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Abstract: Introduction: Z-disk proteins play an essential role in stabilizing the contractile muscle apparatus. Genetic variants in these proteins are associated with cardiomyopathies, a group of genetic cardiac diseases linked to sudden cardiac death. Several studies have identified pathogenic variants in two prominent Z-disk proteins, alpha-actinin (ACTN2) and filamin C (FLNC). Notably, a 2014 study identified the ACTN2 variant M228T in a family of 11 patients with cardiomyopathy. Similarly, a recent investigation uncovered the FLNC variant M82K in two cardiomyopathy families. Both variants reside within the actin-binding domain of their respective Z-disk proteins, which is crucial for organizing and binding actin-thin filaments. However, the mechanisms by which these variants alter protein structure and function remain undefined. This study investigated the structural alterations and functional consequences associated with the ACTN2-M228T and FLNC-M82K variants. Methods: To explore the structural modifications in the ACTN2-M228T and FLNC-M82K mutant proteins, structural modelling approaches were employed. Additionally, recombinant proteins were expressed in a bacterial system and subsequently purified. A comprehensive array of biophysical techniques was utilized to detect alterations in protein structure. Functional assessments were performed using induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) that incorporated the ACTN2-M228T variant. Results: Structural modeling predictions revealed that both ACTN2-M228T and FLNC-M82K variants share a common mechanism that adversely impact the regulatory function of the actin-binding domain. This hypothesis was further evaluated using actin-binding assays. The mutant proteins exhibited increased aggregation identified through mass photometry and size exclusion chromatography coupled with either multi-angle light scattering or small-angle X-ray scattering (SAXS). Moreover, both variants demonstrated reduced solubility and structural stability based on solubility and enzymatic digestion assays. The thermal stability of mutant proteins was also compromised, as assessed through SAXS and differential scanning fluorimetry. To correlate the identified structural alterations with disease mechanisms, the functional implications of ACTN2-M228T variant were assessed using an iPSC-CM model. Mutant cardiomyocytes showed increased ACTN2 aggregate formation and upregulation of hypertrophy, fibrosis, and autophagy markers. Furthermore, significant degradation of ACTN2 was observed through biochemical fractionation assays. ACTN2 destabilization was further evaluated using proteasome and protease inhibitors targeting the ubiquitin-proteosome system and the autophagy-lysosomal pathway, to elucidate their potential roles in the protein degradation process. Conclusion: Collectively, this study provides valuable insights into the effects of Z-disk variants on protein structure and function. The integration of structural and functional approaches represents a crucial step towards understanding disease pathways for cardiomyopathy-linked Z-disk variants.

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Abstract: N-utilization substance A (NusA) is a regulatory factor with pleiotropic functions in gene expression in bacteria. Archaea encode two conserved small proteins, NusA1 and NusA2, with domains orthologous to the two RNA binding K Homology (KH) domains of NusA. Here, we report the crystal structures of NusA2 from Sulfolobus acidocaldarius and Saccharolobus solfataricus obtained at 3.1 Å and 1.68 Å, respectively. NusA2 comprises an N-terminal zinc finger followed by two KH-like domains lacking the GXXG signature. Despite the loss of the GXXG motif, NusA2 binds single-stranded RNA. Mutations in the zinc finger domain compromise the structural integrity of NusA2 at high temperatures and molecular dynamics simulations indicate that zinc binding provides an energy barrier preventing the domain from reaching unfolded states. A structure-guided phylogenetic analysis of the KH-like domains supports the notion that the NusA2 clade is ancestral to the ribosomal protein eS7 in eukaryotes, implying a potential role of NusA2 in translation.

