UBL3 Interacts with PolyQ-Expanded Huntingtin Fragments and Modifies Their Intracellular Sorting.

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.

Alpha-Synuclein Interaction with UBL3 Is Upregulated by Microsomal Glutathione S-Transferase 3, Leading to Increased Extracellular Transport of the Alpha-Synuclein under Oxidative Stress.

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.

UBL3 Interaction with α-Synuclein Is Downregulated by Silencing MGST3.

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.

UBL3 Interacts with Alpha-Synuclein in Cells and the Interaction Is Downregulated by the EGFR Pathway Inhibitor Osimertinib.

Ubiquitin-like 3 (UBL3) acts as a post-translational modification (PTM) factor and regulates protein sorting into small extracellular vesicles (sEVs). sEVs have been reported as vectors for the pathology propagation of neurodegenerative diseases, such as α-synucleinopathies. Alpha-synuclein (α-syn) has been widely studied for its involvement in α-synucleinopathies. However, it is still unknown whether UBL3 interacts with α-syn, and is influenced by drugs or compounds. In this study, we investigated the interaction between UBL3 and α-syn, and any ensuing possible functional and pathological implications. We found that UBL3 can interact with α-syn by the Gaussia princeps based split luciferase complementation assay in cells and immunoprecipitation, while cysteine residues at its C-terminal, which are considered important as PTM factors for UBL3, were not essential for the interaction. The interaction was upregulated by 1-methyl-4-phenylpyridinium exposure. In drug screen results, the interaction was significantly downregulated by the treatment of osimertinib. These results suggest that UBL3 interacts with α-syn in cells and is significantly downregulated by epidermal growth factor receptor (EGFR) pathway inhibitor osimertinib. Therefore, the UBL3 pathway may be a new therapeutic target for α-synucleinopathies in the future.

Tubulin Polyglutamylation by TTLL1 and TTLL7 Regulate Glutamate Concentration in the Mice Brain.

As an important neurotransmitter, glutamate acts in over 90% of excitatory synapses in the human brain. Its metabolic pathway is complicated, and the glutamate pool in neurons has not been fully elucidated. Tubulin polyglutamylation in the brain is mainly mediated by two tubulin tyrosine ligase-like (TTLL) proteins, TTLL1 and TTLL7, which have been indicated to be important for neuronal polarity. In this study, we constructed pure lines of Ttll1 and Ttll7 knockout mice. Ttll knockout mice showed several abnormal behaviors. Matrix-assisted laser desorption/ionization (MALDI) Imaging mass spectrometry (IMS) analyses of these brains showed increases in glutamate, suggesting that tubulin polyglutamylation by these TTLLs acts as a pool of glutamate in neurons and modulates some other amino acids related to glutamate.

A New Potential Therapeutic Target for Cancer in Ubiquitin-Like Proteins-UBL3.

Ubiquitin-like proteins (Ubls) are involved in a variety of biological processes through the modification of proteins. Dysregulation of Ubl modifications is associated with various diseases, especially cancer. Ubiquitin-like protein 3 (UBL3), a type of Ubl, was revealed to be a key factor in the process of small extracellular vesicle (sEV) protein sorting and major histocompatibility complex class II ubiquitination. A variety of sEV proteins that affects cancer properties has been found to interact with UBL3. An increasing number of studies has implied that UBL3 expression affects cancer cell growth and cancer prognosis. In this review, we provide an overview of the relationship between various Ubls and cancers. We mainly introduce UBL3 and its functions and summarize the current findings of UBL3 and examine its potential as a therapeutic target in cancers.

Cinnamomum verum J. Presl Bark Contains High Contents of Nicotinamide Mononucleotide.

The global population is aging, and intervention strategies for anti-aging and the prevention of aging-related diseases have become a topic actively explored today. Nicotinamide adenine dinucleotide (NAD+) is an important molecule in the metabolic process, and its content in tissues and cells decreases with age. The supplementation of nicotinamide mononucleotide (NMN), an important intermediate and precursor of NAD+, has increased NAD+ levels, and its safety has been demonstrated in rodents and human studies. However, the high content of NMN in natural plants has not been fully explored as herbal medicines for drug development. Here, we identified that the leaf of Cinnamomum verum J. Presl (C. verum) was the highest NMN content among the Plant Extract Library (PEL) with food experience, using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). To validate this result, the extraction and quantitative analysis of bark, leaf, root, and stem of fresh C. verum was conducted. The results revealed that the bark had the highest NMN content in C. verum (0.471 mg/100 g). Our study shed light on the prospects of developing natural plants in the context of NMN as drugs for anti-aging and prevention of aging-related diseases. The future should focus on the development and application of C. verum pharmaceutical formulations.

Ubiquitin-like 3 as a new protein-sorting factor for small extracellular vesicles.

Ubiquitin-like 3 (UBL3) is a well-conserved ubiquitin-like protein (UBL) in eukaryotes and regulates the ubiquitin cascade, but the significant roles of UBL3 in cellular processes remained unknown. Recently, UBL3 was elucidated to be a post-translational modification factor that promotes protein sorting to small extracellular vesicles (sEVs). Proteins sorted into sEVs have been studied as etiologies of sEV-related diseases. Also, there have been attempts to construct drug delivery systems (DDSs) by loading proteins into sEVs. In this review, we introduce the new concept that UBL3 has a critical role in the protein-sorting system and compare structure conservation between UBL3 and other UBLs from an evolutionary perspective. We conclude with future perspectives for the utility of UBL3 in sEV-related diseases and DDS.Key words: UBL3, small extracellular vesicles, protein sorting, ubiquitin-like protein, post-translational modification.