PSMB8 Encoding the β5i Proteasome Subunit Is Mutated in Joint Contractures, Muscle Atrophy, Microcytic Anemia, and Panniculitis-Induced Lipodystrophy Syndrome
2010; Elsevier BV; Volume: 87; Issue: 6 Linguagem: Inglês
10.1016/j.ajhg.2010.10.031
ISSN1537-6605
AutoresAnil K. Agarwal, Chao Xing, George Demartino, Dario Mizrachi, M.D. Hernández, Ana Berta Sousa, Laura Martínez de Villarreal, Heloísa G. dos Santos, Abhimanyu Garg,
Tópico(s)Endoplasmic Reticulum Stress and Disease
ResumoWe performed homozygosity mapping in two recently reported pedigrees from Portugal and Mexico with an autosomal-recessive autoinflammatory syndrome characterized by joint contractures, muscle atrophy, microcytic anemia, and panniculitis-induced lipodystrophy (JMP). This revealed only one homozygous region spanning 2.4 Mb (5818 SNPs) on chromosome 6p21 shared by all three affected individuals from both families. We directly sequenced genes involved in immune response located in this critical region, excluding the HLA complex genes. We found a homozygous missense mutation c.224C>T (p.Thr75Met) in the proteasome subunit, beta-type, 8 (PSMB8) gene in affected patients from both pedigrees. The mutation segregated in an autosomal-recessive fashion and was not detected in 275 unrelated ethnically matched healthy subjects. PSMB8 encodes a catalytic subunit of the 20S immunoproteasomes called β5i. Immunoproteasome-mediated proteolysis generates immunogenic epitopes presented by major histocompatibility complex (MHC) class I molecules. Threonine at position 75 is highly conserved and its substitution with methionine disrupts the tertiary structure of PSMB8. As compared to normal lymphoblasts, those from an affected patient showed significantly reduced chymotrypsin-like proteolytic activity mediated by immunoproteasomes. We conclude that mutations in PSMB8 cause JMP syndrome, most probably by affecting MHC class I antigen processing. We performed homozygosity mapping in two recently reported pedigrees from Portugal and Mexico with an autosomal-recessive autoinflammatory syndrome characterized by joint contractures, muscle atrophy, microcytic anemia, and panniculitis-induced lipodystrophy (JMP). This revealed only one homozygous region spanning 2.4 Mb (5818 SNPs) on chromosome 6p21 shared by all three affected individuals from both families. We directly sequenced genes involved in immune response located in this critical region, excluding the HLA complex genes. We found a homozygous missense mutation c.224C>T (p.Thr75Met) in the proteasome subunit, beta-type, 8 (PSMB8) gene in affected patients from both pedigrees. The mutation segregated in an autosomal-recessive fashion and was not detected in 275 unrelated ethnically matched healthy subjects. PSMB8 encodes a catalytic subunit of the 20S immunoproteasomes called β5i. Immunoproteasome-mediated proteolysis generates immunogenic epitopes presented by major histocompatibility complex (MHC) class I molecules. Threonine at position 75 is highly conserved and its substitution with methionine disrupts the tertiary structure of PSMB8. As compared to normal lymphoblasts, those from an affected patient showed significantly reduced chymotrypsin-like proteolytic activity mediated by immunoproteasomes. We conclude that mutations in PSMB8 cause JMP syndrome, most probably by affecting MHC class I antigen processing. We recently reported a distinct autosomal-recessive syndrome presenting with joint contractures, muscle atrophy, microcytic anemia, and panniculitis-induced childhood-onset lipodystrophy (JMP) in three patients from two pedigrees.1Garg A. Hernandez M.D. Sousa A.B. Subramanyam L. de Villarreal L.M. Dos Santos H.G. Barboza O. An autosomal recessive syndrome of joint contractures, muscular atrophy, microcytic anemia, and panniculitis-associated lipodystrophy.J. Clin. Endocrinol. Metab. 