Truncating plakophilin-2 mutations in arrhythmogenic cardiomyopathy are associated with protein haploinsufficiency in both myocardium and epidermis.

BACKGROUND: Arrhythmogenic cardiomyopathy (AC) is a hereditary cardiac condition associated with ventricular arrhythmias, heart failure, and sudden death. The disease is most often caused by mutations in the desmosomal gene for plakophilin-2 (PKP2), which is expressed in both myocardial and epidermal tissue. This study aimed to investigate protein expression in myocardial tissue of patients with AC carrying PKP2 mutations and elucidate whether keratinocytes of the same individuals exhibited a similar pattern of protein expression. METHODS AND RESULTS: Direct sequencing of 5 AC genes in 71 unrelated patients with AC identified 10 different PKP2 mutations in 12 index patients. One patient, heterozygous for a PKP2 nonsense mutation, developed severe heart failure and underwent cardiac transplantation. Western blotting and immunohistochemistry of the explanted heart showed a significant decrease in PKP2 protein expression without detectable amounts of truncated PKP2 protein. Cultured keratinocytes of the patient showed a similar reduction in PKP2 protein expression. Nine additional PKP2 mutations were investigated in both cultured keratinocytes and endomyocardial biopsies from affected individuals. It was evident that PKP2 mutations introducing a premature termination codon in the reading frame were associated with PKP2 transcript and protein levels reduced to ≈50%, whereas a missense variant did not seem to affect the amount of PKP2 protein. CONCLUSIONS: The results of this study showed that truncating PKP2 mutations in AC are associated with low expression of the mutant allele and that the myocardial protein expression of PKP2 is mirrored in keratinocytes. These findings indicate that PKP2 haploinsufficiency contributes to pathogenesis in AC

Two Crystal Structures Demonstrate Large Conformational Changes in the Eukaryotic Ribosomal Translocase

Two crystal structures of yeast translation elongation factor 2 (eEF2) were determined: the apo form at 2.9 Å resolution and eEF2 in the presence of the translocation inhibitor sordarin at 2.1 Å resolution. The overall conformation of apo eEF2 is similar to that of its prokaryotic homolog elongation factor G (EF-G) in complex with GDP. Upon sordarin binding, the three tRNA-mimicking C-terminal domains undergo substantial conformational changes, while the three N-terminal domains containing the nucleotide-binding site form an almost rigid unit. The conformation of eEF2 in complex with sordarin is entirely different from known conformations observed in crystal structures of EF-G or from cryo-EM studies of EF-G–70S complexes. The domain rearrangements induced by sordarin binding and the highly ordered drug-binding site observed in the eEF2–sordarin structure provide a high-resolution structural basis for the mechanism of sordarin inhibition. The two structures also emphasize the dynamic nature of the ribosomal translocase

Structure of mycobacterial maltokinase, the missing link in the essential GlgE-pathway

A novel four-step pathway identified recently in mycobacteria channels trehalose to glycogen synthesis and is also likely involved in the biosynthesis of two other crucial polymers: intracellular methylglucose lipopolysaccharides and exposed capsular glucan. The structures of three of the intervening enzymes - GlgB, GlgE, and TreS - were recently reported, providing the first templates for rational drug design. Here we describe the structural characterization of the fourth enzyme of the pathway, mycobacterial maltokinase (Mak), uncovering a eukaryotic-like kinase (ELK) fold, similar to methylthioribose kinases and aminoglycoside phosphotransferases. The 1.15 Å structure of Mak in complex with a non-hydrolysable ATP analog reveals subtle structural rearrangements upon nucleotide binding in the cleft between the N- and the C-terminal lobes. Remarkably, this new family of ELKs has a novel N-terminal domain topologically resembling the cystatin family of protease inhibitors. By interfacing with and restraining the mobility of the phosphate-binding region of the N-terminal lobe, Mak's unusual N-terminal domain might regulate its phosphotransfer activity and represents the most likely anchoring point for TreS, the upstream enzyme in the pathway. By completing the gallery of atomic-detail models of an essential pathway, this structure opens new avenues for the rational design of alternative anti-tubercular compounds