No abstract

Neutrophils are commonly regarded as the first line of immune response during infection or in tissue injury-induced inflammation. The rapid influx of these cells results in the release of host defense proteins (HDPs) or formation of neutrophil extracellular traps (NETs). As a second wave during inflammation or infection, circulating monocytes arrive at the site. Earlier studies showed that HDPs LL-37 and Lactoferrin (LTF) activate monocytes while neutrophil elastase facilitates the formation of extracellular traps (ETs) in monocytes. However, the knowledge about the impact of HDPs on monocytes remains sparse. In the present study, we investigated the effect of LL-37 and LTF on blood-derived CD14+ monocytes. Both HDPs triggered a significant release of TNFα, nucleosomes, and monocyte ETs. Microscopic analysis indicated that ET formation by LL-37 depends on storage-operated calcium entry (SOCE), mitogen-activated protein kinase (MAPK), and ERK1/2, whereas the LTF-mediated ET release is not affected by any of the here used inhibitors. Quantitative proteomics mass spectrometry analysis of the neutrophil granular content (NGC) revealed a high abundance of Lactoferrin. The stimulation of CD14+ monocytes with NGC resulted in a significant secretion of TNFα and nucleosomes, and the formation of monocyte ETs. The findings of this study provide new insight into the complex interaction of HDPs, neutrophils, and monocytes during inflammation.

Mutations in the gene encoding cardiac myosin-binding protein-C (MyBPC), a thick filament assembly protein that stabilizes sarcomeric structure and regulates cardiac function, are a common cause for the development of hypertrophic cardiomyopathy. About 10% of carriers of the Δ25bp variant of MYBPC3, which is common in individuals from South Asia, are also carriers of the D389V variant on the same allele. Compared with noncarriers and those with MYBPC3Δ25bp alone, indicators for the development of hypertrophic cardiomyopathy occur with increased frequency in MYBPC3Δ25bp/D389V carriers. Residue D389 lies in the IgI-like C2 domain that is part of the N-terminal region of MyBPC. To probe the effects of mutation D389V on structure, thermostability, and protein–protein interactions, we produced and characterized wild-type and mutant constructs corresponding to the isolated 10 kDa C2 domain and a 52 kDa N-terminal fragment that includes subdomains C0 to C2. Our results show marked reductions in the melting temperatures of D389V mutant constructs. Interactions of construct C0–C2 D389V with the cardiac isoforms of myosin-2 and actin remain unchanged. Molecular dynamics simulations reveal changes in the stiffness and conformer dynamics of domain C2 caused by mutation D389V. Our results suggest a pathomechanism for the development of HCM based on the toxic buildup of misfolded protein in young MYBPC3Δ25bp/D389V carriers that is supplanted and enhanced by C-zone haploinsufficiency at older ages.

Cardiac myosin-binding protein C (MyBPC) is a thick-filament associated regulatory protein in the sarcomere. It regulates the sensitive contractile system of the myocardium by acting as a mechanical tether, sensitizing the thin filament or modulating myosin motor activity. Mutations in the MYBPC3 gene are a frequent cause for the development of hypertrophic cardiomyopathy, the most frequent cardiac disorder. Recently, the monoallelic double mutation MYBPC3Δ25bp/D389V has been discovered as a subset of the common MYBPC3Δ25bp variant in South Asia. MYBPC3Δ25bp/D389V carriers exhibit hyperdynamic features, which are considered an early finding for the development of hypertrophic cardiomyopathy. Using correlation-guided molecular dynamics simulations sampling, we show that the D389V mutation shifts the conformational distribution of the C2 domain of MyBPC. We further applied biochemical approaches to probe the effects of the D389V mutation on structure, thermostability and protein-protein interactions of MyBPC C2. The melting temperature (Tm) of MyBPC C2 D389V is decreased by 4 to 7 °C compared to wild type while the interaction of the C0-C2 domains with myosin and actin remains unchanged. Additionally, we utilized steered molecular dynamics (SMD) simulations to investigate the altered unfolding pathway of MyBPC C2 D389V. Based on our data, we propose a pathomechanism for the development of HCM in MYBPC3Δ25bp and MYBPC3Δ25bp/D389V carriers.