2010; 95: E58-E63Crossref PubMed Scopus (73) Google Scholar The lipodystrophy affected the face, arms, and thorax initially and more severely than the abdomen and lower extremities, which were affected later (Figure S1 available online). Although the patients did not have acanthosis nigricans, diabetes, or hyperinsulinemia, mild metabolic disturbances such as borderline hypertriglyceridemia, markedly low levels of high-density lipoprotein cholesterol, and mild elevations of liver enzymes were noted. All the patients had hepato-splenomegaly and hypergammaglobulinemia. Joint contractures initially affected the hands and feet, which were more severely deformed than the other joints. Similar manifestations have been reported previously from Japan in three patients who in addition presented with fever, elevated erythrocyte sedimentation rate, macroglossia, mental retardation, and calcification of basal ganglia.2Tanaka M. Miyatani N. Yamada S. Miyashita K. Toyoshima I. Sakuma K. Tanaka K. Yuasa T. Miyatake T. Tsubaki T. Hereditary lipo-muscular atrophy with joint contracture, skin eruptions and hyper-gamma-globulinemia: A new syndrome.Intern. Med. 1993; 32: 42-45Crossref PubMed Scopus (34) Google Scholar, 3Yamada S. Toyoshima I. Mori S. Tsubaki T. [Sibling cases with lipodystrophic skin change, muscular atrophy, recurrent skin eruptions, and deformities and contractures of the joints. A possible new clinical entity].Rinsho Shinkeigaku. 1984; 24: 703-710PubMed Google Scholar, 4Oyanagi K. Sasaki K. Ohama E. Ikuta F. Kawakami A. Miyatani N. Miyatake T. Yamada S. An autopsy case of a syndrome with muscular atrophy, decreased subcutaneous fat, skin eruption and hyper gamma-globulinemia: Peculiar vascular changes and muscle fiber degeneration.Acta Neuropathol. 1987; 73: 313-319Crossref PubMed Scopus (20) Google Scholar, 5Horikoshi A. Iwabuchi S. Iizuka Y. Hagiwara T. Amaki I. [A case of partial lipodystrophy with erythema, dactylic deformities, calcification of the basal ganglia, immunological disorders, and low IQ level (author's transl)].Rinsho Shinkeigaku. 1980; 20: 173-180PubMed Google Scholar The genetic basis of the JMP syndrome was unknown and we had hypothesized that it belongs to the group of inherited autoinflammatory diseases.6Henderson C. Goldbach-Mansky R. Monogenic autoinflammatory diseases: New insights into clinical aspects and pathogenesis.Curr. Opin. Rheumatol. 2010; 22: 567-578PubMed Google Scholar By using genome-wide homozygosity mapping, we now report a homozygous missense mutation in the proteasome subunit, β-type, 8 (PSMB8 [MIM 177046]) gene, which encodes the β5i subunit (also known as large multifunctional protease 7, LMP7), a catalytic subunit of immunoproteasomes in patients with JMP syndrome. Affected individuals and their family members were recruited into our ongoing study of genetic basis of lipodystrophies (Figure S2). The protocol was approved by the Institutional Review Board of UT Southwestern and Hospital de Santa Maria, and all the patients, their family members, and healthy controls gave the written informed consent. Genomic DNA was extracted from buffy coat or from Epstein-Barr virus-transformed lymphoblastoid cell lines via the Easy-DNA kit (Invitrogen, Carlsbad, CA). Genotyping was performed with the Illumina HumanOmni1-Quad Beadchip by the Microarray Core Facility at UT Southwestern. Allele calls were generated with GenomeStudio version 1.5.16. Consanguinity was confirmed in family JMP100 and suspected in family JMP200, suggesting a recessive disease. Therefore, we carried out both classic homozygosity mapping via linkage analysis7Lander E.S. Botstein D. Homozygosity mapping: A way to map human recessive traits with the DNA of inbred children.Science. 