Limb girdle muscular disease caused by HMGCR mutation and statin myopathy treatable with mevalonolactone.

Myopathy is the main adverse effect of the widely prescribed statin drug class. Statins exert their beneficial effect by inhibiting HMG CoA-reductase, the rate-controlling enzyme of the mevalonate pathway. The mechanism of statin myopathy is yet to be resolved, and its treatment is insufficient. Through homozygosity mapping and whole exome sequencing, followed by functional analysis using confocal microscopy and biochemical and biophysical methods, we demonstrate that a distinct form of human limb girdle muscular disease is caused by a pathogenic homozygous loss-of-function missense mutation in HMG CoA reductase (HMGCR), encoding HMG CoA-reductase. We biochemically synthesized and purified mevalonolactone, never administered to human patients before, and establish the safety of its oral administration in mice. We then show that its oral administration is effective in treating a human patient with no significant adverse effects. Furthermore, we demonstrate that oral mevalonolactone resolved statin-induced myopathy in mice. We conclude that HMGCR mutation causes a late-onset severe progressive muscular disease, which shows similar features to statin-induced myopathy. Our findings indicate that mevalonolactone is effective both in the treatment of hereditary HMGCR myopathy and in a murine model of statin myopathy. Further large clinical trials are in place to enable the clinical use of mevalonolactone both in the rare orphan disease and in the more common statin myopathy.

Structure-function studies of the magnetite-biomineralizing magnetosome-associated protein MamC.

Magnetotactic bacteria are Gram-negative bacteria that navigate along geomagnetic fields using the magnetosome, an organelle that consists of a membrane-enveloped magnetic nanoparticle. Magnetite formation and its properties are controlled by a specific set of proteins. MamC is a small magnetosome-membrane protein that is known to be active in iron biomineralization but its mechanism has yet to be clarified. Here, we studied the relationship between the MamC magnetite-interaction loop (MIL) structure and its magnetite interaction using an inert biomineralization protein-MamC chimera. Our determined structure shows an alpha-helical fold for MamC-MIL with highly charged surfaces. Additionally, the MamC-MIL induces the formation of larger magnetite crystals compared to protein-free and inert biomineralization protein control experiments. We suggest that the connection between the MamC-MIL structure and the protein's charged surfaces is crucial for magnetite binding and thus for the size control of the magnetite nanoparticles.

The FtsZ-like protein FtsZm of Magnetospirillum gryphiswaldense likely interacts with its generic homolog and is required for biomineralization under nitrate deprivation.

Midcell selection, septum formation, and cytokinesis in most bacteria are orchestrated by the eukaryotic tubulin homolog FtsZ. The alphaproteobacterium Magnetospirillum gryphiswaldense (MSR-1) septates asymmetrically, and cytokinesis is linked to splitting and segregation of an intracellular chain of membrane-enveloped magnetite crystals (magnetosomes). In addition to a generic, full-length ftsZ gene, MSR-1 contains a truncated ftsZ homolog (ftsZm) which is located adjacent to genes controlling biomineralization and magnetosome chain formation. We analyzed the role of FtsZm in cell division and biomineralization together with the full-length MSR-1 FtsZ protein. Our results indicate that loss of FtsZm has a strong effect on microoxic magnetite biomineralization which, however, could be rescued by the presence of nitrate in the medium. Fluorescence microscopy revealed that FtsZm-mCherry does not colocalize with the magnetosome-related proteins MamC and MamK but is confined to asymmetric spots at midcell and at the cell pole, coinciding with the FtsZ protein position. In Escherichia coli, both FtsZ homologs form distinct structures but colocalize when coexpressed, suggesting an FtsZ-dependent recruitment of FtsZm. In vitro analyses indicate that FtsZm is able to interact with the FtsZ protein. Together, our data suggest that FtsZm shares key features with its full-length homolog but is involved in redox control for magnetite crystallization.

Unexpected implications of STAT3 acetylation revealed by genetic encoding of acetyl-lysine.

The signal transducer and activator of transcription 3 (STAT3) protein is activated by phosphorylation of a specific tyrosine residue (Tyr705) in response to various extracellular signals. STAT3 activity was also found to be regulated by acetylation of Lys685. However, the molecular mechanism by which Lys685 acetylation affects the transcriptional activity of STAT3 remains elusive. By genetically encoding the co-translational incorporation of acetyl-lysine into position Lys685 and co-expression of STAT3 with the Elk receptor tyrosine kinase, we were able to characterize site-specifically acetylated, and simultaneously acetylated and phosphorylated STAT3. We measured the effect of acetylation on the crystal structure, and DNA binding affinity and specificity of Tyr705-phosphorylated and non-phosphorylated STAT3. In addition, we monitored the deacetylation of acetylated Lys685 by reconstituting the mammalian enzymatic deacetylation reaction in live bacteria. Surprisingly, we found that acetylation, per se, had no effect on the crystal structure, and DNA binding affinity or specificity of STAT3, implying that the previously observed acetylation-dependent transcriptional activity of STAT3 involves an additional cellular component. In addition, we discovered that Tyr705-phosphorylation protects Lys685 from deacetylation in bacteria, providing a new possible explanation for the observed correlation between STAT3 activity and Lys685 acetylation.

