The Immunoproteasome in Human Lens Epithelial Cells During Oxidative Stress

Abstract

Citation: Petersen A, Zetterberg M. The immunoproteasome in human lens epithelial cells during oxidative stress. Invest Ophthalmol Vis Sci. 2016;57:5038-5045. DOI:10.1167/ iovs.16-19536 PURPOSE. The immunoproteasome is known to generate peptides for antigen presentation. However, it has also been proposed to have additional functions such as stress response. The propensity of the immunoproteasome for degradation of oxidatively damaged proteins and peptides makes it interesting in the context of cataract formation and prevention. This study hypothesized that the immunoproteasome is present in human cataractous lenses and that oxidative stress will induce its expression, affect its proteolytic activity and its intracellular location in native cultured human lens epithelial cells (HLECs).METHODS. The expression of the immunoproteasome and the constitutive proteasome subunits b1i/b1, b2i/b2, and b5i/b5 were studied by using Western blotting. The chymotrypsin-like activity was investigated for possible oxidative stress response. Inhibitors specific for the immuno-and constitutive proteasome, ONX-0914 and MG-132, respectively, were used to study their relative contributions to total proteasome activity. The intracellular location of the proteasomal subunits b5i and b5 was studied by immunocytochemistry.RESULTS. Immunoproteasome subunits were detected both in the lens epithelium and in the lens fibers derived from cataract surgery. Oxidative stress to cultured HLECs upregulated the immunoproteasome but not the constitutive proteasome. The chymotrypsin-like activity decreased with increased oxidative stress and the two proteasome types contributed equally to total proteasome activity. Immunocytochemical labeling of subunit b5i showed mainly cytosolic localization, whereas subunit b5 was localized predominantly in the nucleus. H 2 O 2 -induced challenge increased the expression of the immunoproteasome.CONCLUSIONS. The present findings indicate a role for the immunoproteasome in oxidative stress management in the lens.

PA28αβ overexpression enhances learning and memory of female mice without inducing 20S proteasome activity

“…Cell debris was removed by centrifugation at 5000 g for 10 min and protein concentration was determined using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). Eye lenses were lysed by sonication (Branson Ultrasonic Corp., Danbury, CT, USA) in PBS [ 53 ]. Samples were prepared for SDS–PAGE as described [ 54 ], separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), transferred onto a nitrocellulose membrane (Invitrogen, Bleiswjik, the Netherlands) and probed with rabbit mAb PA28α (#9643; Cell Signaling Technology, Inc., Leiden, the Netherlands), rabbit pAb PA28β (#2409), rabbit mAb GluA2 (#13607), rabbit pAb N-Cadherin (#4061), rabbit pAb GluA1 (#13185), rabbit pAb β5/PSMB5 (#11903), rabbit mAb Phospho-CaMKII Thr286 (#12716), rabbit mAb Phospho-CREB Ser133 (#9198), rabbit pAb polyubiquitin K48-linkage specific (ab190061; Abcam, Cambridge UK), goat pAb PSD-95 (ab12093), rabbit pAb GluA1 (ab31232), rabbit mAb Synaptophysin (ab32127), rabbit pAb Spinophilin (ab203275), rabbit mAb NeuN (ab177487), rabbit pAb Estrogen Receptor beta phospho S105 (ab62257), rabbit pAb Rpt2/S4 (ab3317), or rabbit pAb β5i/LMP7 (ab3329).…”

Multikinase inhibitors modulate non-constitutive proteasome expression in colorectal cancer cells

“…Increased expression of non-constitutive proteasomes might help to deal with the consequences of stress-induced build-up of potentially toxic protein aggregates and facilitate their adaptation ( Grune et al, 2011 ; Pickering et al, 2012 ; Johnston-Carey et al, 2015 ). Though we did not specifically address the mechanism that stands behind activation of immunoproteasome subunit expression following the incubation with the MKIs, it is well established that non-constitutive proteasome expression is increased in various stress conditions including oxidative stress ( Johnston-Carey et al, 2015 ; Petersen and Zetterberg, 2016 ; Raynes et al, 2016 ; Wang et al, 2023 ). MKIs were demonstrated to induce various types of stress ( Wang et al, 2023 ).…”

Proteasome activator 28γ (PA28γ) allosterically activates trypsin-like proteolysis by binding to the α-ring of the 20S proteasome

“…It is known that immune function is not impacted when PA28γ is knocked out ( 44 ). In addition, since the cellular localization of the immunoproteasome is under contention and seemingly cell specific ( 45 , 46 ), it is not clear if it could colocalize with the nuclear PA28γ. However, if PA28γ does bind to immunoproteasomes that contain β2i subunits, we could not predict what the affect could be since B2 is substantially different from β2i, as they only share a 58.3% identity.…”