A Novel Role for ATM in Regulating Proteasome-Mediated Protein Degradation through Suppression of the ISG15 Conjugation Pathway

Ataxia Telangiectasia (A-T) is an inherited immunodeficiency disorder wherein mutation of the ATM kinase is responsible for the A-T pathogenesis. Although the precise role of ATM in A-T pathogenesis is still unclear, its function in responding to DNA damage has been well established. Here we demonstrate that in addition to its role in DNA repair, ATM also regulates proteasome-mediated protein turnover through suppression of the ISG15 pathway. This conclusion is based on three major pieces of evidence: First, we demonstrate that proteasome-mediated protein degradation is impaired in A-T cells. Second, we show that the reduced protein turnover is causally linked to the elevated expression of the ubiquitin-like protein ISG15 in A-T cells. Third, we show that expression of the ISG15 is elevated in A-T cells derived from various A-T patients, as well as in brain tissues derived from the ATM knockout mice and A-T patients, suggesting that ATM negatively regulates the ISG15 pathway. Our current findings suggest for the first time that proteasome-mediated protein degradation is impaired in A-T cells due to elevated expression of the ISG15 conjugation pathway, which could contribute to progressive neurodegeneration in A-T patients

Mechanism of Action of Camptothecin

Camptothecin (CPT) class of compounds has been demonstrated to be effective against a broad spectrum of tumors. Their molecular target has been firmly established to be human DNA topoisomerase I (topo I). CPT inhibits topo I by blocking the rejoining step of the cleavage/religation reaction of topo-I, resulting in accumulation of a covalent reaction intermediate, the cleavable complex. The primary mechanism of cell killing by CPT is S-phase-specific killing through potentially lethal collisions between advancing replication forks and topo-I cleavable complexes. Collisions with the transcription machinery have also been shown to trigger the formation of long-lived covalent topo-I DNA complexes, which contribute to CPT cytotoxicity. Two novel repair responses to topo-I-mediated DNA damage involving covalent modifications of topo-I have been discovered. The first involves activation of the ubiquitin/26S proteasome pathway, leading to degradation of topo-I (CPT-induced topo-I downregulation). The second involves SUMO conjugation to topo-I. The potential roles of these new mechanisms for repair of topo-I-mediated DNA damage in determining CPT sensitivity/resistance in tumor cells are discussed

Transcription-Dependent Degradation of Topoisomerase I-DNA Covalent Complexes

Topoisomerase I (Top I)-DNA covalent complexes represent a unique type of DNA lesion whose repair and processing remain unclear. In this study, we show that Top I-DNA covalent complexes transiently arrest RNA transcription in normal nontransformed cells. Arrest of RNA transcription is coupled to activation of proteasomal degradation of Top I and the large subunit of RNA polymerase II. Recovery of transcription occurs gradually and depends on both proteasomal degradation of Top I and functional transcription-coupled repair (TCR). These results suggest that arrest of the RNA polymerase elongation complex by the Top I-DNA covalent complex triggers a 26S proteasome-mediated signaling pathway(s) leading to degradation of both Top I and the large subunit of RNA polymerase II. We propose that proteasomal degradation of Top I and RNA polymerase II precedes repair of the exposed single-strand breaks by TCR

Suppression of the Macrophage Proteasome by Ethanol Impairs MHC Class I Antigen Processing and Presentation

<div><p>Alcohol binge-drinking (acute ethanol consumption) is immunosuppressive and alters both the innate and adaptive arms of the immune system. Antigen presentation by macrophages (and other antigen presenting cells) represents an important function of the innate immune system that, in part, determines the outcome of the host immune response. Ethanol has been shown to suppress antigen presentation in antigen presenting cells though mechanisms of this impairment are not well understood. The constitutive and immunoproteasomes are important components of the cellular proteolytic machinery responsible for the initial steps critical to the generation of MHC Class I peptides for antigen presentation. In this study, we used an <i>in-vitro</i> cell culture model of acute alcohol exposure to study the effect of ethanol on the proteasome function in RAW 264.7 cells. Additionally, primary murine peritoneal macrophages obtained by peritoneal lavage from C57BL/6 mice were used to confirm our cell culture findings. We demonstrate that ethanol impairs proteasome function in peritoneal macrophages through suppression of chymotrypsin-like (Cht-L) proteasome activity as well as composition of the immunoproteasome subunit LMP7. Using primary murine peritoneal macrophages, we have further demonstrated that, ethanol-induced impairment of the proteasome function suppresses processing of antigenic proteins and peptides by the macrophage and in turn suppresses the presentation of these antigens to cells of adaptive immunity. The results of this study provide an important mechanism to explain the immunosuppressive effects of acute ethanol exposure.</p> </div

