ABSTRACT
Title
Glucocorticoid-induced activation of caspase-8 protects the glucocorticoid-induced protein Gilz from proteasomal degradation and induces its binding to SUMO-1 in murine thymocytes
Authors
S. Pierangeli, N. Pozzesi, S. Spinicelli, C. Riccardi, D.V. Delfino
Section of Pharmacology Toxicology and Chemotherapy, Department of Clinical and Experimental Medicine, University of Perugia, Perugia, Italy
Section of Pharmacology Toxicology and Chemotherapy, Department of Clinical and Experimental Medicine, University of Perugia, Perugia, Italy
Abstract
Apoptosis has a crucial role in the thymus as it controls the immune repertoire by selective elimination of unwanted thymocytes.
Glucocorticoids (GCs) induce thymocyte apoptosis by protein synthesis and caspases activation.
GILZ is a protein transcriptionally induced by GCs and able to induce thymocyte apoptosis by Bcl-xL downregulation and caspases-8 and caspases -3 activation.
We investigatedthe possible cross-talk between glucocorticoid (GC)-induced leucine zipper (Gilz) and caspase-8.
By suppressing caspases activation in dexamethasone (DEX)-treated thymocytes with specific caspase-8 or -9 inhibitors, we determined that expression of Dex-induced Gilz protein was reduced when caspase-8 but not caspase-9activity was inhibited.
The GILZ mRNA and protein analyses showed that this caspase-8-dependent abrogation of Gilz was partially due to increased protein degradationby proteasome. Only the inhibition of proteasome but not lysosomes abrogated, in fact, the reduction in Gilz expression, suggesting that DEX-induced caspase-8 activation could protect DEX-induced GILZ protein from its proteasomal degradation.
The ubiquitin/proteasome pathway is an important machinery involved in the maintenance of cellular homeostasis regulating multiple cellular processes including cell-cycle progression and apoptosis; post-transaltional modification by ubiquitin plays a central role in targeting proteins for this extralysosomalproteolytic degradation.
In addition to ubiquitin, there are several ubiquitin-like proteins (UBLs), such as small ubiquitin-like modifier (SUMO), which has been shown to covalently modify a large number of proteins using an enzymatic pathway similar to the ubiquitination.In some cases, SUMO and ubiquitin may directly compete for modification of the same lysines, thus inhibiting each other’s binding to target proteins.
As a putative SUMO-binding site was identified in the Gilz amino-acid sequence, we hypothesized that the caspase-8 dependent protection of Gilz could be due to caspase-8-driven sumoylation that inhibits ubiquitination on the same lysine acceptor site.
We identified a 30-kDa protein that was compatible with the size of a Gilz–SUMO-1 complex and was recognized by the anti-SUMO-1 and anti-Gilz antibodies. In addition, Gilz bound to Ubc9, the specific SUMO-1 E2-conjugating enzyme, in vitro and coimmunoprecipitated with Ubc9 in vivo. Furthermore, Gilz coimmunoprecipitated with SUMO-1 both in vitro and in vivo, and this interaction depended on caspase-8 activation. This requirement for caspase-8 was further supported by the evaluation of caspase-8-deficientthymocytes and lymphocytes in which Gilz expression was reduced.
In summary, our results suggest that caspase-8 activation protects Gilz from proteasomal degradation and induces its binding to SUMO-1 in GC-treated thymocytes.
Glucocorticoids (GCs) induce thymocyte apoptosis by protein synthesis and caspases activation.
GILZ is a protein transcriptionally induced by GCs and able to induce thymocyte apoptosis by Bcl-xL downregulation and caspases-8 and caspases -3 activation.
We investigatedthe possible cross-talk between glucocorticoid (GC)-induced leucine zipper (Gilz) and caspase-8.
By suppressing caspases activation in dexamethasone (DEX)-treated thymocytes with specific caspase-8 or -9 inhibitors, we determined that expression of Dex-induced Gilz protein was reduced when caspase-8 but not caspase-9activity was inhibited.
The GILZ mRNA and protein analyses showed that this caspase-8-dependent abrogation of Gilz was partially due to increased protein degradationby proteasome. Only the inhibition of proteasome but not lysosomes abrogated, in fact, the reduction in Gilz expression, suggesting that DEX-induced caspase-8 activation could protect DEX-induced GILZ protein from its proteasomal degradation.
The ubiquitin/proteasome pathway is an important machinery involved in the maintenance of cellular homeostasis regulating multiple cellular processes including cell-cycle progression and apoptosis; post-transaltional modification by ubiquitin plays a central role in targeting proteins for this extralysosomalproteolytic degradation.
In addition to ubiquitin, there are several ubiquitin-like proteins (UBLs), such as small ubiquitin-like modifier (SUMO), which has been shown to covalently modify a large number of proteins using an enzymatic pathway similar to the ubiquitination.In some cases, SUMO and ubiquitin may directly compete for modification of the same lysines, thus inhibiting each other’s binding to target proteins.
As a putative SUMO-binding site was identified in the Gilz amino-acid sequence, we hypothesized that the caspase-8 dependent protection of Gilz could be due to caspase-8-driven sumoylation that inhibits ubiquitination on the same lysine acceptor site.
We identified a 30-kDa protein that was compatible with the size of a Gilz–SUMO-1 complex and was recognized by the anti-SUMO-1 and anti-Gilz antibodies. In addition, Gilz bound to Ubc9, the specific SUMO-1 E2-conjugating enzyme, in vitro and coimmunoprecipitated with Ubc9 in vivo. Furthermore, Gilz coimmunoprecipitated with SUMO-1 both in vitro and in vivo, and this interaction depended on caspase-8 activation. This requirement for caspase-8 was further supported by the evaluation of caspase-8-deficientthymocytes and lymphocytes in which Gilz expression was reduced.
In summary, our results suggest that caspase-8 activation protects Gilz from proteasomal degradation and induces its binding to SUMO-1 in GC-treated thymocytes.