The proteasome-associated autoinflammatory syndrome (PRAAS) is a spectrum of autoinflammatory disorders. The cause of the PRAAS is an impairment of the immunoproteasome activity. The immunoproteasome (IP) is a special type of proteasome which is inducible under inflammatory conditions and constitutively expressed in hematopoietic cells. MECL-1 (β2i), LMP2 (β1i), and LMP7 (β5i) are the proteolytically active subunits of the immunoproteasome, and they are known to shape the antigenic repertoire presented on major histocompatibility complex (MHC) class I molecules. Because of catalytic subunits mutations, the immunoproteasome activity is reduced in PRAAS patients, leading to a continuum of clinical manifestations depending on the mutation carried and which often include spiking fevers, skin rashes and erythema, and lipodystrophy.
To better understand the molecular basis of this spectrum of disorders, we exploited lentivirus (LTVs) carrying two different mutations of PSMB8, the gene coding for LMP7: cG315T and the cA313C. The expression of these mutated gene sequences has been placed under the control of a doxycycline inducible system carried by a p21 plasmid. The CRISPR-Cas9 technique has been exploited to selectively inactivate the normal PSMB8 gene in the promonocytic U937 cell strain. We studied the impact of the two mutations on immunoproteasome activity and the differences among the c.G315T, a putative dominant-negative mutation detected in a family with un undefined inflammatory syndrome and the c.A313C mutation, an already described PRAAS-causing PSMB8 mutant.
Here, we show that the c.A313C mutant may have a lower impact on the immunoproteasome activity compared to the c.G315T mutant. We also show that in both cells from patients carrying the c.G315T variant and in cell lines expressing mutant constructs, we can clearly distinguish two probable PSMB8 protein variants with two different molecular weights. These findings suggest that it could be possible to exploit the differences among the wild type and the mutated PSMB8 proteins to better understand if it is possible to develop new therapeutic strategies for affected patients.