Understanding the Biomineralization Role of Magnetite-Interacting Components (MICs) From Magnetotactic Bacteria.

Biomineralization is a process that takes place in all domains of life and which usually helps organisms to harden soft tissues by creating inorganic structures that facilitate their biological functions. It was shown that biominerals are under tight biological control via proteins that are involved in nucleation initiation and/or which act as structural skeletons. Magnetotactic bacteria (MTB) use iron biomineralization to create nano-magnetic particles in a specialized organelle, the magnetosome, to align to the geomagnetic field. A specific set of magnetite-associated proteins (MAPs) is involved in regulating magnetite nucleation, size, and shape. These MAPs are all predicted to contain specific 17-22 residue-long sequences involved in magnetite formation. To understand the mechanism of magnetite formation, we focused on three different MAPs, MamC, Mms6 and Mms7, and studied the predicted iron-binding sequences. Using nuclear magnetic resonance (NMR), we differentiated the recognition mode of each MAP based on ion specificity, affinity, and binding residues. The significance of critical residues in each peptide was evaluated by mutation followed by an iron co-precipitation assay. Among the peptides, MamC showed weak ion binding but created the most significant effect in enhancing magnetite particle size, indicating the potency in controlling magnetite particle shape and size. Alternatively, Mms6 and Mms7 had strong binding affinities but less effect in modulating magnetite particle size, representing their major role potentially in initiating nucleation by increasing local metal concentration. Overall, our results explain how different MAPs affect magnetite synthesis, interact with Fe2+ ions and which residues are important for the MAPs functions.

The importance of the helical structure of a MamC-derived magnetite-interacting peptide for its function in magnetite formation.

Biomineralization is the process of mineral formation by organisms and involves the uptake of ions from the environment in order to produce minerals, with the process generally being mediated by proteins. Most proteins that are involved in mineral interactions are predicted to contain disordered regions containing large numbers of negatively charged amino acids. Magnetotactic bacteria, which are used as a model system for iron biomineralization, are Gram-negative bacteria that can navigate through geomagnetic fields using a specific organelle, the magnetosome. Each organelle comprises a membrane-enveloped magnetic nanoparticle, magnetite, the formation of which is controlled by a specific set of proteins. One of the most abundant of these proteins is MamC, a small magnetosome-associated integral membrane protein that contains two transmembrane α-helices connected by an ∼21-amino-acid peptide. In vitro studies of this MamC peptide showed that it forms a helical structure that can interact with the magnetite surface and affect the size and shape of the growing crystal. Our results show that a disordered structure of the MamC magnetite-interacting component (MamC-MIC) abolishes its interaction with magnetite particles. Moreover, the size and shape of magnetite crystals grown in in vitro magnetite-precipitation experiments in the presence of this disordered peptide were different from the traits of crystals grown in the presence of other peptides or in the presence of the helical MIC. It is suggested that the helical structure of the MamC-MIC is important for its function during magnetite formation.

Structure prediction of magnetosome-associated proteins.

Magnetotactic bacteria (MTB) are Gram-negative bacteria that can navigate along geomagnetic fields. This ability is a result of a unique intracellular organelle, the magnetosome. These organelles are composed of membrane-enclosed magnetite (Fe3O4) or greigite (Fe3S4) crystals ordered into chains along the cell. Magnetosome formation, assembly, and magnetic nano-crystal biomineralization are controlled by magnetosome-associated proteins (MAPs). Most MAP-encoding genes are located in a conserved genomic region - the magnetosome island (MAI). The MAI appears to be conserved in all MTB that were analyzed so far, although the MAI size and organization differs between species. It was shown that MAI deletion leads to a non-magnetic phenotype, further highlighting its important role in magnetosome formation. Today, about 28 proteins are known to be involved in magnetosome formation, but the structures and functions of most MAPs are unknown. To reveal the structure-function relationship of MAPs we used bioinformatics tools in order to build homology models as a way to understand their possible role in magnetosome formation. Here we present a predicted 3D structural models' overview for all known Magnetospirillum gryphiswaldense strain MSR-1 MAPs.