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. 2013 Feb 26;110(9):3489-94.
doi: 10.1073/pnas.1222732110. Epub 2013 Jan 28.

Recessive loss of function of the neuronal ubiquitin hydrolase UCHL1 leads to early-onset progressive neurodegeneration

Kaya Bilguvar  1 , Navneet K Tyagi, Cigdem Ozkara, Beyhan Tuysuz, Mehmet Bakircioglu, Murim Choi, Sakir Delil, Ahmet O Caglayan, Jacob F Baranoski, Ozdem Erturk, Cengiz Yalcinkaya, Murat Karacorlu, Alp Dincer, Michele H Johnson, Shrikant Mane, Sreeganga S Chandra, Angeliki Louvi, Titus J Boggon, Richard P Lifton, Arthur L Horwich, Murat Gunel
Affiliations

Recessive loss of function of the neuronal ubiquitin hydrolase UCHL1 leads to early-onset progressive neurodegeneration

Kaya Bilguvar et al. Proc Natl Acad Sci U S A. .

Abstract

Ubiquitin C-terminal hydrolase-L1 (UCHL1), a neuron-specific de-ubiquitinating enzyme, is one of the most abundant proteins in the brain. We describe three siblings from a consanguineous union with a previously unreported early-onset progressive neurodegenerative syndrome featuring childhood onset blindness, cerebellar ataxia, nystagmus, dorsal column dysfuction, and spasticity with upper motor neuron dysfunction. Through homozygosity mapping of the affected individuals followed by whole-exome sequencing of the index case, we identified a previously undescribed homozygous missense mutation within the ubiquitin binding domain of UCHL1 (UCHL1(GLU7ALA)), shared by all affected subjects. As demonstrated by isothermal titration calorimetry, purified UCHL1(GLU7ALA), compared with WT, exhibited at least sevenfold reduced affinity for ubiquitin. In vitro, the mutation led to a near complete loss of UCHL1 hydrolase activity. The GLU7ALA variant is predicted to interfere with the substrate binding by restricting the proper positioning of the substrate for tunneling underneath the cross-over loop spanning the catalytic cleft of UCHL1. This interference with substrate binding, combined with near complete loss of hydrolase activity, resulted in a >100-fold reduction in the efficiency of UCHL1(GLU7ALA) relative to WT. These findings demonstrate a broad requirement of UCHL1 in the maintenance of the nervous system.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Identification of the GLU7ALA (E7A) mutation in UCHL1 in kindred NG 1024. (A) Simplified pedigree is shown; affected subjects are denoted by filled symbols (arrow indicates the index case). (B) Coronal MR images of NG 1024–3 (Right) and a control subject (Left) reveal diffuse cerebral and optic chiasmal atrophy (red arrowhead) in the index case. Cortical atrophy is indicated by cerebral volume loss with increase in the subarachnoid and intersulci spaces over the brain, which are filled with cerebrospinal fluid (black in these images). (C) Axial orbital MR images of a control subject (Left) and NG 1024–3 (Right) show optic nerve (red arrowhead) atrophy. (D) Sagittal MR images of a control subject (Left) and NG 1024–3 (Right) reveal cerebral cortical, cerebellar (red circle), and optic tract (red arrowheads) atrophy. (E) Exome sequencing reveals an A to C change. The WT sequence in blue is shown on top, with the mutant base in red below. There was 35-fold coverage, with all reads revealing the substitution. (F) Sanger sequencing confirms the mutation that results in glutamic acid (E, in blue) to alanine (A, in red) change. The unaffected parents are heterozygous for the variant (Right).
Fig. 2.
Fig. 2.
Structural and functional studies of UCHL1 carrying the GLU7ALA mutation. (A) Structural mapping of GLU7ALA (E7A) onto the crystal structure of UCHL1 in complex with the ubiquitin substrate mimic, UbVMe (13) (PDB ID: 3IFW). UCHL1 is colored cyan, with the L8 loop colored orange. UbVMe is colored yellow. Residue E7 is colored red. (Inset) Close-up of the hydrogen-bonding network at the threshold of the substrate tunnel. Panel made using the program Pymol (www.pymol.org). (B) Comparison of the enzymatic activity of WT UCHL1 and the variants I93M, C90S, and E7A. Real-time release of fluorescent AMC is shown for WT-UCHL1 (black), I93M (blue), E7A (red), and C90S (green). (C) Binding isotherms of the titration of WT-UCHL1 and respective mutants with ubiquitin. Binding of ubiquitin (500 μM) to corresponding proteins (50 μM) is shown. (Upper) Raw data. (Lower) Integrated heat data as enthalpy as a function of molar ratio of ligand to protein. The solid line in the lower panel represents the best curve fit to the data by using a one-site binding model.
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