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Mol Neurobiol. 2017 May;54(4):2928-2938. doi: 10.1007/s12035-016-9862-x. Epub 2016 Mar 29.

The Interaction of UDP-N-Acetylglucosamine 2-Epimerase/N-Acetylmannosamine Kinase (GNE) and Alpha-Actinin 2 Is Altered in GNE Myopathy M743T Mutant.

Author information

1
Goldyne Savad Institute of Gene Therapy, Hadassah Hebrew University Medical Center, Jerusalem, Israel.
2
Department of Plant and Environmental Sciences, Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel.
3
Core Research Facility, Institute for Drug Research, School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem, Israel.
4
Laboratory of Biochemistry, Department of Life Sciences and Technology, Beuth University of Applied Sciences Berlin, Berlin, Germany.
5
Goldyne Savad Institute of Gene Therapy, Hadassah Hebrew University Medical Center, Jerusalem, Israel. stella@mail.huji.ac.il.

Abstract

UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) is the gene mutated in GNE myopathy. In an attempt to elucidate GNE functions that could account for the muscle pathophysiology of this disorder, the interaction of GNE with α-actinins has been investigated. Surface plasmon resonance and microscale thermophoresis analysis revealed, that in vitro, GNE interacts with α-actinin 2, and that this interaction has a 10-fold higher affinity compared to the GNE-α-actinin 1 interaction. Further, GNE carrying the M743T mutation, the most frequent mutation in GNE myopathy, has a 10-fold lower binding affinity to α-actinin 2 than intact GNE. It is possible that this decrease eventually affects the interaction, thus causing functional imbalance of this complex in skeletal muscle that could contribute to the myopathy phenotype. In vivo, using bi-molecular fluorescent complementation, we show the specific binding of the two proteins inside the intact cell, in a unique interaction pattern between the two partners. This interaction is disrupted in the absence of the C-terminal calmodulin-like domain of α-actinin 2, which is altered in α-actinin 1. Moreover, the binding of GNE to α-actinin 2 prevents additional binding of α-actinin 1 but not vice versa. These results suggest that the interaction between GNE and α-actinin 1 and α-actinin 2 occur at different sites in the α-actinin molecules and that for α-actinin 2 the interaction site is located at the C-terminus of the protein.

KEYWORDS:

BIFC; GNE; GNE myopathy ; HIBM; MST; SPR; α-Actinin 1; α-Actinin 2

PMID:
27023225
DOI:
10.1007/s12035-016-9862-x
[Indexed for MEDLINE]

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