Site-specific ubiquitination of MLKL targets it to endosomes and targets Listeria and Yersinia to the lysosomes.

Phosphorylation of the pseudokinase mixed lineage kinase domain-like protein (MLKL) by the protein kinase RIPK3 targets MLKL to the cell membrane, where it triggers necroptotic cell death. We report that conjugation of K63-linked polyubiquitin chains to distinct lysine residues in the N-terminal HeLo domain of phosphorylated MLKL (facilitated by the ubiquitin ligase ITCH that binds MLKL via a WW domain) targets MLKL instead to endosomes. This results in the release of phosphorylated MLKL within extracellular vesicles. It also prompts enhanced endosomal trafficking of intracellular bacteria such as Listeria monocytogenes and Yersinia enterocolitica to the lysosomes, resulting in decreased bacterial yield. Thus, MLKL can be directed by specific covalent modifications to differing subcellular sites, whence it signals either for cell death or for non-deadly defense mechanisms.

Augmented Reality Assisted Surgical Navigation System for Epidural Needle Intervention.

An augmented reality (AR)-assisted surgical navigation system was developed for epidural needle intervention. The system includes three components: a virtual reality-based surgical planning software, a patient and tool tracking system, and an AR-based surgical navigation system. A three-dimensional (3D) path plan for the epidural needle was established on the preoperative computed tomography (CT) image. The plan is then registered to the intraoperative space by 3D models of the target vertebrae using skin markers and real-time tracking information. In the procedure, the plan and tracking information are transmitted to the head-mounted display (HMD) through a wireless network such that the device directly visualizes the plan onto the back surface of the patient. The physician determines the entry point and inserts the needle into the target based on the direct visual guidance of the system. An experiment was conducted to validate the system using two torso phantoms that mimic human respiration. The experimental results demonstrated that the time and the number of X-rays required for needle insertion were significantly decreased by the proposed method (43.6±20.55sec, 2.9±1.3times) compared to those of the conventional fluoroscopy-guided approach (124.5 ± 46.7s, 9.3±2.4times), whereas the average targeting errors were similar in both cases. The proposed system may potentially decrease ionizing radiation exposure not only to the patient but also to the medical team.

Reduction in MLKL-mediated endosomal trafficking enhances the TRAIL-DR4/5 signal to increase cancer cell death.

Mixed lineage kinase domain-like (MLKL) is an essential molecule of necroptosis, a cell death process that is initiated by direct disruption of the plasma membrane. During necroptosis, MLKL is phosphorylated by receptor interacting protein kinase-3 (RIPK3 or RIP3), and then translocates to the plasma membrane to disrupt membrane integrity. Recent data suggest that MLKL also has a RIP3-indendent function in the generation of intraluminal and extracellular vesicles (EVs), as well as in myelin sheath breakdown when promoting sciatic nerve regeneration. Here we show that depletion of MLKL enhances TRAIL-induced cell death in a RIP3-independent manner. Depletion of MLKL leads to prolonged cytotoxic signals that increase TRAIL-induced cell death. Initially, TRAIL binds to DR5 at the cell surface and is endocytosed at similar rates in MLKL-expressing and MLKL-depleted cells, eventual degradation of intracellular TRAIL by the lysosome is delayed in MLKL-depleted cells, corresponding with prolonged/enhanced intracellular signals such as p-ERK and p-p38 in these cells. Colocalization of TRAIL with the marker of early endosomes, EEA1 suggests that TRAIL is accumulated in early endosomes in MLKL-depleted cells compared to MLKL-expressing cells. This indicates that depletion of MLKL reduces receptor-ligand endosomal trafficking leading to increased TRAIL-cytotoxicity. An MLKL mutant that compromises its necroptotic function and its function in the generation of EVs was sufficient to rescue MLKL deficiency, suggesting that the N-terminal structural elements necessary for these functions are not required for the function of MLKL in the intracellular trafficking associated with regulating death receptor cytotoxicity. A reduction in MLKL expression in cancer cells would therefore be expected to result in enhanced TRAIL-induced therapeutic efficacy.

MLKL, the Protein that Mediates Necroptosis, Also Regulates Endosomal Trafficking and Extracellular Vesicle Generation.

Activation of the pseudokinase mixed lineage kinase domain-like (MLKL) upon its phosphorylation by the protein kinase RIPK3 triggers necroptosis, a form of programmed cell death in which rupture of cellular membranes yields release of intracellular components. We report that MLKL also associated with endosomes and controlled the transport of endocytosed proteins, thereby enhancing degradation of receptors and ligands, modulating their induced signaling and facilitating the generation of extracellular vesicles. This role was exerted on two quantitative grades: a constitutive one independent of RIPK3, and an enhanced one, triggered by RIPK3, where the association of MLKL with the endosomes was enhanced, and it was found to bind endosomal sorting complexes required for transport (ESCRT) proteins and the flotillins and to be excluded, together with them, from cells within vesicles. We suggest that release of phosphorylated MLKL within extracellular vesicles serves as a mechanism for self-restricting the necroptotic activity of this protein.

IEX-1-induced cell death requires BIM and is modulated by MCL-1.