1987; 236: 1567-1570Crossref PubMed Scopus (686) Google Scholar and modern homozygosity mapping via identifying regions of extended homozygosity. We chose 7296 single-nucleotide polymorphisms (SNPs) from among the ∼1 million SNPs from the HumanOmni1-Quad chip to construct a sparse linkage map, in which SNPs were spaced ∼2 per centi-Morgan. In family JMP200, we performed linkage analysis with the sparse map under a recessive model by assuming individuals JMP200.1 and JMP200.2 were first cousins, as proposed by Hildebrandt et al.8Hildebrandt F. Heeringa S.F. Ruschendorf F. Attanasio M. Nurnberg G. Becker C. Seelow D. Huebner N. Chernin G. Vlangos C.N. et al.A systematic approach to mapping recessive disease genes in individuals from outbred populations.PLoS Genet. 2009; 5: e1000353Crossref PubMed Scopus (133) Google Scholar The analysis was done with MERLIN.9Abecasis G.R. Cherny S.S. Cookson W.O. Cardon L.R. Merlin—rapid analysis of dense genetic maps using sparse gene flow trees.Nat. Genet. 2002; 30: 97-101Crossref PubMed Scopus (2780) Google Scholar We also screened for regions of extended homozygosity in all individuals via PLINK.10Purcell S. Neale B. Todd-Brown K. Thomas L. Ferreira M.A. Bender D. Maller J. Sklar P. de Bakker P.I. Daly M.J. et al.PLINK: A tool set for whole-genome association and population-based linkage analyses.Am. J. Hum. Genet. 2007; 81: 559-575Abstract Full Text Full Text PDF PubMed Scopus (19629) Google Scholar The genome-wide linkage scan in family JMP200 identified only one region on 6p21 spanning ∼16.7 Mb (∼13.4 cM) with a positive lod score of 0.93 (Figure S3). Across the genome, there was only one homozygous region spanning 2.4 Mb (5818 SNPs) shared by all three affected individuals from both families (Figure 1A ) but not by any unaffected. This homozygous region was located under the linkage peak. There was no indication of copy number variation in the region according to the Log R Ratio. Excluding the HLA complex, ∼200 genes are located within this region, including those that are predicted or are of unknown function. Because we had hypothesized that the JMP syndrome is an autoinflammatory syndrome, we initially selected genes that might affect the immune response (Figure 1B). These included PSMB8 and PSMB9 (proteasomal subunits also called β5i and β1i, respectively; MIM 177046 and 177045, respectively), TAP1 and TAP2 (transporters 1 and 2, ATP-binding cassette, subfamily B associated with antigen processing; MIM 170260 and 170261, respectively), and TAPBP (TAP binding protein; MIM 601962). We also selected PBX2 (pre-B cell leukemia homeobox 2, a transcription factor; MIM 176311), BTNL2 (butyrophilin-like 2, a member of the B7 family of costimulatory molecules; MIM 606000), AIF1 (allograft inflammatory factor 1, which is associated with rheumatoid arthritis; MIM 601833), and AGPAT1 (acylglycerol phosphate acyltransferase, isoform 1, involved in triglyceride biosynthesis; MIM 603099). The coding regions and the splice site junctions of the genes in the critical region were amplified with gene-specific primers located in intronic regions (Table S1). Polymerase chain reaction (PCR) was assembled as described earlier.11Agarwal A.K. Arioglu E. de Almeida S. Akkoc N. Taylor S.I. Bowcock A.M. Barnes R.I. Garg A. AGPAT2 is mutated in congenital generalized lipodystrophy linked to chromosome 9q34.Nat. Genet. 2002; 31: 21-23Crossref PubMed Scopus (419) Google Scholar The PCR product was purified to remove primers and dNTPs and sequenced with ABI Prism 3100. We found a homozygous c.224C>T mutation in PSMB8 (NM_148919.3; MIM 177046) in the affected subjects from both pedigrees (Figures 1C–1F). This mutation is predicted to result in substitution of threonine at position 75 with methionine (p.Thr75Met; numbering according to transcript 1B; NP_683720.2). The available parents and unaffected siblings harbored the heterozygous mutation. No other disease-causing variant was found on sequencing of candidate genes in the region (Table