Nondegradative Ubiquitination of Apoptosis Inducing Factor (AIF) by X-Linked Inhibitor of Apoptosis at a Residue Critical for AIF-Mediated Chromatin Degradation

Abstract

Apoptosis inducing factor (AIF) is a mediator of caspase-independent cell death that is also necessary for mitochondrial energy production. How these seemingly opposite cellular functions of AIF are controlled is poorly understood. X-linked inhibitor of apoptosis (XIAP) is an endogenous inhibitor of caspases that also regulates several caspase-independent signaling pathways. The RING domain of XIAP possesses E3 ubiquitin ligase activity, though the importance of this function to signal regulation remains incompletely defined. XIAP binds and ubiquitinates AIF, and in this study, we determined the functional consequences of XIAP-mediated AIF ubiquitination. Unlike canonical ubiquitination, XIAP-dependent AIF ubiquitination did not lead to proteasomal degradation of AIF. Experiments using ubiquitin mutants demonstrated that the XIAP-dependent ubiquitin linkage was not formed through the commonly used lysine 48, suggesting a noncanonical ubiquitin linkage is employed. Further studies demonstrated that only lysine 255 of AIF was a target of XIAP-dependent ubiquitination. Using recombinant AIF, we determined that mutating lysine 255 of AIF interferes with the ability of AIF not only to bind DNA but also to degrade chromatin in vitro. These data indicate that XIAP regulates the death-inducing activity of AIF through nondegradative ubiquitination, further defining the role of XIAP in controlling AIF and caspase-independent cell death pathways.

Defining NADH-Driven Allostery Regulating Apoptosis-Inducing Factor

“…Binding and import of mitochondrial import factor CHCHD4 relies on AIF-CTC and may enable AIF-CTC architectures via protein interaction (Hangen et al, 2015). In contrast, cell death regulator heat-shock protein 70 (Hsp70) (Schmitt et al., 2003) and E3-ubiquitin ligase X-linked inhibitor of apoptosis protein (XIAP) (Lewis et al, 2011) target residues near AIF’s Cβ-clasp (K192, K194) and dimerization interface (K255), respectively, suggesting a requirement for the closed, monomeric architecture of oxidized AIF. The recent discovery of added mitochondrial binding partners of AIF (Lenhausen et al, 2016; Nakao et al, 2015) and a PARP-1-dependent AIF-associated nuclease (Wang et al., 2016) opens further opportunities for finding how the mechanism for architectural switching defined here regulates AIF’s mitochondrial and cell death functions in vivo .…”

The Enzymatic Activity of Apoptosis-inducing Factor Supports Energy Metabolism Benefiting the Growth and Invasiveness of Advanced Prostate Cancer Cells

“…A second possibility is that the death-inducing activity of AIF is blocked by up-regulation of inhibitory molecules. We have recently shown that through a ubiquitinationdependent mechanism, the pro-survival protein X-linked inhibitor of apoptosis (XIAP) is capable of preventing AIF-mediated DNA degradation (56). XIAP is also elevated in prostate cancer (35,57), raising the possibility that XIAP blocks AIF death induction and allows the mitochondrial functions of AIF to support cancer cell survival.…”

The TREX1 C-terminal Region Controls Cellular Localization through Ubiquitination

“…All constructs were confirmed by sequencing (GENEWIZ). The pCW7-His-Myc-ubiquitin plasmids (WT, K6R, K11R, K27R, K29R, K48R, and K63R) have been described (43).…”

“…Cell Lysis and Immunoprecipitation-Cell lysates were prepared in radioimmune precipitation assay buffer, urea lysis buffer, or CHAPS buffer (43,44). All lysis buffers were supplemented with 1 mM PMSF and one Complete Mini protease inhibitor mixture tablet (Roche Applied Science) prior to use.…”

“…Beads were recovered by centrifugation and washed with CHAPS buffer, and precipitated proteins were eluted by the addition of NuPAGE lithium dodecyl sulfate sample buffer (Novex) and heating for 5 min at 95°C. Urea lysates for nickel-nitrilotriacetic acid (Ni-NTA) precipitation were prepared as described (43). Immunoprecipitations were performed multiple times from independent transfections with similar results.…”