Disease-associated mutations impacting BC-loop flexibility trigger long-range transthyretin tetramer destabilization and aggregation

Hereditary transthyretin amyloidosis (ATTR) is an autosomal dominant disease characterized by the extracellular deposition of the transport protein transthyretin (TTR) as amyloid fibrils. Despite the progress achieved in recent years, understanding why different TTR residue substitutions lead to different clinical manifestations remains elusive. Here, we studied the molecular basis of disease-causing missense mutations affecting residues R34 and K35. R34G and K35T variants cause vitreous amyloidosis, whereas R34T and K35N mutations result in amyloid polyneuropathy and restrictive cardiomyopathy. All variants are more sensitive to pH-induced dissociation and amyloid formation than the wild-type (WT)-TTR counterpart, specifically in the variants deposited in the eyes amyloid formation occurs close to physiological pHs. Chemical denaturation experiments indicate that all the mutants are less stable than WT-TTR, with the vitreous amyloidosis variants, R34G and K35T, being highly destabilized. Sequence-induced stabilization of the dimer–dimer interface with T119M rendered tetramers containing R34G or K35T mutations resistant to pH-induced aggregation. Because R34 and K35 are among the residues more distant to the TTR interface, their impact in this region is therefore theorized to occur at long range. The crystal structures of double mutants, R34G/T119M and K35T/T119M, together with molecular dynamics simulations indicate that their strong destabilizing effect is initiated locally at the BC loop, increasing its flexibility in a mutation-dependent manner. Overall, the present findings help us to understand the sequence-dynamic-structural mechanistic details of TTR amyloid aggregation triggered by R34 and K35 variants and to link the degree of mutation-induced conformational flexibility to protein aggregation propensity.     

Regulatory volume decrease in cardiomyocytes is modulated by calcium influx and reactive oxygen species

We investigated the role of Ca²⁺ in generating reactive oxygen species (ROS) induced by hyposmotic stress (Hypo) and its relationship to regulatory volume decrease (RVD) in cardiomyocytes. Hypo-induced increases in cytoplasmic and mitochondrial Ca²⁺. Nifedipine (Nife) inhibited both Hypo-induced Ca²⁺ and ROS increases. Overexpression of catalase (CAT) induced RVD and a decrease in Hypo-induced blebs. Nife prevented CAT-dependent RVD activation. These results show a dual role of Hypo-induced Ca²⁺ influx in the control of cardiomyocyte viability. Hypo-induced an intracellular Ca²⁺ increase which activated RVD and inhibited necrotic blebbing thus favoring cell survival, while simultaneously increasing ROS generation, which in turn inhibited RVD and induced necrosis.     

Dietary fatty acid composition differently influences retinoylation reaction in rat testes mitochondria

All-trans-retinoic acid (atRA) is incorporated covalently into proteins of rat testes mitochondria. In this study, the effect of three diets with different fatty acid composition on the retinoylation of proteins of rat testes mitochondria has been investigated. Different groups of rats were fed on a basal diet supplemented with 15% of either coconut oil (CO), olive oil (OO) or fish oil (FO). We found that, when compared with CO, the binding of retinoic acid was decreased in FO- and OO-fed rats. Mitochondrial phospholipids composition was differently influenced by dietary treatments; minor changes were observed in fatty acid composition of phospholipids. Few differences were observed in the Arrhenius plots among the three groups of rats. Kinetic analysis revealed a decrease in the V _max value in FO- and OO- as compared with CO-fed rats. No difference among the three groups were observed in the K _M value. The retinoylation reaction was inhibited by 13-cis-RA and 9-cis-RA.