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The difference in the intracellular Arg/Lys-rich and EHLVY motifs contributes to distinct subcellular distribution of HAI-1 versus HAI-2

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Abstract

The integral membrane, Kunitz-type, serine protease inhibitors, HAI-1 and HAI-2, closely resemble one another structurally and with regard to their specificity and potency against proteases. Structural complementarity between the Kunitz domains and serine protease domains renders the membrane-associated serine proteases, matriptase and prostasin, the primary target proteases of the HAIs. The shared biochemical enzyme–inhibitor relationships are, however, at odds with their behavior at the cellular level, where HAI-1 appears to be the default inhibitor of these proteases and HAI-2 a cell-type-selective inhibitor, even though they are widely co-expressed. The limited motility of these proteins caused by their membrane anchorages may require their co-localization within a certain distance to allow the establishment of a cellular level functional relationship between the proteases and the inhibitors. The differences in their subcellular localization with HAI-1 both inside the cell and on the cell surface, compared to HAI-2 predominately in intracellular granules has, therefore, been implicated in the differential manner of their control of matriptase and prostasin proteolysis. The targeting signals present in the intracellular domains of the HAIs are systematically investigated herein. Studies involving domain swap and point mutation, in combination with immunocytochemistry and cell surface biotinylation/avidin depletion, reveal that the different subcellular localization between the HAIs can largely be attributed to differences in the intracellular Arg/Lys-rich and EHLVY motifs. These intrinsic differences in the targeting signal render the HAIs as two independent rather than redundant proteolysis regulators.

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Acknowledgements

This study was supported by National Cancer Institute (NCI) Grant R01 CA 123223 (to MDJ and CYL), Grant (MND-MAB-110-045) from the Ministry of National Defense Medical Affairs Bureau, Taiwan (to JKW), and Grants (CMNDMC10807; CMNDMC10905) from Chi-Mei Medical Center, Tainan, Taiwan (to S-M Huang). We also acknowledge the assistance provided by the Microscopy and Imaging Shared Resource and the Tissue Culture Shared Resource, which are supported in part by the Lombardi Comprehensive Cancer Center support grant (NIH/NCI grant P30-CA051008). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Authors and Affiliations

  1. Lombardi Comprehensive Cancer Center, Department of Oncology, Georgetown University, W412, W416 Research Building 3970 Reservoir Road NW, Washington, DC, 20057, USA

    Nanxi Huang, Robert B. Barndt, Dajun D. Lu, Qiaochu Wang, Michael D. Johnson & Chen-Yong Lin

  2. Department of Biochemistry, National Defense Medical Center, Taipei, 114, Taiwan, ROC

    Shih-Ming Huang & Jehng-Kang Wang

  3. Division of Hematology/Oncology, Department of Internal Medicine, Tri-Service General Hospital, Taipei, Taiwan, ROC

    Ping-Ying Chang

  4. School of Medicine, National Defense Medical Center, Taipei, Taiwan, ROC

    Ping-Ying Chang, Chao-Yang Chen & Je-Ming Hu

  5. Division of Colorectal Surgery, Department of Surgery, Tri-Service General Hospital Taipei, Taipei, Taiwan, ROC

    Chao-Yang Chen & Je-Ming Hu

  6. Graduate Institute of Medical Sciences, National Defense Medical Center, Taipei, Taiwan, ROC

    Je-Ming Hu

  7. Department of Pharmacy, Chi-Mei Medical Center, Tainan, Taiwan, ROC

    Hui-Chen Su & Chen-Yong Lin

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  1. Nanxi Huang
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Huang, N., Barndt, R.B., Lu, D.D. et al. The difference in the intracellular Arg/Lys-rich and EHLVY motifs contributes to distinct subcellular distribution of HAI-1 versus HAI-2. Human Cell 35, 163–178 (2022). https://doi.org/10.1007/s13577-021-00632-x

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