上田 洋司

Small extracellular vesicles (sEVs) mediate cell-to-cell communication by carrying RNAs and proteins. Ubiquitin-like 3 (UBL3) functions as a posttranslational modification factor, regulating protein sorting to sEVs. Programmed cell death ligand 1 (PD-L1) binds to programmed cell death 1 (PD-1) on immune cells, suppressing their function. Although immune checkpoint inhibitors, anti-PD-L1 and anti-PD-1 antibodies, have improved cancer treatment, efficacy remains limited (~ 25%). Per recent studies, PD-L1-containing sEVs are elevated in cancer patients, contributing to impaired immunotherapy responses. Herein, we discovered that PD-L1 is modified by UBL3 and that its sorting to sEVs is regulated by UBL3. Furthermore, we found that statins, commonly prescribed for hypercholesterolemia, inhibit UBL3 modification, thereby reducing PD-L1 sorting to sEVs. Among patients with a high tumor proportion score, serum levels of PD-L1-containing sEVs were significantly lower in those using statins. Consistently, bioinformatic analysis revealed that UBL3 and PD-L1 expression levels affect lung cancer survival. Integrating statins into existing combination therapies may therefore offer a promising strategy to enhance immunotherapy efficacy.

Transient memories are converted to persistent memories at the synapse and circuit/systems levels. The synapse-level consolidation parallels electrophysiological transition from early- to late-phase long-term potentiation of synaptic transmission (E-/L-LTP). While glutamate signaling upregulations coupled with dendritic spine enlargement are common underpinnings of E-LTP and L-LTP, synaptic mechanisms conferring persistence on L-LTP remain unclear. Here, we show that L-LTP induced at the perforant path-hippocampal dentate gyrus (DG) synapses accompanies cytoskeletal remodeling that involves actin and the septin subunit SEPT3. L-LTP in DG neurons causes fast spine enlargement, followed by SEPT3-dependent smooth endoplasmic reticulum (sER) extension into enlarged spines. Spines containing sER show greater Ca2+ responses upon synaptic input and local synaptic activity. Consistently, Sept3 knockout in mice (Sept3-/-) impairs memory consolidation and causes a scarcity of sER-containing spines. These findings indicate a concept that sER extension into active spines serves as a synaptic basis of memory consolidation.

Exosomes are small extracellular vesicles (sEVs) secreted via multivesicular bodies (MVBs)/late endosomes and mediators of cell-cell communication. We previously reported a novel post-translational modification by ubiquitin-like 3 (UBL3). UBL3 is localized in MVBs and the plasma membrane and released outside as sEVs, including exosomes. Approximately 60% of proteins sorted in sEVs are affected by UBL3 and localized in various organelles, the plasma membrane, and the cytosol, suggesting that its dynamic movement in the cell before entering the MVBs. To examine the intracellular dynamics of UBL3, we constructed a sophisticated visualization system via fusing fluorescent timers that changed from blue to red form over time with UBL3 and by its expression under Tet-on regulation. Intriguingly, we found that after synthesis, UBL3 was initially distributed within the cytosol. Subsequently, UBL3 was localized to MVBs and the plasma membrane and finally showed predominant accumulation in MVBs. Furthermore, by super-resolution microscopy analysis, UBL3 was found to be associated with one of its substrates, α-tubulin, in the cytosol, and the complex was subsequently transported to MVBs. This spatiotemporal visualization system for UBL3 will form a basis for further studies to elucidate when and where UBL3 associates with its substrates/binding proteins before localization in MVBs.

Discovery of novel post-translational modifications provides new insights into changes in protein function, localization, and stability. They are also key elements in understanding disease mechanisms and developing therapeutic strategies. We have previously reported that ubiquitin-like 3 (UBL3) serves as a novel post-translational modifier that is highly expressed in the cerebral cortex and hippocampus, in addition to various other organs, and that 60% of proteins contained in small extracellular vesicles (sEVs), including exosomes, are influenced by UBL3. In this study, we generated transgenic mice expressing biotinylated UBL3 in the forebrain under control of the alpha-CaMKII promoter (Ubl3Tg/+). Western blot analysis revealed that the expression of UBL3 in the cerebral cortex and hippocampus was 6- to 7-fold higher than that in the cerebellum. Therefore, we performed immunoprecipitation of protein extracts from the cerebral cortex of Ubl3+/+ and Ubl3Tg/+ mice using avidin beads to comprehensively discover UBL3 interacting proteins, identifying 35 new UBL3 interacting proteins. Nine proteins were annotated as extracellular exosomes. Gene Ontology (GO) analysis suggested a new relationship between sEVs and RNA metabolism in neurodegenerative diseases. We confirmed the association of endogenous UBL3 with the RNA-binding proteins FUS and HPRT1-both listed in the Neurodegenerative Diseases Variation Database (NDDVD)-and with LYPLA1, which is involved in Huntington's disease, using immunoprecipitation (IP)-western blotting analysis. These UBL3 interacting proteins will accelerate the continued elucidation of sEV research about proteins regulated by novel post-translational modifications by UBL3 in the brain.