| The endoplasmic reticulum (ER) is the site where the synthesized proteins are matured and assembled for them further being delivered to the target organelles, and the homeostasis of the folded and unfolded proteins in ER is the fundamental requirement of the cellular functions. Some extracellular or intracellular insults could result in accumulation of unfolded proteins within ER, leading to distortion of the ER homeostasis and increase of ER stress. In response to ER stress, the coordinately adaptive pathway, so-called the unfolded protein response (UPR), is activated to restore the homeostatic situation. Inositol-requiring protein-1 (IRE1) located on the ER membrane is one of three key UPRs. Although IRE1 has been systematically investigated, there are many mysteries to be explored yet, such as the proteins interacted with IRE1, the downstream of transduction signaling and the possible modifications of IRE1. Setting IRE1 as a studying object, we first surveyed the proteins interacted with IRE1, and further focused on how an interacted IRE1 protein modified IRE1 and regulated the IRE1 relevant signaling. With the effort, we attempted to find and elucidate the new functions and molecular mechanism of IRE1 under ER stress.Using affinity proteomics, we identified several ubiquitination related proteins in the immunoprecipitates of IRE1. We paid a great attention to one of E3 ubiquitination ligases, carboxyl terminus of Hsp70 interacting protein (CHIP), because CHIP occupied relatively high abundance in the IRE1 immunoprecipitates, shared of the binding capacity to Hsp70 with IRE1, and showed some links with oncology. The molecular mechanism underneath CHIP and IRE1, however, remains unclear. In addition, we verified the interaction between IRE1 and CHIP through three approaches. In the cells with normal expression and overexpression of CHIP, the co-existence of IRE1/CHIP was detected by Western blot in the co-immunoprecipitates; using the GST-CHIP as a bait, the endogenous IREl was found to bind with the bait protein; and under confocal microscope, co-localization of IRE1/CHIP was observed.As CHIP is an E3 ubiquitination ligase and is able to interact with IRE1, we inquire to whether IRE1 is a CHIP substrate. Taking geldnamycin (GA) as an ER stress inducer, we found that CHIP could ubiquitinate IRE1, whereas knockdown of CHIP within cells resulted in blocking the IRE1 ubiquitination under ER stress. Moreover, we further sought how the functional sites of CHIP impacted the ubiquitinating to IRE1. The mutation at CHIP K30, a binding site to Hsp70, didn’t alter the IRE1 ubiquitination, nevertheless, the mutation at CHIP H260, a catalytic site, almost abolished the IRE1 ubiquitination within cells.It is generally accepted that the interaction of CHIP/Hsp70 plays a chaperone role, which forms transitional complexes with some proteins and brings the proteins into degradation pathway. Under ER stress condition, we surprisingly observed that CHIP did not accelerate the IREl degradation, whereas benefited the IRE1 stability somehow. It was reported that the different ubiquitin linked manner to protein rendered the substrate functions. We therefore implemented the site-directed mutagenesis to figure out the possible manners of ubiquitin linked with IRE1, and found that IRE1 was ubiquitinated in a K63-linked manner, implying the IRE1 ubiquitination likely to participate in regulation of the IRE1 signaling. Furthermore, we systematically examined how the ubiquitinated IRE1 by CHIP affected its relevant signaling. When CHIP was knockdown, the phosphorylation of IRE1 as well as of the IRE1 downstream effectors, JNK and p38, and the interaction of IRE1/TRAF2 were significantly attenuated. When the deubiquitinase, OTUB1, was transfected into the cells, the IRE1 ubiquitination and its relevant signaling appeared as the similar changes as CHIP knockdown. The evidence demonstrated that CHIP plays an important role to regulate the IRE1 signal pathway. Also, we identified the ubiquitinated sites on IRE1, the ubiqutination at K545 involved in the IRE1 phosphorylation, while the ubiquitination on K828 implicated the interaction of IRE1/TRAF2.The involvement of the IRE1 ubiqutination ligated by CHIP and the IRE1 signaling to the cell behavior was further investigated under ER stress. IRE1 knockdown enhanced the SA-β-gal staining ratio in the cells, and inhibition towards phosphorylation of JNK and p38 also strengthened the senescence signals, indicating that IRE1 could participate in cell senescence. When the CHIP abundance was controlled in cells, we realized that the senescence phenotype was negatively correlated with the CHIP expression level. We thus inferred that under ER stress the ubiquitination of IRE1 converted by CHIP takes part in senescence process.Herein, the interrelationship of CHIP and IRE1 was systematically investigated under ER stress, and IRE1 ubiquitinated by CHIP was identified at the first time. Taking all the evidence together, we have proposed a new model of the IRE1 related signaling, in which the IRE1 ubiquitination directly influences its downstream signal transduction, subsequently regulates the senescence process. |