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Abstract: Harnessing the potential beneficial effects of kinase signalling through the generation of direct kinase activators remains an underexplored area of drug development. This also applies to the PI3K signalling pathway, which has been extensively targeted by inhibitors for conditions with PI3K overactivation, such as cancer and immune dysregulation. Here we report the discovery of UCL-TRO-1938 (referred to as 1938 hereon), a small-molecule activator of the PI3Kα isoform, a crucial effector of growth factor signalling. 1938 allosterically activates PI3Kα through a distinct mechanism by enhancing multiple steps of the PI3Kα catalytic cycle and causes both local and global conformational changes in the PI3Kα structure. This compound is selective for PI3Kα over other PI3K isoforms and multiple protein and lipid kinases. It transiently activates PI3K signalling in all rodent and human cells tested, resulting in cellular responses such as proliferation and neurite outgrowth. In rodent models, acute treatment with 1938 provides cardioprotection from ischaemia–reperfusion injury and, after local administration, enhances nerve regeneration following nerve crush. This study identifies a chemical tool to directly probe the PI3Kα signalling pathway and a new approach to modulate PI3K activity, widening the therapeutic potential of targeting these enzymes through short-term activation for tissue protection and regeneration. Our findings illustrate the potential of activating kinases for therapeutic benefit, a currently largely untapped area of drug development.

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Abstract: The regular reappearance of coronavirus (CoV) outbreaks over the past 20 years has caused significant health consequences and financial burdens worldwide. The most recent and still ongoing novel CoV pandemic, caused by Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) has brought a range of devastating consequences. Due to the exceptionally fast development of vaccines, the mortality rate of the virus has been curbed to a significant extent. However, the limitations of vaccination efficiency and applicability, coupled with the still high infection rate, emphasise the urgent need for discovering safe and effective antivirals against SARS-CoV-2 by suppressing its replication or attenuating its virulence. Non-structural protein 1 (nsp1), a unique viral and conserved leader protein, is a crucial virulence factor for causing host mRNA degradation, suppressing interferon (IFN) expression and host antiviral signalling pathways. In view of the essential role of nsp1 in the CoV life cycle, it is regarded as an exploitable target for antiviral drug discovery. Here, we report a variety of fragment hits against the N-terminal domain of SARS-CoV-2 nsp1 identified by fragment-based screening via X-ray crystallography. We also determined the structure of nsp1 at atomic resolution (0.99 Å). Binding affinities of hits against nsp1 and potential stabilisation were determined by orthogonal biophysical assays such as microscale thermophoresis and thermal shift assays. We identified two ligand-binding sites on nsp1, one deep and one shallow pocket, which are not conserved between the three medically relevant SARS, SARS-CoV-2 and MERS coronaviruses. Our study provides an excellent starting point for the development of more potent nsp1-targeting inhibitors and functional studies on SARS-CoV-2 nsp1.

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Abstract: The organization of the mitochondrial electron transport chain proteins into supercomplexes (SCs) is now undisputed; however, their assembly process, or the role of differential expression isoforms, remain to be determined. In Saccharomyces cerevisiae, cytochrome c oxidase (CIV) forms SCs of varying stoichiometry with cytochrome bc1 (CIII). Recent studies have revealed, in normoxic growth conditions, an interface made exclusively by Cox5A, the only yeast respiratory protein that exists as one of two isoforms depending on oxygen levels. Here we present the cryo-EM structures of the III2-IV1 and III2-IV2 SCs containing the hypoxic isoform Cox5B solved at 3.4 and 2.8 Å, respectively. We show that the change of isoform does not affect SC formation or activity, and that SC stoichiometry is dictated by the level of CIII/CIV biosynthesis. Comparison of the CIV5B- and CIV5A-containing SC structures highlighted few differences, found mainly in the region of Cox5. Additional density was revealed in all SCs, independent of the CIV isoform, in a pocket formed by Cox1, Cox3, Cox12, and Cox13, away from the CIII–CIV interface. In the CIV5B-containing hypoxic SCs, this could be confidently assigned to the hypoxia-induced gene 1 (Hig1) type 2 protein Rcf2. With conserved residues in mammalian Hig1 proteins and Cox3/Cox12/Cox13 orthologs, we propose that Hig1 type 2 proteins are stoichiometric subunits of CIV, at least when within a III-IV SC.