N-acetyl-L-cysteine inhibits 26S proteasome function: implications for effects on NF-κB activation

Abstract

Ionizing radiation shares with cytokines, such as TNF-α, an ability to generate free radicals in cells and activate downstream proinflammatory responses through NF-κB-dependent signal transduction pathways. Support for the role of free radicals in triggering such responses comes from the use of free radical scavengers like N-acetyl-L-cysteine (NAC). The nature of the link between free radical generation and NF-κB activation is, however, unclear. In this study, we explore the possibility that scavenging of free radicals by NAC might not be the mechanism by which it inhibits NF-κB activation, but rather that NAC acts through inhibition of proteasome function. The effect of NAC on the chymotryptic function of the 26s and 20s proteasome complex was measured in extracts from EVC 304 bladder carcinoma cells by assessing degradation of fluorogenic substrates. NAC inhibited 26s but not 20s proteasome activity, suggesting that it interferes with 19s regulatory subunit function. NAC blocked radiation-induced NF-κB activity in ECV 304 cells and RAW 264.7 macrophages, as measured by a gel shift assay, at doses that inhibited proteasome activity. This provides a possible mechanism whereby NAC could block NF-κB activation and affect the expression of other molecules that are dependent on the ubiquitin/proteasome system for their degradation, other than by scavenging free radicals.

Introduction

The free radical scavenger N-acetyl-L-cysteine (NAC) is used clinically for a broad spectrum of indications including mucolysis, detoxification after acetaminophen poisoning, adult respiratory distress syndrome (ARDS), hyperoxia-induced pulmonary damage, HIV infection, cancer, and heart disease [1], [2], [3], [4]. Free radicals are critical in the determination of protein structure, regulation of enzyme activity, protein phosphorylation, and control of transcription factor activity and binding, and NAC is often used to explore their role in these effects. However, despite its frequent use and the enormous clinical knowledge about this drug, free radical scavenging is often assumed to be the mechanism by which it brings about its effects although its exact targets are unknown. For example, it is unclear how NAC acts to downregulate expression of the transcription factor NF-κB, which is a major mediator of inflammatory responses and controls expression of a large variety of genes encoding cytokines, growth factors, acute phase proteins, and cell adhesion and immunoregulatory molecules [5], [6].

The findings that NAC prevents NF-κB activation in response to a variety of signals, including TNF-α and ionizing radiation [7], [8], are generally taken as support for the involvement of free radicals in the process. TNF-α is thought to mediate both signal transduction and its cytotoxic effects through reactive oxygen intermediates (ROI). However, there is evidence that the lipoxygenase pathway may be more important than free radicals in mediating the latter [9]. Furthermore, some of the effects of NAC, like the G1-arrest described by Sekharam and colleagues [10], are not easily explained through a simple mechanism involving direct scavenging of free radicals.

NF-κB is a family of homo- or hetero-dimers of proteins of the RelA/NF-κB family. They pre-exist in the cytosol, bound to inhibitor molecules (IκB) that prevent nuclear translocation of the complex. Classically, upon appropriate signaling, IκB is phosphorylated at two serine sites (ser32, ser36) by specific kinases (IKK), poly-ubiquitinated and degraded by the 26s proteasome. This releases NF-κB and allows nuclear translocation followed by initiation of transcription of dependent genes (for a review see [11]). Degradation of IκB by the 26s proteasome is a mandatory step for NF-κB activation in response to most signals. The 26s proteasome is a large protease of 2MDa that consists of a cylindrical 20s core particle formed by four rings each with seven alpha and beta subunits. The inner two rings form the catalytic site of the complex. These exhibit five distinct cleavage activities [12]. Activity is regulated over a wide range by substitution of constitutive beta-subunits by interferon-inducible subunits LMP2, LMP7, and MECL-1 [13], [14] and by 19s regulatory and 11s activator units that control substrate access, de-ubiquitination, and substrate linearization [15].

ATP- and ubiquitin-dependent protein degradation by the 26s proteasome is one of the most important degradation pathways of mammalian cells. The rate of degradation, as well as the rate of synthesis, regulates intracellular levels of proteins like p53, IκB, cJun, cFos, and cyclins A, B, and E, p21 and p27 [16], [17]. It therefore controls cellular responses in many physiological and pathophysiological conditions [12]. It plays an additional important role in the immune system by determining the peptides that are expressed on the cell surface in association with MHC class I molecules [18], [19], [20]. Similarities between many of the effects attributed to NAC and those that follow inhibition of the ubiquitin-proteasome pathway led us to investigate a possible direct effect of NAC on 26s proteasome function using a well-established in vitro model for inflammatory responses. Our findings highlight an additional, and possibly major, pharmacological aspect of this frequently used drug.