The non-obese diabetic (NOD) mouse is an established and widely used model of autoimmune type I diabetes 1. Besides the involvement of class II-and class I-restricted T cells, additional major histocompatibility complex (MHC) and non-MHC-linked genetic factors contribute to disease susceptibility 2. Faustman and colleagues claim that in NOD mice the expression of the IFNγ-inducible large multifunctional proteasome subunit 2 (LMP2; Genome Database designation, PSMB9) is decreased both at the mRNA and protein level 3, 4. In addition, they noted a failure to appropriately activate NFκB in cells from NOD mice due to defective IκB degradation. This observation was attributed to the claimed defect in LMP2 synthesis. While the published data show that activation of NFκB relies on proteasomal proteolysis, there is no independent evidence that such activation would require the specific involvement of LMP2-bearing proteasomes 5.

We examined the expression of LMP2 mRNA by real time PCR and found no evidence for impaired transcription of the LMP2 gene in NOD mice as compared to BALB/c mice (Fig. 1a). Further, immunoblot analysis showed no reduction of LMP2, LMP7 and MECL1 protein levels in NOD mice, compared to other strains (Fig. 1b). The slight differences in mobility of LMP2 and LMP7 proteins on SDS PAGE are attributable to their genetic polymorphism. What about proteasome activity? The assessment of proteasomal proteolysis in crude extracts using fluorogenic substrates is complicated by the presence of other non-proteasomal peptidases. We therefore analyzed directly the content of cell extracts for active proteasome β subunits using a mechanism-based active site directed probe 6. We observed normal proteasome activity in extracts from thymus and spleen of NOD mice (Fig. 1c). Active immunoproteasomes including LMP2 are present at comparable levels in all strains analyzed, and did not appear to be influenced by age, gender or the presence or absence of diabetes (Fig. 1c and data not shown). LMP2, together with LMP7 and MECL1 are essential subunits for the assembly of the immunoproteasome 7. Mere overexpression of a single immunoproteasome subunit does not lead to assembly of functional immunoproteasomes, and conversely, the ablation of either LMP2 or LMP7 suffices to eliminate formation of immunoproteasomes which contain all three interferon-γ-inducible β subunits 8. Thus, the absence of LMP2, as claimed by Faustman and coworkers, should have abolished the activity of immunoproteasomes altogether.