Dominguez, F. | bioRxiv Science

bioRxiv2020

Structural and Functional Characterization of Host FHL1 Protein Interaction with Hypervariable Domain of Chikungunya Virus nsP3 Protein

Decades of insufficient control resulted in unprecedented spread of chikungunya virus (CHIKV) around the globe and millions already suffered from the highly debilitating disease. Nevertheless, the current understanding of CHIKV-host interactions and adaptability of the virus to replication in mosquitoes and mammalian hosts is still elusive. Our new study shows that four-and-a-half LIM domain protein (FHL1) is one of the host factors that interact with hypervariable domain (HVD) of CHIKV nsP3. Unlike G3BPs, FHL1 is not a pre-requisite of CHIKV replication, and many commonly used cell lines do not express FHL1. However, its expression has detectable stimulatory effect(s) on CHIKV replication, and the Fhl1 KO cell lines demonstrate slower infection spread. The NMR-based studies revealed that the binding site of FHL1 in CHIKV nsP3 HVD overlaps with that of another pro-viral host factor, CD2AP. The structural data also demonstrated that FHL1-HVD interaction is mostly determined by LIM1 domain of FHL1. However, it does not mirror binding of the entire protein, suggesting that other LIM domains are involved. In agreement with previously published data, our biological experiments showed that interactions of CHIKV HVD with CD2AP and FHL1 have additive positive effects on the efficiency of CHIKV replication. This study shows that CHIKV mutants with extensive modifications of FHL1- or both FHL1- and CD2AP- binding sites remain viable and develop spreading infection in multiple cell types. Thus, such modifications of HVD may improve live CHIKV vaccine candidates in terms of their safety and stability of the attenuated phenotype. IMPORTANCEReplication of chikungunya virus (CHIKV) is determined by a wide range of host factors. Previously, we have demonstrated that the hypervariable domain (HVD) of CHIKV nsP3 protein contains linear motifs that recruit defined families of host proteins into formation of functional viral replication complexes. Now, using NMR-based structural and biological approaches, we have characterized the binding site of cellular FHL1 protein in CHIKV HVD and defined the biological significance of this interaction. In contrast to previously described binding of G3BP to CHIKV HVD, the FHL1-HVD interaction was found to not be a prerequisite of viral replication. However, the presence of FHL1 has a stimulatory effect on CHIKV infectivity and subsequently, the infection spread. FHL1 and CD2AP proteins were found to have overlapping binding sites in CHIKV HVD and additive pro-viral functions. Elimination of FHL1-binding site in nsP3 HVD can be used for the development of stable, live attenuated vaccine candidates.

microbiology↗

bioRxiv2020

Nanomechanical phenotypes in cMyBP-C mutants that cause hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is a disease of the myocardium caused by mutations in sarcomeric proteins with mechanical roles, such as the molecular motor myosin. Around half of the HCM-causing genetic variants target contraction modulator cardiac myosin-binding protein C (cMyBP-C), although the underlying pathogenic mechanisms remain unclear since many of these mutations cause no alterations in protein structure and stability. As an alternative pathomechanism, here we have examined whether pathogenic mutations perturb the nanomechanics of cMyBP-C, which would compromise its modulatory mechanical tethers across sliding actomyosin filaments. Using single-molecule atomic force spectroscopy, we have quantified mechanical folding and unfolding transitions in cMyBP-C mutant domains. Our results show that domains containing mutation R495W are mechanically weaker than wild-type at forces below 40 pN, and that R502Q mutant domains fold faster than wild-type. None of these alterations are found in control, non-pathogenic variants, suggesting that nanomechanical phenotypes induced by pathogenic cMyBP-C mutations contribute to HCM development. We propose that mutation-induced nanomechanical alterations may be common in mechanical proteins involved in human pathologies.

biophysics↗

bioRxiv2020

Conserved cysteines in titin sustain the mechanical function of cardiomyocytes

The protein titin determines cardiomyocyte contraction and truncating variants in the titin gene (TTN) are the most common cause of dilated cardiomyopathy (DCM). Different to truncations, missense variants in TTN are currently classified as variants of uncertain significance due to their high frequency in the population and the absence of functional annotation. Here, we report the regulatory role of conserved, mechanically active titin cysteines, which, contrary to current views, we uncover to be reversibly oxidized in basal conditions leading to isoform- and force-dependent modulation of titin stiffness and dynamics. Building on our functional studies, we demonstrate that missense mutations targeting a conserved titin cysteine alter myocyte contractile function and cause DCM in humans. Our findings have a direct impact on genetic counselling in clinical practice. One sentence summaryMutations targeting cysteines key to the mechanoredox control of titin cause human dilated cardiomyopathy

physiology↗