S2). Genotyping DNA of 275 healthy ethnically matched subjects of Hispanic origin from Dallas revealed no one harboring the c.224C>T variant in PSMB8. We aligned genotypes of the three affected from both families in the homozygous region and identified a segment of 0.22 Mb covering PSMB8 shared by all three affected (Table S3). This suggests that the haplotypes shared by the affected subjects in both families are identical-by-descent; however, the origin of the mutation seems to be ancient. The threonine at position 75 of PSMB8 is highly conserved across species (Figure 2A ). Prediction of the structure and conserved regions between highly homologous (>75%) human PSMB8 (protein ID NP_683720.2) and yeast PSMB5 (ortholog of human β5, protein ID NP_015428.1) via Clustal W analysis allowed us to manually exchange the amino acids in yeast PSMB5 (PDB identification number 1z7q, chain L)12Forster A. Masters E.I. Whitby F.G. Robinson H. Hill C.P. The 1.9 A structure of a proteasome-11S activator complex and implications for proteasome-PAN/PA700 interactions.Mol. Cell. 2005; 18: 589-599Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar, 13Unno M. Mizushima T. Morimoto Y. Tomisugi Y. Tanaka K. Yasuoka N. Tsukihara T. Structure determination of the constitutive 20S proteasome from bovine liver at 2.75 A resolution.J. Biochem. 2002; 131: 171-173Crossref PubMed Scopus (43) Google Scholar with those represented exclusively in human PSMB8 via the PyMol program (PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC). For further optimization protocol, we used multiple step energy minimization and molecular dynamics (MD) simulation to fully relax our initial model. All simulations were carried out with the Amber 9 program14Case D.A. Cheatham 3rd, T.E. Darden T. Gohlke H. Luo R. Merz Jr., K.M. Onufriev A. Simmerling C. Wang B. Woods R.J. The Amber biomolecular simulation programs.J. Comput. Chem. 2005; 26: 1668-1688Crossref PubMed Scopus (6519) Google Scholar with force field (ff99) and periodic boundary conditions.15Ponder J.W. Case D.A. Force fields for protein simulations.Adv. Protein Chem. 2003; 66: 27-85Crossref PubMed Scopus (1490) Google Scholar To simulate behavior in solution, the protein was solvated with a 10 Å TIP3P octagon water cap. Prior to MD simulations, two steps of minimization were performed. First, the human PSMB8 was fixed and only the positions occupied by water were minimized followed by the minimization of the entire simulation system. After energy minimization, a 100 picosecond MD simulation was carried out under constant volume conditions, and temperature was heated up continuously from 0°K to 300°K. The time step of the simulations was 2.0 femtoseconds with a cutoff of 12 Å for nonbonded interactions. The final conformations were obtained when the total energy reached the minimum. The best structure was examined with the Profile-3D16Luthy R. Bowie J.U. Eisenberg D. Assessment of protein models with three-dimensional profiles.Nature. 1992; 356: 83-85Crossref PubMed Scopus (2563) Google Scholar and Procheck17Laskowski R.A. Moss D.S. Thornton J.M. Main-chain bond lengths and bond angles in protein structures.J. Mol. Biol. 1993; 231: 1049-1067Crossref PubMed Scopus (1080) Google Scholar programs. Protein modeling of the Thr75Met substitution showed that it relaxes PSMB8 by 1.2 Å and may affect the proteolytic processing of peptides (Figure 2B). Proteasome activity was determined in the cell lysates of Epstein-Barr virus-transformed lymphoblasts from a control subject and an affected patient (JMP200.4). Lymphoblasts were washed with PBS, pelleted by centrifugation, and lysed in four volumes of buffer consisting of 50 mM Tris-HCl (pH 7.8), 5 mM 2-mercaptoethanol, 1 mM ATP, and 5 mM MgCl2. Lysates were cleared of cell debris by centrifugation at 5000 × g for 5 min. Proteasome activity was