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Brugada Syndrome (BrS) is a rare inherited cardiac arrhythmia causing potentially fatal ventricular tachycardia or fibrillation, mainly occurring during rest or sleep in young individuals without heart structural issues. It increases the risk of sudden cardiac death, and its characteristic feature is an abnormal ST segment elevation on the ECG. While BrS has diverse genetic origins, a subset of cases can be conducted to mutations in the SCN5A gene, which encodes for the Nav1.5 sodium channel. Our study focused on three novel SCN5A mutations (p.A344S, p.N347K, and p.D349N) found in unrelated BrS families. Using patch clamp experiments, we found that these mutations disrupted sodium currents: p.A344S reduced current density, while p.N347K and p.D349N completely abolished it, leading to altered voltage dependence and inactivation kinetics when co-expressed with normal channels. We also explored the effects of mexiletine treatment, which can modulate ion channel function. Interestingly, the p.N347K and p.D349N mutations responded well to the treatment, rescuing the current density, while p.A344S showed a limited response. Structural analysis revealed these mutations were positioned in key regions of the channel, impacting its stability and function. This research deepens our understanding of BrS by uncovering the complex relationship between genetic mutations, ion channel behavior, and potential therapeutic interventions.

Light chain amyloidosis (AL) is caused by the aberrant overproduction of immunoglobulin light chains (LCs). The resulting abnormally high LC concentrations in blood lead to deposit formation in the heart and other target organs. Organ damage is caused not only by the accumulation of bulky amyloid deposits, but extensive clinical data indicate that circulating soluble LCs also exert cardiotoxic effects. The nematode C. elegans has been validated to recapitulate LC soluble toxicity in vivo, and in such a model a role for copper ions in increasing LC soluble toxicity has been reported. Here, we applied microscale thermophoresis, isothermal calorimetry and thermal melting to demonstrate the specific binding of Cu2+ to the variable domain of amyloidogenic H7 with a sub-micromolar affinity. Histidine residues present in the LC sequence are not involved in the binding, and yet their mutation to Ala reduces the soluble toxicity of H7. Copper ions bind to and destabilize the variable domains and induce a limited stabilization in this domain. In summary, the data reported here, elucidate the biochemical bases of the Cu2+-induced toxicity; moreover, they also show that copper binding is just one of the several biochemical traits contributing to LC soluble in vivo toxicity.

Neuroserpin (NS) is a member of the serine protease inhibitors superfamily. Specific point mutations are responsible for its accumulation in the endoplasmic reticulum of neurons that leads to a pathological condition named familial encephalopathy with neuroserpin inclusion bodies (FENIB). Wild-type NS presents two N-glycosylation chains and does not form polymers in vivo, while non-glycosylated NS causes aberrant polymer accumulation in cell models. To date, all in vitro studies have been conducted on bacterially expressed NS, de facto neglecting the role of glycosylation in the biochemical properties of NS. Here, we report the expression and purification of human glycosylated NS (gNS) using a novel eukaryotic expression system, LEXSY. Our results confirm the correct N-glycosylation of wild-type gNS. The fold and stability of gNS are not altered compared to bacterially expressed NS, as demonstrated by the circular dichroism and intrinsic tryptophan fluorescence assays. Intriguingly, gNS displays a remarkably reduced polymerisation propensity compared to non-glycosylated NS, in keeping with what was previously observed for wild-type NS in vivo and in cell models. Thus, our results support the relevance of gNS as a new in vitro tool to study the molecular bases of FENIB.

Beta-2 microglobulin (β2m) is a protein responsible for a pathologic condition, known as dialysis-related amyloidosis (DRA), caused by its aggregation and subsequent amyloid formation. A naturally occurring mutation of β2m, D76N, presents a higher amyloidogenic propensity compared to the wild type counterpart. Since the three-dimensional structure of the protein is essentially unaffected by the mutation, the increased aggregation propensity of D76N has been generally ascribed to its lower thermodynamic stability and increased dynamics. In this study we compare the equilibrium unfolding and the aggregation propensity of wild type β2m and D76N variant at different experimental conditions. Our data revealed a surprising effect of the D76N mutation in the residual structure of the denatured state, which appears less compact than that of the wild type protein. A careful investigation of the structural malleability of the denatured state of wild type β2m and D76N pinpoint a clear role of the denatured state in triggering the amyloidogenic propensity of the protein. The experimental results are discussed in the light of the previous work on β2m and its role in disease.