Revisiting the role of the immunoproteasome in the activation of the canonical NF-κB pathway

Abstract

The discovery of NF-κB signaling pathways has greatly enhanced our understanding of inflammatory and immune responses. In the canonical NF-κB pathway, the proteasomal degradation of IκBα, an inhibitory protein of NF-κB, is widely accepted to be a key regulatory step. However, contradictory findings have been reported as to whether the immunoproteasome plays an obligatory role in the degradation of IκBα and activation of the canonical NF-κB pathway. Such results were obtained mainly using traditional gene deletion strategies. Here, we have revisited the involvement of the immunoproteasome in the canonical NF-κB pathway using small molecule inhibitors of the immunoproteasome, namely UK-101 and LKS01 targeting β1i and β5i, respectively. H23 and Panc-1 cancer cells were pretreated with UK-101, LKS01 or epoxomicin (a prototypic inhibitor targeting both the constitutive proteasome and immunoproteasome). We then examined whether these pretreatments lead to any defect in activating the canonical NF-κB pathway following TNFα exposure by monitoring the phosphorylation and degradation of IκBα, nuclear translocation of NF-κB proteins and DNA binding and transcriptional activity of NF-κB. Our results consistently indicated that there is no defect in activating the canonical NF-κB pathway following selective inhibition of the immunoproteasome catalytic subunits β1i, β5i or both using UK-101 and LKS01, in contrast to epoxomicin. In summary, our current results using chemical genetic approaches strongly support that the catalytic activity of the immunoproteasome subunits β1i and β5i is not required for canonical NF-κB activation in lung and pancreatic adenocarcinoma cell line models.

Immunoproteasome Deficiency Modifies the Alternative Pathway of NFκB Signaling

“…Our results showing that activation of the Classical Pathway is not affected by the absence of i-proteasome subunits is consistent with a recent study that used a chemical genetic approach [36]. The authors observed no defects in TNFα-induced activation of the Classical Pathway (i.e., degradation of IκBα, nuclear translocation of p65/p50) using small molecule inhibitors that specifically target either the LMP2 or LMP7 subunits.…”

PSMB9 Codon 60 Polymorphisms Have No Impact on the Activity of the Immunoproteasome Catalytic Subunit B1i Expressed in Multiple Types of Solid Cancer

“…The fluorogenic substrate Suc-LLVY-AMC used by Mishto et al [17] is commonly used to measure the overall chymotrypsin-like activity of the proteasome. However, since this substrate can be hydrolyzed by both the immunoproteasome (β1i and β5i) and the constitutive proteasome (β5) [25], the results obtained using Suc-LLVY-AMC cannot tease out the direct contribution of the β1i genetic polymorphism. Our current results were obtained using the recently reported substrate Ac-PAL-AMC that is selectively cleaved by the β1i subunit (Figure 2) [20].…”

Immunoproteasome subunit deficiency has no influence on the canonical pathway of NF-κB activation

“…A study conducted with small molecule inhibitors targeting LMP2 or LMP7 further supports the conception of NF-B activation being independent of proteasome composition. Inhibition of LMP2, LMP7, or even both had no influence on IB␣ degradation in cells stimulated with TNF-␣ (Jang et al, 2012). Given that neither genetic deletion nor chemical inhibition of immunoproteasome subunits affects canonical NF-B activation, the elucidation of alternative mechanisms how the immunoproteasome influences cytokine production, T helper cell differentiation, and autoimmunity requires substantial research efforts in the future.…”