determined by hydrolysis of peptide substrates as described previously.18Wójcik C. DeMartino G.N. Analysis of Drosophila 26S proteasome using RNA interference.J. Biol. Chem. 2002; 277: 6188-6197Crossref PubMed Scopus (93) Google Scholar We employed three different peptides that are specific, preferred substrates for three classes of peptidases having chymotrypsin-like, trypsin-like, and peptidyl glutamyl peptide-hydrolyzing (PGPH) activity. In brief, cell extracts were incubated with 50 μM substrates (succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin, succinyl-Val-Leu-Arg-7-amino-4-methylcoumarin, or benzyloxycarbonyl-Leu-Leu-Glu-7-amino-4-methylcoumarin) for 20 min at 37°C. The rate of amino-4-methylcoumarin production, a direct measure of proteasome activity, was monitored continuously by fluorescence detection at 360 nm excitation/480 nm emission, with a BioTek Synergy MX Plate reader. Steady-state rates of proteolysis were expressed as arbitrary fluorescence units produced per min of incubation and were normalized per μg of protein. Protein concentration was determined with the Bradford method.19Bradford M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (215632) Google Scholar We observed markedly reduced chymotrypsin-like activity but not trypsin-like or PGPH-like activity in lymphoblast cell lysates from the patient with JMP syndrome (Figure 2C). A marked reduction in the proteasomal activity was observed in the presence of MG132, a specific inhibitor of S26 proteasomes, when tested with substrate succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin, consistent with the presence of proteasomal activity (Figure S4). This observation was further confirmed by determining the presence of key subunits of proteasome/immunoproteasome by western blots. For western blotting, 40 μg of total cellular protein from lymphoblast cell lysates from the patient and control was resolved on 10% SDS-PAGE and transferred to nitrocellulose membranes for immunoblotting with various primary antibodies against Rpt2 (an ATPase of the PA700, a 19S proteasome regulatory particle), Rpn12 (a subunit of 19S proteasome regulatory subunit), and α2, β5i, and β5 (subunits of 20S proteasome). All of the antibodies were raised in-house as described previously,18Wójcik C. DeMartino G.N. Analysis of Drosophila 26S proteasome using RNA interference.J. Biol. Chem. 2002; 277: 6188-6197Crossref PubMed Scopus (93) Google Scholar, 20Fabunmi R.P. Wigley W.C. Thomas P.J. DeMartino G.N. Interferon gamma regulates accumulation of the proteasome activator PA28 and immunoproteasomes at nuclear PML bodies.J. Cell Sci. 2001; 114: 29-36Crossref PubMed Google Scholar detected with peroxidase-labeled secondary antibody, and exposed to X-ray film. β-actin was used as a housekeeping gene and loading control. No difference was observed in the expression of various components of the proteasomal units, including β5i subunit in the cell lysates from the control and patient (Figure 2D). Therefore, this study suggests that a homozygous missense loss-of-function mutation in PSMB8 causes the JMP syndrome. This conclusion is supported by the following observations: (1) evidence of linkage to the 6p21.2 region and homozygosity in the same region; (2) a homozygous c.224C>T missense variant in PSMB8 in the two pedigrees; (3) segregation of the c.224C>T mutation in other members of the pedigrees in an autosomal-recessive fashion; (4) no previous report of this variant as a SNP in any database; (5) lack of this variant in ethnically matched healthy controls; (6) substitution of methionine for the highly conserved threonine at position 75 of PSMB8 disrupts the tertiary structure of the protein; and (7) reduced chymotrypsin-like proteasomal activity in cell lysates of lymphoblasts from an affected patient. To gain insight into the functional effects of the