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. 2024 Oct 22;16(6):1175-1188.
doi: 10.3390/neurolint16060089.

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

Soho Oyama  1 , Hengsen Zhang  1   2 , Rafia Ferdous  1 , Yuna Tomochika  1 , Bin Chen  1 , Shuyun Jiang  1   3 , Md Shoriful Islam  1 , Md Mahmudul Hasan  1 , Qing Zhai  1 , A S M Waliullah  1 , Yashuang Ping  1 , Jing Yan  1 , Mst Afsana Mimi  1 , Chi Zhang  1 , Shuhei Aramaki  1 , Yusuke Takanashi  4 , Tomoaki Kahyo  1   5 , Yoshio Hashizume  6 , Daita Kaneda  5 , Mitsutoshi Setou  1   7
Affiliations

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

Soho Oyama et al. Neurol Int. .

Abstract

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.

Keywords: huntingtin protein; huntington’s disease; interaction; polyglutamine; sorting; ubiquitin-like 3.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Distribution of UBL3 in the striatum site of postmortem brain tissue in Huntington patients and control (Scale bars: HE-staining 10 mm, 1C2-staining 10 μm, UBL3 left 100 μm and right 50 μm, Arrows: UBL3-Positive Inclusions) The first column provides an overview of hematoxylin- and eosin (HE)-stained striatum regions of the brain. The second column shows the presence of polyglutamine in the nuclei of neurons. Subsequent staining with the UBL3 antibody reveals that UBL3 is distributed as inclusions in the cytoplasm and nuclei of neurons within the striatum of HD patients. In contrast, UBL3 exhibits a more diffuse, dot-like distribution in the striatum of the control sample.
Figure 2
Figure 2
UBL3 interacts with expanded polyglutamine-containing N-terminal huntingtin fragments: (A) Schematic of expanded polyglutamine-containing N-terminal huntingtin fragments (nHTTpolyQ78) and split Gluc-tagged proteins, including NGluc-UBL3, NGluc-UBL3∆5, and nHTTpolyQ78-CGluc. (B,C) Luminescence of the culture medium and cell lysate from the transfected HEK293 cells with various combinations. The luminescence values are presented as mean ± S.D. from three independent experiments. (D) Co-immunoprecipitated Flag-UBL3 and Flag-UBL3∆5 interact with MYC-nHTTpolyQ78. The input lanes represent 1% of the sample before Co-IP, and the Co-IP lanes represent 20% of the Co-IP products.
Figure 3
Figure 3
UBL3 alters the intracellular sorting of expanded polyglutamine-containing N-terminal huntingtin fragments (* p < 0.05, ** p < 0.01, *** p < 0.001, *** p < 0.0001): (A) Schematic of mHTT-HiBiT. (B,C) Luminescence of the culture medium and cell lysate from the transfected HEK293 cells with various combinations. The luminescence ± SD in quadruplicate experiments is shown. (D) Relative ratio of luminescence of culture media divided by cell lysate.
Figure 4
Figure 4
UBL3 colocalizes with expanded polyglutamine-containing N-terminal huntingtin fragments (Scale bars: 10 μm): (A) Immunocytochemistry (ICC)-staining representative images of HEK293 cells transfected or co-transfected with Flag-UBL3, Flag-UBL3∆5, and MYC-HTTpolyQ78. (B) Immunocytochemistry (ICC)-staining representative images of HEK293 cells transfected or co-transfected with Flag-UBL3, Flag-UBL3∆5, and nHTTpolyQ72-HiBiT.
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References

    1. Downes B.P., Saracco S.A., Lee S.S., Crowell D.N., Vierstra R.D. MUBs, a Family of Ubiquitin-fold Proteins That Are Plasma Membrane-anchored by Prenylation. J. Biol. Chem. 2006;281:27145–27157. doi: 10.1074/jbc.M602283200. - DOI - PubMed
    1. Ageta H., Ageta-Ishihara N., Hitachi K., Karayel O., Onouchi T., Yamaguchi H., Kahyo T., Hatanaka K., Ikegami K., Yoshioka Y., et al. UBL3 modification influences protein sorting to small extracellular vesicles. Nat. Commun. 2018;9:3936. doi: 10.1038/s41467-018-06197-y. - DOI - PMC - PubMed
    1. Takanashi Y., Kahyo T., Kamamoto S., Zhang H., Chen B., Ping Y., Mizuno K., Kawase A., Koizumi K., Satou M., et al. Ubiquitin-like 3 as a new protein-sorting factor for small extracellular vesicles. Cell Struct. Funct. 2022;47:1–18. doi: 10.1247/csf.21078. - DOI - PMC - PubMed
    1. Chen B., Hasan M., Zhang H., Zhai Q., Waliullah A.S.M., Ping Y., Zhang C., Oyama S., Mimi M.A., Tomochika Y., et al. UBL3 Interacts with Alpha-Synuclein in Cells and the Interaction Is Downregulated by the EGFR Pathway Inhibitor Osimertinib. Biomedicines. 2023;11:1685. doi: 10.3390/biomedicines11061685. - DOI - PMC - PubMed
    1. Yan J., Zhang H., Tomochika Y., Chen B., Ping Y., Islam S., Aramaki S., Sato T., Nagashima Y., Nakamura T., et al. UBL3 Interaction with α-Synuclein Is Downregulated by Silencing MGST3. Biomedicines. 2023;11:2491. doi: 10.3390/biomedicines11092491. - DOI - PMC - PubMed

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