20S chymotrypsin-like (Cht-L) proteasome activity is suppressed in ethanol-treated RAW 264.7 cells. A.

<p>Murine RAW 264.7 (2×10⁵ cells/ml) cells were treated with 50 mM or 100 mM ethanol (EtOH) for 24 h or 48 h. 0 mM (control) cells received no treatment. Cell lysates obtained at the indicated time points were analyzed for 20S Cht-L proteasome activity by fluorigenic assay as described in Methods. <b>B.</b> RAW 264.7 cells were treated with MG132 (0.5 uM) as a positive control of proteasome suppression. Cell lysates were analyzed at the indicated time points for proteasome activity as described in panel A. <b>C.</b> Ethanol-treated RAW 264.7 cells were stimulated with heat-killed <i>K.pneumoniae</i> (<i>Kp</i>), 6 hours after initial EtOH exposure. Cell lysates obtained at the indicated time points were analyzed as described above in panel A. <b>D.</b> RAW 264.7 cells were treated with MG132 (0.5 uM) followed by challenge with <i>Kp</i> as described in panel C. Cells were lysed at the indicated time points and analyzed for proteasome activity as described above. Proteasome activity data was plotted as mean relative fluorescence units (R.F.U.) ± SEM (three replicates per experiment) and is representative of three independent experiments. Higher R.F.U. values indicate higher proteasome activity. *p<0.05 vs. control (0 mM) at the respective time point. **p<0.05 vs. control (0 mM) and 50 mM EtOH at the respective time point.</p

Ovalbumin antigen MHC-Class I processing and presentation is suppressed in ethanol-treated murine primary peritoneal macrophages.

<p><b>A.</b> Schematic representation of experimental setup as described in Methods. Briefly, murine peritoneal macrophages were treated 50 mM or 100 mM EtOH for 24, 48 or 72 h. Proteasome inhibitors MG132 (5 uM) or lactacystin (10 uM) were used as positive controls of proteasome inhibition. EtOH-exposed cells were treated with full-length chicken ovalbumin (2 mg/ml) for 2 hours. Macrophages washed with PBS and co-incubated with OT-1 mouse CD8+ cytotoxic T lymphocytes (Ratio 2∶1) for 18 h. Cell supernatants were collected and analyzed for cytokine IL-2. <b>B.</b> CD8+ T cell derived IL-2 levels from proteasome inhibitor-treated peritoneal macrophage groups is shown. <b>C.</b> CD8+ T cell derived IL-2 levels from 24 h EtOH-treated peritoneal macrophage groups is shown. <b>D.</b> CD8+ T cell derived IL-2 levels from 48 h EtOH-treated peritoneal macrophage groups is shown. <b>E.</b> CD8+ T cell derived IL-2 levels from 72 h EtOH-treated peritoneal macrophage groups. Data plotted is mean levels of cytokine IL-2 (pg/ml) ± SEM (three replicates per experiment) and is representative of three independent experiments. *p<0.05 vs. control (0 mM) at respective time point. **p<0.05 vs. control (0 mM) and 50 mM EtOH at respective time point.</p

Ethanol suppresses 20S Cht-L proteasome activity in murine primary peritoneal macrophages. A.