p.Thr75Met variant, we measured serum interleukin (IL) 1β, 2, 4, 5, 6, 8, 10, 12, IL-2 receptor, interferon-γ, and tumor necrosis factor-α (ARUP Laboratories, Salt Lake City, UT) in two affected patients from JMP200 pedigree, which revealed that both affected patients had 7- to 19-fold increased levels of IL-6 and 2.8- to 3.5-fold elevated levels of interferon γ (Table 1). Whereas serum levels of IL-8 were >9-fold elevated in JMP 200.3, in JMP200.4 only 1.6-fold elevation was observed. The levels of other cytokines were not elevated in both patients. Marked elevation of erythrocyte sedimentation rate and serum γ globulins, interferon-γ, and IL-6 levels without elevation in other cytokines such as IL-1 and TNF-α suggest not only an ongoing inflammation but also a peculiar biomarker signature present in the JMP syndrome. Ongoing inflammation was clearly evident in the patient JMP200.4 who had many active skin lesions at the time of examination (Figure S1), which were biopsy proven to be consistent with panniculitis.Table 1Serum Cytokine Levels in Affected PatientsSerum CytokineJMP 200.3JMP 200.4Reference Intervals in (pg/mL)IL-1β27180–36IL-28<50–12IL-2 receptor80613160–1033IL-46<50–5IL-5650–5IL-637790–5IL-84680–5IL-109180–18IL-12860–6IL-13<5<50–5Interferon-γ14170–5TNF-α6<50–22IL, interleukin; TNF, tumor necrosis factor. Abnormally high levels are indicated in bold. Open table in a new tab IL, interleukin; TNF, tumor necrosis factor. Abnormally high levels are indicated in bold. PSMB8 encodes β5i, a catalytic subunit of the immunoproteasome.21Rivett A.J. Hearn A.R. Proteasome function in antigen presentation: Immunoproteasome complexes, peptide production, and interactions with viral proteins.Curr. Protein Pept. Sci. 2004; 5: 153-161Crossref PubMed Scopus (113) Google Scholar Immunoproteasome-mediated proteolysis generates immunogenic epitopes presented by major histocompatibility complex (MHC) class I molecules. This degradation follows covalent modification of the proteins with polyubiquitin,22Bochtler M. Ditzel L. Groll M. Hartmann C. Huber R. The proteasome.Annu. Rev. Biophys. Biomol. Struct. 1999; 28: 295-317Crossref PubMed Scopus (423) Google Scholar, 23Kloetzel P.M. Antigen processing by the proteasome.Nat. Rev. Mol. Cell Biol. 2001; 2: 179-187Crossref PubMed Scopus (494) Google Scholar which targets them to the 26S proteasome. The 26S proteasome is composed of two subcomplexes: the 20S proteasome that contributes protease function and PA700 (19S regulator) that mediates translocation of the attached substrates to the 20S proteasome. The 20S proteasome is a 700,000 dalton complex composed of four axially stacked heptameric rings. Each of the two identical outer rings contains seven different α type subunits, and each of the two identical inner rings contains seven different β type subunits.23Kloetzel P.M. Antigen processing by the proteasome.Nat. Rev. Mol. Cell Biol. 2001; 2: 179-187Crossref PubMed Scopus (494) Google Scholar The β1, β2, and β5 subunits display different specificities for peptide bond hydrolysis and have predominantly PGPH-, trypsin-, and chymotrypsin-like activities, respectively.22Bochtler M. Ditzel L. Groll M. Hartmann C. Huber R. The proteasome.Annu. Rev. Biophys. Biomol. Struct. 1999; 28: 295-317Crossref PubMed Scopus (423) Google Scholar, 24Groll M. Heinemeyer W. Jager S. Ullrich T. Bochtler M. Wolf D.H. Huber R. The catalytic sites of 20S proteasomes and their role in subunit maturation: A mutational and crystallographic study.Proc. Natl. Acad. Sci. USA. 1999; 96: 10976-10983Crossref PubMed Scopus (240) Google Scholar Higher eukaryotes contain isoforms of β1, β2, and β5, termed β1i, β2i, and β5i, respectively, which are selectively incorporated into immunoproteasomes. Immunoproteasomes are constitutively expressed in cells of hematopoietic origin, particularly lymphocytes and