Currently, little is known about the genetic background of restrictive cardiomyopathy (RCM). Herein, we screened an index patient with RCM in combination with atrial fibrillation using a next generation sequencing (NGS) approach and identified the heterozygous mutation DES-c.735G>C. As DES-c.735G>C affects the last base pair of exon-3, it is unknown whether putative missense or splice site mutations are caused. Therefore, we applied nanopore amplicon sequencing revealing the expression of a transcript without exon-3 in the explanted myocardial tissue of the index patient. Western blot analysis verified this finding at the protein level. In addition, we performed cell culture experiments revealing an abnormal cytoplasmic aggregation of the truncated desmin form (p.D214-E245del) but not of the missense variant (p.E245D). In conclusion, we show that DES-c.735G>C causes a splicing defect leading to exon-3 skipping of the DES gene. DES-c.735G>C can be classified as a pathogenic mutation associated with RCM and atrial fibrillation. In the future, this finding might have relevance for the genetic understanding of similar cases.

Lysosomal pH dysregulation is a critical element of the pathophysiology of neurodegenerative diseases, cancers, and lysosomal storage disorders (LSDs). To study the role of lysosomes in pathophysiology, probes to analyze lysosomal size, positioning, and pH are indispensable tools. Here, we developed and characterized a ratiometric genetically encoded lysosomal pH probe, RpH-ILV, targeted to a subpopulation of lysosomal intraluminal vesicles. This subpopulation behaves similarly to the general population of LAMP1-positive vesicles in terms of pH response to pharmacological stresses. In addition, RpH-ILV, which is trafficked to the lysosome via a different cytosolic motif than our previous ratiometric sensor, RpH-LAMP1, is well tolerated by the model organism Drosophila melanogaster , exhibits minimal plasma membrane fluorescence, and reveals sensitivity to the lysosomal damaging agent LLOMe, adding a valuable tool to our repertoire of lysosomal pH sensors.

Gram-negative bacteria such as Aeromonas and Yersinia spp. have developed mechanisms to inhibit the immune defense of their host. Effector proteins are directly injected into the host cytoplasm from the bacterial cytosol via type III secretion systems (T3SSs), where they modulate the cytoskeleton and signaling of the cell. Assembly of, and secretion via, T3SSs is tightly regulated by a number of bacterial proteins, including SctX (AscX in Aeromonas), the secretion of which is essential for T3SS function. Here, crystal structures of AscX in complex with SctY chaperones from Yersinia or Photorhabdus spp. carrying homologous T3SSs are described. There are crystal pathologies in all cases, with one crystal form diffracting anisotropically and the other two exhibiting strong pseudotranslation. The new structures reveal that the positioning of the substrate is very similar on different chaperones. However, the two C-terminal SctX helices that cap the N-terminal tetratricopeptide repeat of SctY shift and tilt depending on the identity of the chaperone. Moreover, the C-terminus of the α3 helix of AscX exhibits an unprecedented kink in two of the structures. In previous structures, the C-terminus of SctX protrudes beyond the chaperone as a straight helix: a conformation that is required for binding to the nonameric export gate SctV but that is unfavorable for binary SctX–SctY complexes due to the hydrophobicity of helix α3 of SctX. A kink in helix α3 may allow the chaperone to shield the hydrophobic C-terminus of SctX in solution.

BACKGROUND: The DES gene encodes the IF (intermediate filament) protein desmin, which connects different multiprotein complexes, such as the cardiac desmosomes, and is highly important for the structural integrity of cardiomyocytes. Pathogenic DES mutations cause filament assembly defects leading to cardiomyopathies. However, most DES variants listed in genetic disease databases are currently classified as variants of unknown significance. Here, we characterized 21 different DES variants of unknown significance and 18 additional proline variants, localized in a highly conserved stretch at the C terminus of the desmin coil-2 subdomain. METHODS: We inserted desmin variants via site-directed mutagenesis and investigated the filament assembly in transfected cell lines and cardiomyocytes derived from induced pluripotent stem cells by confocal microscopy. In addition, we purified recombinant wild-type and mutant desmin and analyzed the filament formation by atomic force microscopy. Coexpression with wild-type desmin delivered by adeno-associated virus was used to model the heterozygous status of cardiomyopathy patients. RESULTS: Twelve DES variants of unknown significance formed cytoplasmic aggregates, which were likewise verified by atomic force microscopy. Of note, these 12 variants disturb the filament assembly even when coexpressed with wild-type desmin. Using a proline screen, we showed that proline residues localized at nearly each of the positions in this stretch cause filament assembly defects. By modeling the tetrameric structure of desmin, we demonstrated that specific heptad positions, as well as positions of intramolecular and intermolecular ion bridge sites, are particularly susceptible to mutations that promote desmin aggregation. CONCLUSIONS: In summary, our study demonstrated that the highly conserved stretch at the C terminus of the coil-2 subdomain is a hotspot region, where several pathogenic DES mutations cause an aberrant desmin aggregation. Based on our molecular data, we suggest reclassifying the aggregate-forming variants as likely pathogenic mutations rather than variants of unknown significance. Our study may have relevance for the genetic counseling of cardiomyopathy patients with similar DES variants.