Altered chaperone–nonmuscle myosin II interactions drive pathogenicity of the UNC45A c.710T>C variant in osteo-oto-hepato-enteric syndrome
Stephanie Waich, Karin Kreidl, Julia Vodopiutz, Arzu Meltem Demir, Adam R. Pollio, Vojtěch Dostál, Kristian Pfaller, Marianna Parlato, Nadine Cerf-Bensussan, Rüdiger Adam, Georg F. Vogel, Holm H. Uhlig, Frank M. Ruemmele, Thomas Müller, Michael W. Hess, Andreas R. Janecke, Lukas A. Huber, Taras Valovka
Stephanie Waich, Karin Kreidl, Julia Vodopiutz, Arzu Meltem Demir, Adam R. Pollio, Vojtěch Dostál, Kristian Pfaller, Marianna Parlato, Nadine Cerf-Bensussan, Rüdiger Adam, Georg F. Vogel, Holm H. Uhlig, Frank M. Ruemmele, Thomas Müller, Michael W. Hess, Andreas R. Janecke, Lukas A. Huber, Taras Valovka
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Research Article Cell biology Genetics

Altered chaperone–nonmuscle myosin II interactions drive pathogenicity of the UNC45A c.710T>C variant in osteo-oto-hepato-enteric syndrome

Abstract

The osteo-oto-hepato-enteric (O2HE) syndrome is a severe autosomal recessive disease ascribed to loss-of-function mutations in the Unc-45 myosin chaperone A (UNC45A) gene. The clinical spectrum includes bone fragility, hearing loss, cholestasis, and life-threatening diarrhea associated with microvillus inclusion disease–like enteropathy. Here, we present molecular and functional analysis of the UNC45A c.710T>C (p.Leu237Pro) missense variant, which revealed a unique pathogenicity compared with other genetic variants causing UNC45A deficiency. The UNC45A p.Leu237Pro mutant retained chaperone activity, prevented myosin aggregation, and supported proper nonmuscle myosin II (NMII) filament formation in patient fibroblasts and human osteosarcoma (U2OS) cells. However, the mutant formed atypically stable oligomers and prevented chaperone-myosin complex dissociation, thereby inhibiting NMII functions. Similar to biallelic UNC45A deficiency, this resulted in impaired intracellular trafficking, defective recycling, and abnormal retention of transferrin at various endocytic sites. In particular, coexpression of wild-type protein attenuated the pathogenic effects of the variant by inhibiting excessive oligomer formation. Our results elucidate the pathogenic mechanisms and recessive characteristics of this variant and may aid in the development of targeted therapies.

Authors

Stephanie Waich, Karin Kreidl, Julia Vodopiutz, Arzu Meltem Demir, Adam R. Pollio, Vojtěch Dostál, Kristian Pfaller, Marianna Parlato, Nadine Cerf-Bensussan, Rüdiger Adam, Georg F. Vogel, Holm H. Uhlig, Frank M. Ruemmele, Thomas Müller, Michael W. Hess, Andreas R. Janecke, Lukas A. Huber, Taras Valovka

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Figure 3

Epifluorescence microscopy showing the UNC45A mutant to retain chaperone activity and support NMIIA folding and assembly.

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Epifluorescence microscopy showing the UNC45A mutant to retain chaperone...
(A) Patient P2.1 and control fibroblasts were treated either with vehicle (DMSO) or with 10 μM MG-132 proteasome inhibitor for 4 hours and stained with an antibody recognizing NMIIA. Normal NMIIA patterns were seen under both experimental conditions in all genotypes. Representative image sections were 10× magnified. Nuclei were detected with HOECHST. Scale bars, 20 μm. (B) U2OS parental, UNC45A-KO, and stably complemented UNC45A wild-type and mutant cells, treated with 10 μM MG-132 for 4 hours or left untreated, were labeled with an NMIIA-specific antibody (red) and a nucleus marker (HOECHST; blue). Representative image sections were 10× magnified. Yellow arrowheads in untreated UNC45A-KO cells indicate faint and dispersed NMIIA staining and fragmented NMIIA-positive bundles. MG-132–treated UNC45A-KO cells exhibit NMIIA-specific aggregates (yellow arrowheads). In p.Leu237Pro UNC45A–expressing cells, NMIIA filament assembly was rescued to a large extent, and no NMIIA-positive aggregates appeared upon proteasome inhibition. Scale bars, 20 μm.