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Molecular characterization of wild-type and HSAN2B-linked FAM134B

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Abstract

Background

Family with sequence similarity 134, member B (FAM134B), also known as Reticulophagy regulator 1 (RETREG1), is an ER-phagy receptor involved in ER homeostasis. Congenital mutations in the FAM134B gene have been reported to be associated with hereditary sensory and autonomic neuropathy type 2B (HSAN2B); however, the molecular differences between wild-type and HSAN2B-linked FAM134B are not fully understood.

Methods and results

We prepared several human FAM134B constructs, such as the HSAN2B-linked mutant, and compared their features with those of wild-type FAM134B by transfecting these constructs into FAM134B-deficient Neuro2a cells. Although intrinsic FAM134B protein expression in wild-type Neuro2a cells was affected by the supply of amino acids in the culture medium, the expression of each HSAN2B-linked mutant FAM134B protein was hardly affected by serum and amino acid deprivation. On the other hand, the intracellular localization of GFP-tagged HSAN2B-linked mutants, except for P7Gfs133X, overlapped well with ER-localized SP-RFPKDEL and did not differ from that of GFP-tagged wild-type FAM134B. However, analysis of protein‒protein interactions using the NanoBiT reporter assay revealed the difference between wild-type and C-terminal truncated mutant FAM134B. Furthermore, this NanoBiT assay demonstrated that both wild-type and G216R FAM134B interacted with LC3/GABARAPL1 to the same extent, but the FAM134B construct with mutations near the LC3-interacting region (LIR) did not. Similar to the NanoBiT assay, the C-terminal-truncated FAM134B showed lower ER-phagy activities, as assessed by the cotransfection of GFP-tagged reporters.

Conclusions

We showed that wild-type and HSAN2B-linked FAM134B have different molecular characteristics by transfecting cells with various types of constructs. Thus, this study provides new insights into the molecular mechanisms underlying HSAN2B as well as the regulation of ER-phagy.

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Data availability

The data generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Abbreviations

CRISPR/Cas9:

Clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins 9

ER:

Endoplasmic reticulum

ERAD:

ER stress associated degradation

ERSE:

ER stress response element

FAM134B:

Family with sequence similarity 134, member B

GADD153:

Growth arrest and DNA-damage-inducible protein 153

G3PDH:

Glyceraldehyde 3-phosphate dehydrogenase

HSAN2B:

Hereditary sensory and autonomic neuropathy type 2B

LC3:

Microtubule associated protein 1 light chain 3α

LIR:

LC3-interacting region

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Acknowledgements

This study was partially supported by Grants-in-aid from the Japan Society for the Promotion of Science (JSPS, Japan, KAKENHI, Nos. 19H04030 and 20K21751 to K.O.). We are grateful to Dr. George Church and Dr. Qiming Sun for providing the hCas9, human FAM134B and human Sec61B genes, respectively.

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Authors and Affiliations

  1. Graduate School of Natural Science and Technology, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan

    Akane Kanamori, Shohei Hinaga, Yoko Hirata & Kentaro Oh-hashi

  2. Department of Chemistry and Biomolecular Science, Faculty of Engineering, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan

    Yoko Hirata & Kentaro Oh-hashi

  3. United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan

    Yoko Hirata & Kentaro Oh-hashi

  4. Department of Pain Management and Palliative Care Medicine, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamikyo-Ku, Kyoto, 602-0841, Japan

    Fumimasa Amaya

  5. Center for One Medicine Innovative Translational Research (COMIT), Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan

    Kentaro Oh-hashi

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  1. Akane Kanamori
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  2. Shohei Hinaga
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Contributions

AK, SH and KO performed the experiments; KO and YH confirmed the results; and KO and FA designed and prepared the manuscript.

Corresponding author

Correspondence to Kentaro Oh-hashi.

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Supplementary Information

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11033_2023_8517_MOESM1_ESM.pdf

Supplementary Fig. S1: Establishment of FAM134B-deficient Neuro2a cells. Neuro2a cells were transfected with the constructs for the doner gene, gRNA and hCas9 and selected with the appropriate concentration of hygromycin to establish a FAM134B-deficient line. Expression of the indicated proteins in parental wild-type (wt) Neuro2a and FAM134B-deficient cells was detected as described in the “Materials and methods” section. Supplementary Fig. S2: Measurement of NanoBiT activity of wild-type and G216R FAM134B in live Neuro2a cells. Forty-two hours after transfection of the indicated NanoBiT-tagged genes into FAM134B-deficient cells, the culture medium was replaced with fresh OPTI-MEM. After the addition of diluted NanoBiT reagent into each well, the luciferase activity of each sample was measured as described in the “Materials and methods” section. Each value represents the mean ± SEM from three independent cultures. Supplementary Fig. S3: The cleavage of SP-RFP-GFPKDEL protein by co-transfection of FAM134B in Neuro2a cells. Forty-eight hours after transfection of empty or the indicated Flag-tagged FAM134B gene together with SP-RFP-GFPKDEL into FAM134B-deficient cells, expression of the indicated proteins was measured as described in the “Materials and methods” section. For the evaluation of ER-phagy, the levels of each cleaved GFP fragment were measured using an anti-GFP antibody. The representative result of four independent experiments is shown. Supplementary Fig. S4: Effects of MG132 and CMA treatments on the LC3 expression in cells cultured with serum- and amino acid-starved medium. Wild-type Neuro2a cells were cultured in normal culture medium or serum- and amino acid-starved medium (− S/− AA) supplemented with or without essential amino acids (E-AA) in the presence or absence of MG132 (MG, 10 μM) and CMA (50 nM) for 12 h. The representative result of two independent experiments is shown. (PDF 1248 kb)

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Kanamori, A., Hinaga, S., Hirata, Y. et al. Molecular characterization of wild-type and HSAN2B-linked FAM134B. Mol Biol Rep 50, 6005–6017 (2023). https://doi.org/10.1007/s11033-023-08517-y

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  • DOI: https://doi.org/10.1007/s11033-023-08517-y

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