<p>Murine peritoneal macrophages (2.5×10⁵ cells/ml) were treated with 50 mM or 100 mM EtOH for 24 h or 48 h. 0 mM (control) cells received no treatment. Cell lysates obtained at the indicated time points were analyzed for 20S Cht-L proteasome activity by fluorigenic assay as described in Methods. <b>B.</b> Peritoneal macrophages were treated with MG132 (5 uM), as a positive control of proteasome suppression. Cell lysates were obtained at the indicated time points and analyzed as described in Panel A. <b>C.</b> Ethanol-treated peritoneal macrophages were stimulated with heat-killed <i>Kp</i>, 6 hours after initial EtOH exposure. Cell lysates obtained at indicated time points were analyzed for proteasome activity as described in panel A. <b>D.</b> MG132-treated peritoneal macrophages were stimulated with <i>Kp</i> as described in Panel C. Cell lysates were obtained at the indicated time points and analyzed for proteasome activity as in Panel A. Proteasome activity data plotted as mean relative fluorescence units (R.F.U.) ± SEM (three replicates per experiment) and is representative of three independent experiments. Higher R.F.U. values indicate higher proteasome activity. *p<0.05 vs. control at the respective time point. **p<0.05 vs. control and 50 mM EtOH at the respective time points.</p

Ethanol does not alter steady-state protein levels of constitutive proteasome subunit beta 5 (PSMB5), but suppresses steady-state protein levels of immunoproteasome subunit LMP7.

<p><b>A.</b> Murine unstimulated RAW 264.7 cells (2×10⁵ cells/ml) were treated with 50 mM (lane 3) or 100 mM (Lane 4) ethanol (EtOH) for 48 h. Cells were also treated with MG132 (0.5 uM) (lane 2) as a positive control of proteasome inhibition. 0 mM (control) cells received no treatment (lane1). Cell lysates were obtained 48 h post EtOH exposure. The lysates of triplicate samples were pooled for gel loading, analyzed by 15% SDS-PAGE and immunoblotted with anti-PSMB5 antibody as described in the methods (upper panel). The membrane was stripped and reprobed with anti-beta-actin antibody to ensure equal protein loading as described in the methods section (lower panel). The bar graph represents densitometry quantitation of PSMB5 levels between treatment groups and is represented as INT/mm². The depicted gel blot is representative of two independent experiments. Each band of the blot represents the pooled lysates from three independently treated samples. <b>B.</b> Unstimulated RAW 264.7 cells (2×10⁵ cells/ml) were treated with 50 mM (lane 3) or 100 mM (lane 4) EtOH for 24 h (Left panel) or 48 h (Right Panel). Cells were also treated with MG132 (0.5 uM) (lanes 2) as a positive control of proteasome inhibition. 0 mM (control) cells received no treatment (lane1). Cell lysates were obtained 24 h or 48 h post-EtOH exposure. The lysates of triplicate samples were pooled for gel loading, analyzed by 15% SDS-PAGE and immunoblotted with anti-LMP7 antibody as described in the methods (24 h and 48 h upper panels). The membrane was stripped and reprobed with anti-beta-actin antibody to ensure equal protein loading as described in the methods section (24 h and 48 h lower panel). The bar graphs beneath each immunoblot represent densitometry quantitation of LMP7 levels between treatment groups at 24 h and 48 h respectively and is represented as INT/mm². The depicted gel blots are representative of two independent experiments. Each band of the blot represents the pooled lysates from three independently treated samples.</p

Antigen presentation of C-terminal extended SIINFEKLTE is suppressed in ethanol-treated murine primary peritoneal macrophages.

<p>Experimental setup is the same as detailed in Fig. 4A, except that instead of full length OVA the C-terminal extended SIINTEKLTE peptide was used. <b>A.</b> CD8+ T cell-derived IL-2 levels from 24 h EtOH-treated peritoneal macrophage groups is shown. <b>B.</b> CD8+ T cell-derived IL-2 levels from 48 h EtOH-treated macrophage groups is shown. Data plotted is mean levels of cytokine IL-2 (pg/ml) ± SEM (three replicates per experiment) and is representative of three independent experiments. **p<0.05 vs. control and 50 mM EtOH at respective time point.</p