Ubr1-induced selective endo-phagy/autophagy protects against the endosomal and Ca2+-induced proteostasis disease stress

The defence mechanisms against endo-lysosomal homeostasis stress remain incompletely understood. Here, we identify Ubr1 as a protein quality control (QC) ubiquitin ligase that counteracts proteostasis stress by enhancing cargo selective autophagy for lysosomal degradation. Astrocyte regulatory cluster membrane protein MLC1 mutations increased intracellular Ca2+ and caused endosomal compartment stress by fusion and enlargement. Endosomal protein QC pathway using ubiquitin QC ligases CHIP and Ubr1 with ESCRT-machinery was able to target only a fraction of MLC1-mutants for lysosomal degradation. As a consequence of the endosomal stress, we found an alternative QC route dependent on Ubr1, SQSTM1/p62 and arginylation to bypass MLC1-mutants to endosomal autophagy (endo-phagy). Significantly, this unfolded a general biological endo-lysosomal QC pathway for arginylated Ubr1-SQSTM1/p62 autophagy targets during Ca2+-assault. Conversely, the loss of Ubr1 with the absence of arginylation elicited endosomal compartment stress. These findings underscore the critical housekeeping role of Ubr1-dependent endo-phagy/autophagy in constitutive and provoked endo-lysosomal proteostasis stress, and link Ubr1 to Ca2+-homeostasis and proteins implicated in various diseases including cancers and brain disorders.

Brain I3 Binding Protein regulates K-Ras4B membrane localization and signaling

Membrane localization of Ras proteins is necessary for their biological functions and oncogenic activity. We report here on the identification of Brain I3 Binding Protein (BRI3BP) as a novel binding partner for Ras. We show that K-Ras4B plasma membrane localization and biological function are reduced in the absence of BRI3BP. BRI3BP interacts with K-Ras4B and K-Ras4A and our data suggest that BRI3BP operates within the recycling endosomal compartment to regulate K-Ras localization to the plasma membrane. This study uncovers a new regulatory protein for Ras membrane localization.

Dynamic control of nucleic-acid-sensing Toll-like receptors by the endosomal compartment

Nucleic acid (NA)-sensing Toll-like receptors (TLRs) are synthesized in the endoplasmic reticulum and mature with chaperones, such as Unc93B1 and the protein associated with TLR4 A (PRAT4A)–gp96 complex. The TLR–Unc93B1 complexes move to the endosomal compartment, where proteases such as cathepsins activate their responsiveness through proteolytic cleavage of the extracellular domain of TLRs. Without proteolytic cleavage, ligand-dependent dimerization of NA-sensing TLRs is prevented by the uncleaved loop in the extracellular domains. Additionally, the association of Unc93B1 inhibits ligand-dependent dimerization of TLR3 and TLR9 and, therefore, Unc93B1 is released from these TLRs before dimerization. Ligand-activated NA-sensing TLRs induce the production of proinflammatory cytokines and act on the endosomal compartment to initiate anterograde trafficking to the cell periphery for type I interferon production. In the endosomal compartment, DNA and RNA are degraded by DNases and RNases, respectively, generating degradation products. DNase 2A and RNase T2 generate ligands for TLR9 and TLR8, respectively. In this mechanism, DNases and RNases control innate immune responses to NAs in endosomal compartments. NA-sensing TLRs and the endosomal compartment work together to monitor environmental cues through endosomes and decide to launch innate immune responses.

Endosomal PI(3)P regulation by the COMMD/CCDC22/CCDC93 (CCC) complex controls membrane protein recycling

Protein recycling through the endolysosomal system relies on molecular assemblies that interact with cargo proteins, membranes, and effector molecules. Among them, the COMMD/CCDC22/CCDC93 (CCC) complex plays a critical role in recycling events. While CCC is closely associated with retriever, a cargo recognition complex, its mechanism of action remains unexplained. Herein we show that CCC and retriever are closely linked through sharing a common subunit (VPS35L), yet the integrity of CCC, but not retriever, is required to maintain normal endosomal levels of phosphatidylinositol-3-phosphate (PI(3)P). CCC complex depletion leads to elevated PI(3)P levels, enhanced recruitment and activation of WASH (an actin nucleation promoting factor), excess endosomal F-actin and trapping of internalized receptors. Mechanistically, we find that CCC regulates the phosphorylation and endosomal recruitment of the PI(3)P phosphatase MTMR2. Taken together, we show that the regulation of PI(3)P levels by the CCC complex is critical to protein recycling in the endosomal compartment.

Co-delivery of D-(KLAKLAK)2 Peptide and Chlorin e6 using a Liposomal Complex for Synergistic Cancer Therapy

Nanotechnology-based photo-chemo combination therapy has been extensively investigated to improve therapeutic outcomes in anticancer treatment. Specifically, with the help of a singlet oxygen generated by the photosensitizer, the endocytosed nanoparticles are allowed to escape from the endosomal compartment, which is currently an obstacle in nanotechnology-based anticancer therapy. In this study, a liposomal complex system (Lipo (Pep, Ce6)), composed of a chlorin e6-conjugated di-block copolymer (PEG-PLL(-g-Ce6)) and a D-(KLAKLAK)2 peptide loading liposome (Lipo (Pep)), was developed and evaluated for its anticancer activity. Due to the membrane lytic ability of the D-(KLAKLAK)2 peptide and the membrane disruptive effect of the singlet oxygen generated from chlorin e6, Lipo (Pep, Ce6) accelerated the disruption of the endosomal compartment, and exhibited strong synergistic anticancer activity in vitro. The prepared liposomal complex system could potentially maximize the efficacy of the nanotechnology-based photo-chemo combination therapy, and can be regarded as a novel, versatile strategy in advanced tumor therapy.