Ubiquitin-like 3 (UBL3) is a membrane-anchored protein that plays a crucial role in sorting proteins into small extracellular vesicles. Aggregations of alpha-synuclein (α-syn) are associated with the pathology of neurodegenerative diseases such as Parkinson’s disease. Recently, the interaction between UBL3 and α-syn was discovered, with potential implications in clearing excess α-syn from neurons and its role in disease spread. However, the regulator that can mediate the interaction between UBL3 and α-syn remains unclear. In this study, using the split gaussian luciferase complementation assay and RNA interference technology, we identified that QSOX2, HTATIP2, UBE3C, MGST3, NSF, HECTD1, SAE1, and ATG3 were involved in downregulating the interaction between UBL3 and α-syn. Notably, silencing MGST3 had the most significant impact. Immunocytochemistry staining confirmed the impact of MGST3 silencing on the co-localization of UBL3 and α-syn in cells. MGST3 is a part of the antioxidant system, and silencing MGST3 is believed to contribute to oxidative stress. We induced oxidative stress with hydrogen peroxide, observing its effect on the UBL3-α-syn interaction, and showing that 800 µM of H₂O₂ downregulated this interaction. In conclusion, silencing MGST3 downregulates the interaction between UBL3 and α-syn.

Aberrant aggregation of misfolded alpha-synuclein (α-syn), a major pathological hallmark of related neurodegenerative diseases such as Parkinson’s disease (PD), can translocate between cells. Ubiquitin-like 3 (UBL3) is a membrane-anchored ubiquitin-fold protein and post-translational modifier. UBL3 promotes protein sorting into small extracellular vesicles (sEVs) and thereby mediates intercellular communication. Our recent studies have shown that α-syn interacts with UBL3 and that this interaction is downregulated after silencing microsomal glutathione S-transferase 3 (MGST3). However, how MGST3 regulates the interaction of α-syn and UBL3 remains unclear. In the present study, we further explored this by overexpressing MGST3. In the split Gaussia luciferase complementation assay, we found that the interaction between α-syn and UBL3 was upregulated by MGST3. While Western blot and RT-qPCR analyses showed that silencing or overexpression of MGST3 did not significantly alter the expression of α-syn and UBL3, the immunocytochemical staining analysis indicated that MGST3 increased the co-localization of α-syn and UBL3. We suggested roles for the anti-oxidative stress function of MGST3 and found that the effect of MGST3 overexpression on the interaction between α-syn with UBL3 was significantly rescued under excess oxidative stress and promoted intracellular α-syn to extracellular transport. In conclusion, our results demonstrate that MGST3 upregulates the interaction between α-syn with UBL3 and promotes the interaction to translocate intracellular α-syn to the extracellular. Overall, our findings provide new insights and ideas for promoting the modulation of UBL3 as a therapeutic agent for the treatment of synucleinopathy-associated neurodegenerative diseases.

Background/Objectives: UBL3 (Ubiquitin-like 3) is a protein that plays a crucial role in post-translational modifications, particularly in regulating protein transport within small extracellular vesicles. While previous research has predominantly focused on its interactions with α-synuclein, this study investigates UBL3’s role in Huntington’s disease (HD). HD is characterized by movement disorders and cognitive impairments, with its pathogenesis linked to toxic, polyglutamine (polyQ)-expanded mutant huntingtin fragments (mHTT). However, the mechanisms underlying the interaction between UBL3 and mHTT remain poorly understood. Methods: To elucidate this relationship, we performed hematoxylin and eosin (HE) staining and immunohistochemistry (IHC) on postmortem brain tissue from HD patients. Gaussia princeps-based split-luciferase complementation assay and co-immunoprecipitation were employed to confirm the interaction between UBL3 and mHTT. Additionally, we conducted a HiBiT lytic detection assay to assess the influence of UBL3 on the intracellular sorting of mHTT. Finally, immunocytochemical staining was utilized to validate the colocalization and distribution of these proteins. Results: Our findings revealed UBL3-positive inclusions in the cytoplasm and nuclei of neurons throughout the striatum of HD patients. We discovered that UBL3 colocalizes and interacts with mHTT and modulates its intracellular sorting. Conclusions: These results suggest that UBL3 may play a significant role in the interaction and sorting of mHTT, contributing to the understanding of its potential implications in the pathophysiology of Huntington’s disease.

Acute and chronic inflammation are common in patients with end-stage kidney disease (ESKD). So, the adsorption of pro-inflammatory cytokines by the hollow fiber of the dialysis membrane has been expected to modify the inflammatory dysregulation in ESKD patients. However, it remains to be determined in detail what molecules of fiber materials can preferably adsorb proteins from the circulating circuit. We aimed this study to analyze directly the adsorbed proteins in the polymethyl methacrylate (PMMA) and polyethersulfone (PES) membranes in patients on predilution online hemodiafiltration (OL-HDF). To compare the adsorbed proteins in the PMMA and PES hemodiafilters membrane, we initially performed predilution OL-HDF using the PES (MFX-25Seco) membrane while then switched to the PMMA (PMF™-A) membrane under the same condition in three patients. We extracted proteins from the collected hemodiafilters by extraction, then SDS-PAGE of the extracted sample, protein isolation, in-gel tryptic digestion, and nano-LC MS/MS analyses. The concentrations of adsorbed proteins from the PMMA and PES membrane extracts were 35.6±7.9 μg/μL and 26.1±9.2 μg/μL. SDS-PAGE analysis revealed distinct variations of adsorbed proteins mainly in the molecular weight between 10 to 25 kDa. By tryptic gel digestion and mass spectrometric analysis, the PMMA membrane exhibited higher adsorptions of β2 microglobulin, dermcidin, retinol-binding protein-4, and lambda-1 light chain than those from the PES membrane. In contrast, amyloid A-1 protein was adsorbed more potently in the PES membrane. Western blot analyses revealed that the PMMA membrane adsorbed interleukin-6 (IL-6) approximately 5 to 118 times compared to the PES membrane. These findings suggest that PMMA-based OL-HDF therapy may be useful in controlling inflammatory status in ESKD patients.