monocytes, but can be induced in nonhematopoietic cells after exposure to inflammatory cytokines such as interferon-γ.21Rivett A.J. Hearn A.R. Proteasome function in antigen presentation: Immunoproteasome complexes, peptide production, and interactions with viral proteins.Curr. Protein Pept. Sci. 2004; 5: 153-161Crossref PubMed Scopus (113) Google Scholar Proteasomes and immunoproteasomes are functionally very similar, but not identical. Immunoproteasomes stimulate cleavage after hydrophobic, basic, and branched chain residues, while suppressing cleavage after acidic residues,25Toes R.E. Nussbaum A.K. Degermann S. Schirle M. Emmerich N.P. Kraft M. Laplace C. Zwinderman A. Dick T.P. Muller J. et al.Discrete cleavage motifs of constitutive and immunoproteasomes revealed by quantitative analysis of cleavage products.J. Exp. Med. 2001; 194: 1-12Crossref PubMed Scopus (276) Google Scholar, 26Gaczynska M. Rock K.L. Spies T. Goldberg A.L. Peptidase activities of proteasomes are differentially regulated by the major histocompatibility complex-encoded genes for LMP2 and LMP7.Proc. Natl. Acad. Sci. USA. 1994; 91: 9213-9217Crossref PubMed Scopus (274) Google Scholar, 27Diez-Rivero C.M. Lafuente E.M. Reche P.A. Computational analysis and modeling of cleavage by the immunoproteasome and the constitutive proteasome.BMC Bioinformatics. 2010; 11: 479Crossref PubMed Scopus (37) Google Scholar and thus can potentially enhance the generation of some antigenic epitopes differently from the proteasomes. However, a clear distinction of the role of proteasomes and immunoproteasomes in immune response has not yet emerged. Targeted gene deletion of Psmb8 in mice causes reduced MHC class I cell surface expression and inefficient presentation of the endogenous male antigen HY.28Fehling H.J. Swat W. Laplace C. Kuhn R. Rajewsky K. Muller U. von Boehmer H. MHC class I expression in mice lacking the proteasome subunit LMP-7.Science. 1994; 265: 1234-1237Crossref PubMed Scopus (442) Google Scholar These mice are healthy, have normal T and B lymphocyte counts in their lymphoid organs,28Fehling H.J. Swat W. Laplace C. Kuhn R. Rajewsky K. Muller U. von Boehmer H. MHC class I expression in mice lacking the proteasome subunit LMP-7.Science. 1994; 265: 1234-1237Crossref PubMed Scopus (442) Google Scholar and respond normally to challenge with lymphocytic choriomeningitis virus,29Nussbaum A.K. Rodriguez-Carreno M.P. Benning N. Botten J. Whitton J.L. Immunoproteasome-deficient mice mount largely normal CD8+ T cell responses to lymphocytic choriomeningitis virus infection and DNA vaccination.J. Immunol. 2005; 175: 1153-1160Crossref PubMed Scopus (41) Google Scholar but are susceptible to infection with Toxoplasma gondii.30Tu L. Moriya C. Imai T. Ishida H. Tetsutani K. Duan X. Murata S. Tanaka K. Shimokawa C. Hisaeda H. et al.Critical role for the immunoproteasome subunit LMP7 in the resistance of mice to Toxoplasma gondii infection.Eur. J. Immunol. 2009; 39: 3385-3394Crossref PubMed Scopus (34) Google Scholar The patients with JMP syndrome, however, are not known to be prone to infections. The lack of resemblance of the phenotype of Psmb8−/− mice to that observed in patients with JMP syndrome may indicate different biology of the PSMB8 in humans and mice. Further to this, our patients have only missense mutation, whereas in the mice the whole gene is deleted. Nonetheless, further studies are required to fully understand the underlying reasons for discrepancy in the phenotype of patients with JMP syndrome and Psmb8−/− mice. Recently, studies with PR-957, a selective inhibitor of β5i (LMP7) that binds to the active site and specifically inhibits chymotrypsin-like peptidase activity, have revealed a unique role of PSMB8 in cytokine production.31Muchamuel T. Basler M. Aujay M.A. Suzuki E. Kalim K.W. Lauer C. Sylvain C. Ring E.R. Shields J. Jiang J. et al.A