MCL-1 (myeloid cell leukemia-1) is a distinguished and pivotal member of the pro-survival BCL-2 family of proteins, and we isolated IEX-1 (immediate early response gene X-1) as a MCL-1-interacting protein using the yeast two-hybrid system and confirmed their endogenous association in human cells. The underlying mechanisms by which IEX-1 affects cell survival and death are largely unknown. Ectopic expression of IEX-1-induced caspase-dependent apoptosis in 293T cells, and the response was significantly modulated by changes in the MCL-1 expression level in cells. Forced expression of IEX-1 was unable to induce cell death or to perturb mitochondrial membrane potential in BIM-depleted cells. Additionally, knockouts of NOXA or PUMA did not affect the activities of IEX-1, indicating that the pro-death action of IEX-1 specifically requires BIM. Our findings provide insight into a new regulatory circuit that controls cell death and survival by the coordinated action of MCL-1, IEX-1, and BIM.

HIP1R interacts with a member of Bcl-2 family, BCL2L10, and induces BAK-dependent cell death.

The Bcl-2 family members are evolutionally conserved and crucial regulators of apoptosis. BCL2L10 (human Diva or BCL-B) is a member of the Bcl-2 family that has contradictory functions in apoptosis. In the present study, we identified the Huntington-interacting protein 1-related (HIP1R) protein following a search for Diva-interacting proteins using the yeast two-hybrid system. HIP1R is a multi-domain protein that regulates the clathrin-mediated endocytic machinery and actin assembly in cells. Interaction of endogenous proteins of BCL2L10 and HIP1R in 293T cells was determined by immunoprecipitation, and their direct association was confirmed by the Far-Western analysis. The deletion of both the AP180-homology (ANTH) and F-actin-binding the talin-HIP1/R/Sla2p actin-tethering C-terminal homology (THATCH) domains of HIP1R greatly compromised its binding ability to BCL2L10. Ectopic expression of HIP1R resulted in moderate cell death of 293T cells in conjunction with the dissipation of mitochondrial membrane potential and caspase 9 activation. A member of proapoptotic Bcl-2 family, BAK, was required for HIP1R to induce cell death, while BAX was dispensable. In addition, BCL2L10 was associated with endogenous caspase 9, and their binding was augmented by HIP1R overexpression. Thus, this study provided the previously unknown function of HIP1R involved in the intrinsic cell death pathway and further explored possible mechanisms by which HIP1R induces cell death.

Differential expression of clusterin according to histological type of endometrial carcinoma.

OBJECTIVE: Clusterin expression has been associated with various malignancies. Endometrial carcinoma is divided into endometrioid and papillary serous type carcinoma according to the histological characteristics and regarding to the unopposed estrogenic stimulation. In this study, we investigated the expression profiles of clusterin according to the histological types and the effect of estrogen stimulation on its expression in endometrial carcinoma. METHOD: Clusterin expression in endometrial carcinoma tissues was examined by RT-PCR, Western blot analysis, and immunohistochemistry. 63 endometrioid and 10 papillary serous types of fresh cases and 81 endometrioid and 7 papillary serous types of paraffin-embedded cases were studied. Regulation of clusterin expression by beta-estradiol in HEC-1B and HEC-1A cells was investigated using RT-PCR and Western blot analysis. Cell proliferative function of clusterin was examined in 293T cells. RESULTS: We found higher expression of clusterin in endometrioid compared to papillary serous carcinoma using both immunohistochemistry (p=0.033) and Western blot analysis (p=0.024). The mRNA and protein expressions of clusterin in endometrioid carcinoma were higher than in benign endometrium (p=0.002). Forced expression of clusterin promoted 293T cell survival in a concentration-dependent manner, and estradiol treatment increased clusterin expression in HEC-1B but not in HEC-1A cells. CONCLUSIONS: These data suggest that clusterin expression is related to endometrioid carcinoma of endometrium, in which estrogen is involved in the regulatory network of clusterin.

NM23-H2 involves in negative regulation of Diva and Bcl2L10 in apoptosis signaling.

The Bcl-2 family members are evolutionally conserved and crucial regulators of apoptosis. Diva (Boo), an ortholog of Bcl2L10 or Bcl-B, is a member of the Bcl-2 family that has contradictory functions in apoptosis. To understand the signaling mechanisms of Diva, we searched for proteins that interact with Diva using the yeast two-hybrid system. We identified a nucleoside diphosphate kinase isoform, NM23-H2. Here, we show that Diva bound to NM23-H2 in cells in which the transmembrane domain of Diva was required, and both proteins were colocalized in cytoplasm. Of interest, Diva protein level was significantly down-regulated by NM23-H2 as knock down of NM23-H2 restored Diva expression. Overexpression of NM23-H2 induced apoptosis, and the depletion of NM23-H2 led to the increase of Diva's apoptotic activity. Thus, these results indicate the existence of a previously undiscovered mechanism by which NM23-H2 involves in the regulation of Diva-mediated apoptosis.

Transcriptional factor FOXL2 interacts with DP103 and induces apoptosis.

Blepharophimosis-ptosis-epicanthus inversus syndrome type I is an autosomal disorder caused by mutations in FOXL2 gene and associated with premature ovarian failure in women by a dominant inheritance. FOXL2 is a recently identified protein that belongs to forkhead family transcription factor, of which signaling pathways are still unknown. Here, we show that FOXL2 induces apoptosis in both Chinese hamster ovary cells and rat granulosa cells, and it interacts with DP103, a DEAD box-containing protein. Overexpression of DP103 itself did not affect cell viability while its coexpression with FOXL2 led to the potentiation of cell death. Our results present previously undiscovered functions of these proteins, an apoptotic activity of FOXL2 in the ovary and a modulating activity of DP103 by interacting with FOXL2.