Titin domains with reduced core hydrophobicity cause dilated cardiomyopathy.

The underlying genetic defect in most cases of dilated cardiomyopathy (DCM), a common inherited heart disease, remains unknown. Intriguingly, many patients carry single missense variants of uncertain pathogenicity targeting the giant protein titin, a fundamental sarcomere component. To explore the deleterious potential of these variants, we first solved the wild-type and mutant crystal structures of I21, the titin domain targeted by pathogenic variant p.C3575S. Although both structures are remarkably similar, the reduced hydrophobicity of deeply buried position 3575 strongly destabilizes the mutant domain, a scenario supported by molecular dynamics simulations and by biochemical assays that show no disulfide involving C3575. Prompted by these observations, we have found that thousands of similar hydrophobicity-reducing variants associate specifically with DCM. Hence, our results imply that titin domain destabilization causes DCM, a conceptual framework that not only informs pathogenicity assessment of gene variants but also points to therapeutic strategies counterbalancing protein destabilization.

Gluconeogenic precursor availability regulates flux through the glyoxylate shunt in Pseudomonas aeruginosa.

The glyoxylate shunt bypasses the oxidative decarboxylation steps of the tricarboxylic acid (TCA) cycle, thereby conserving carbon skeletons for gluconeogenesis and biomass production. In Escherichia coli, carbon flux is redirected through the first enzyme of the glyoxylate shunt, isocitrate lyase (ICL), following phosphorylation and inactivation of the TCA cycle enzyme, isocitrate dehydrogenase (ICD), by the kinase/phosphatase, AceK. In contrast, mycobacterial species lack AceK and employ a phosphorylation-insensitive isocitrate dehydrogenase (IDH), which is allosterically activated by the product of ICL activity, glyoxylate. However, Pseudomonas aeruginosa expresses IDH, ICD, ICL, and AceK, raising the question of how these enzymes are regulated to ensure proper flux distribution between the competing pathways. Here, we present the structure, kinetics, and regulation of ICL, IDH, and ICD from P. aeruginosa We found that flux partitioning is coordinated through reciprocal regulation of these enzymes, linking distribution of carbon flux to the availability of the key gluconeogenic precursors, oxaloacetate and pyruvate. Specifically, a greater abundance of these metabolites activated IDH and inhibited ICL, leading to increased TCA cycle flux. Regulation was also exerted through AceK-dependent phosphorylation of ICD; high levels of acetyl-CoA (which would be expected to accumulate when oxaloacetate is limiting) stimulated the kinase activity of AceK, whereas high levels of oxaloacetate stimulated its phosphatase activity. In summary, the TCA cycle-glyoxylate shunt branch point in P. aeruginosa has a complex enzymology that is profoundly different from those in other species characterized to date. Presumably, this reflects its predilection for consuming fatty acids, especially during infection scenarios.

Which microbial factors really are important in Pseudomonas aeruginosa infections?

Over the last two decades, tens of millions of dollars have been invested in understanding virulence in the human pathogen, Pseudomonas aeruginosa. However, the top 'hits' obtained in a recent TnSeq analysis aimed at identifying those genes that are conditionally essential for infection did not include most of the known virulence factors identified in these earlier studies. Instead, it seems that P. aeruginosa faces metabolic challenges in vivo, and unless it can overcome these, it fails to thrive and is cleared from the host. In this review, we look at the kinds of metabolic pathways that the pathogen seems to find essential, and comment on how this knowledge might be therapeutically exploited.