selective inhibitor of the immunoproteasome subunit LMP7 blocks cytokine production and attenuates progression of experimental arthritis.Nat. Med. 2009; 15: 781-787Crossref PubMed Scopus (443) Google Scholar In endotoxin-stimulated peripheral blood mononuclear cells from healthy subjects and from patients with rheumatoid arthritis, PR-957 blocked production of IL-23 by ∼80% and of TNF-α and IL-6 by ∼50%.31Muchamuel T. Basler M. Aujay M.A. Suzuki E. Kalim K.W. Lauer C. Sylvain C. Ring E.R. Shields J. Jiang J. et al.A selective inhibitor of the immunoproteasome subunit LMP7 blocks cytokine production and attenuates progression of experimental arthritis.Nat. Med. 2009; 15: 781-787Crossref PubMed Scopus (443) Google Scholar Further, PR-957 ameliorated disease in two mouse models of arthritis.31Muchamuel T. Basler M. Aujay M.A. Suzuki E. Kalim K.W. Lauer C. Sylvain C. Ring E.R. Shields J. Jiang J. et al.A selective inhibitor of the immunoproteasome subunit LMP7 blocks cytokine production and attenuates progression of experimental arthritis.Nat. Med. 2009; 15: 781-787Crossref PubMed Scopus (443) Google Scholar However, our patients had hypergammaglobulinemia and extremely high serum levels of IL-6 and interferon-γ. These data suggest that p.Thr75Met substitution may have additional effects beyond a partial loss of function. This substitution may alter the processing of an exogenous or endogenous antigen so as to trigger an accelerated immune response targeted most directly to adipose tissue, joints, and muscles. So far, the monogenic autoinflammatory diseases have been reported to be due to mutations in the family of PYRIN domain-containing proteins, proteins interacting with pyrin, and others, resulting in activation of the interleukin-1β pathway.6Henderson C. Goldbach-Mansky R. Monogenic autoinflammatory diseases: New insights into clinical aspects and pathogenesis.Curr. Opin. Rheumatol. 2010; 22: 567-578PubMed Google Scholar This report expands the spectrum of autoinflammatory diseases to include JMP syndrome resulting from altered immunoproteasome function. How reduced chymotrypsin-like proteasomal activity resulting from a loss-of-function PSMB8 mutation results in joint contractures, panniculitis-induced lipodystrophy, and the other clinical features of JMP syndrome remains to be elucidated. We thank Sarah Masood and Katie Tunison for help with illustrations and mutational screening; Shyam Murali, Prateek Sharma, and Marquis Harris for sequencing candidate genes in the critical region; Helen Hobbs, M.D., for providing DNA samples of the Hispanic controls; Tommy Hyatt for genotyping; Laurie Davis, Ph.D., for providing control lymphoblasts; David Thompson for help with the proteasomal activity assay; Beverley Adams-Huet, M.S., for statistical analysis; Andrew Zinn, M.D., Ph.D., for helpful suggestions; and Michael S. Brown, M.D., for critical review of the manuscript. This work was supported by the National Institutes of Health grants R01-DK54387 and DK46181 and CTSA Grant UL1 RR024982 and Southwest Medical Foundation. Download .pdf (1.25 MB) Help with pdf files Document S1. Four Figures and Three Tables The URLs for data presented herein are as follows:Human Genome Variation Society, http://www.hgvs.org/mutnomen/MERLIN, http://www.sph.umich.edu/csg/abecasis/Merlin/National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov/Online Mendelian Inheritance in Man (OMIM), http://www.ncbi.nlm.nih.gov/Omim/PLINK, pngu.mgh.harvard.edu/∼purcell/plink/Prochek, http://www.ebi.ac.uk/thornton-srv/software/PROCHECK/Protein, http://www.ncbi.nlm.nih.gov/protein/RCSB Protein Databank, http://www.rcsb.org/pdb/explore/explore.do?structureId=1Z7QSNP database, http://www.ncbi.nlm.nih.gov/snp/University of California, Santa Cruz (UCSC) Genome Browser, http://www.genome.ucsc.edu
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