No abstract

Mutations in the mitochondrially located protein CHCHD10 cause motoneuron disease by an unknown mechanism. In this study, we investigate the mutations p.R15L and p.G66V in comparison to wild-type CHCHD10 and the non-pathogenic variant p.P34S in vitro, in patient cells as well as in the vertebrate in vivo model zebrafish. We demonstrate a reduction of CHCHD10 protein levels in p.R15L and p.G66V mutant patient cells to approximately 50%. Quantitative real-time PCR revealed that expression of CHCHD10 p.R15L, but not of CHCHD10 p.G66V, is already abrogated at the mRNA level. Altered secondary structure and rapid protein degradation are observed with regard to the CHCHD10 p.G66V mutant. In contrast, no significant differences in expression, degradation rate or secondary structure of non-pathogenic CHCHD10 p.P34S are detected when compared with wild-type protein. Knockdown of CHCHD10 expression in zebrafish to about 50% causes motoneuron pathology, abnormal myofibrillar structure and motility deficits in vivo. Thus, our data show that the CHCHD10 mutations p.R15L and p.G66V cause motoneuron disease primarily based on haploinsufficiency of CHCHD10.

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The Shank proteins are crucial scaffolding elements of the post-synaptic density (PSD). One of the best-characterized domains in Shank is the PDZ domain, which binds to C-terminal segments of several other PSD proteins. We carried out a detailed structural analysis of Shank3 PDZ domain-peptide complexes, to understand determinants of binding affinity towards different ligand proteins. Ligand peptides from four different proteins were cocrystallized with the Shank3 PDZ domain, and binding affinities were determined calorimetrically. In addition to conserved class I interactions between the first and third C-terminal peptide residue and Shank3, side chain interactions of other residues in the peptide with the PDZ domain are important factors in defining affinity. Structural conservation suggests that the binding specificities of the PDZ domains from different Shanks are similar. Two conserved buried water molecules in PDZ domains may affect correct local folding of ligand recognition determinants. The solution structure of a tandem Shank3 construct containing the SH3 and PDZ domains showed that the two domains are close to each other, which could be of relevance, when recognizing and binding full target proteins. The SH3 domain did not affect the affinity of the PDZ domain towards short target peptides, and the schizophrenia-linked Shank3 mutation R536W in the linker between the domains had no effect on the structure or peptide interactions of the Shank3 SH3-PDZ unit. Our data show the spatial arrangement of two adjacent Shank domains and pinpoint affinity determinants for short PDZ domain ligands with limited sequence homology.

The post-synapBc density mulBdomain scaffolding proteins of the Shank family are structurally poorly characterised. The Shank family consists of three members, and domain-specific interacBons of Shank are involved in forming a network of proteins at the post-synapBc region for intracellular signalling and cellular scaffolding. While X-ray crystallography has provided some informaBon on individual Shank domains, the structural basis of Shank interacBons is sBll largely unknown. In this study, the producBon and crystallisaBon of the previously uncharacterised Shank3 SH3 domain is presented. The highly twinned crystals diffracted synchrotron X-rays to a resoluBon higher than 0.9 Å, and these crystals will eventually have the potenBal to provide an ultrahigh-resoluBon view into the Shank family SH3 domains and their interacBons.Composition of reservoir solution 0.1 M bis-tris (pH 6.5), 32.5% PEG3350 Volume and ratio of drop 450 nl, drop ratio 2:1 (protein:buffer) Volume of reservoir 50 µl