable protein, whereas the -9G>A mutation in β7-V5 or the β5i-T75M and -G201V mutations caused decreased protein expression. The β7-3aadel-, β5i-G201V-, -K105Q-, and -A92T-V5 versions display altered maturation of these β subunits (see accumulation of preforms or intermediate forms). (C) Incorporation of V5-tagged subunits into the proteasome complexes was assessed by native PAGE analysis and immunoblot against the V5 epitope tag. The β7-3aadel-V5 and β5i-G201V-V5 exhibit the most prominent incorporation defects. (D) With the exception of the -9G>A mutation in PSMB4-V5, all mutant subunit mRNAs were equally expressed in HeLa cells, as evaluated by RT-PCR. Expression of endogenous GAPDH mRNA served as loading control. Transfection efficiency was determined by FACS analyses of cotransfected EGFP vector. (B–D) Representative results from n = 3.