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.

Tyr-Asp inhibition of glyceraldehyde 3-phosphate dehydrogenase affects plant redox metabolism.

How organisms integrate metabolism with the external environment is a central question in biology. Here, we describe a novel regulatory small molecule, a proteogenic dipeptide Tyr-Asp, which improves plant tolerance to oxidative stress by directly interfering with glucose metabolism. Specifically, Tyr-Asp inhibits the activity of a key glycolytic enzyme, glyceraldehyde 3-phosphate dehydrogenase (GAPC), and redirects glucose toward pentose phosphate pathway (PPP) and NADPH production. In line with the metabolic data, Tyr-Asp supplementation improved the growth performance of both Arabidopsis and tobacco seedlings subjected to oxidative stress conditions. Moreover, inhibition of Arabidopsis phosphoenolpyruvate carboxykinase (PEPCK) activity by a group of branched-chain amino acid-containing dipeptides, but not by Tyr-Asp, points to a multisite regulation of glycolytic/gluconeogenic pathway by dipeptides. In summary, our results open the intriguing possibility that proteogenic dipeptides act as evolutionarily conserved small-molecule regulators at the nexus of stress, protein degradation, and metabolism.

LeishIF4E2 is a cap-binding protein that plays a role in Leishmania cell cycle progression.

Leishmania encode six paralogs of the cap-binding protein eIF4E and five eIF4G candidates, forming unique complexes. Two cap-binding proteins, LeishIF4E1 and LeishIF4E2, do not bind any identified LeishIF4Gs, thus their roles are intriguing. Here, we combine structural prediction, proteomic analysis, and interaction assays to shed light on LeishIF4E2 function. A nonconserved C-terminal extension was identified through structure prediction and sequence alignment. m7 GTP-binding assays involving both recombinant and transgenic LeishIF4E2 with and without the C-terminal extension revealed that this extension functions as a regulatory gate, modulating the cap-binding activity of LeishIF4E2. The interactomes of the two LeishIF4E2 versions were investigated, highlighting the role of the C-terminal extension in binding to SLBP2. SLBP2 is known to interact with a stem-loop structure in the 3' UTRs of histone mRNAs. Consistent with the predicted inhibitory effect of SLBP2 on histone expression in Xenopus laevis, a hemizygous deletion mutant of LeishIF4E2, exhibited an upregulation of several histones. We therefore propose that LeishIF4E2 is involved in histone expression, possibly through its interaction between SLBP2 and LeishIF4E2, thus affecting cell cycle progression. In addition, cell synchronization showed that LeishIF4E2 expression decreased during the S-phase, when histones are known to be synthesized. Previous studies in T. brucei also highlighted an association between TbEIF4E2 and SLBP2, and further reported on an interaction between TbIF4E2 and S-phase-abundant mRNAs. Our results show that overexpression of LeishIF4E2 correlates with upregulation of cell cycle and chromosome maintenance proteins. Along with its effect on histone expression, we propose that LeishIF4E2 is involved in cell cycle progression.

A Missense Variation in PHACTR2 Associates with Impaired Actin Dynamics, Dilated Cardiomyopathy, and Left Ventricular Non-Compaction in Humans.

Dilated cardiomyopathy (DCM) with left ventricular non-compaction (LVNC) is a primary myocardial disease leading to contractile dysfunction, progressive heart failure, and excessive risk of sudden cardiac death. Using whole-exome sequencing to investigate a possible genetic cause of DCM with LVNC in a consanguineous child, a homozygous nucleotide change c.1532G>A causing p.Arg511His in PHACTR2 was found. The missense change can affect the binding of PHACTR2 to actin by eliminating the hydrogen bonds between them. The amino acid change does not change PHACTR2 localization to the cytoplasm. The patient's fibroblasts showed a decreased globular to fibrillary actin ratio compared to the control fibroblasts. The re-polymerization of fibrillary actin after treatment with cytochalasin D, which disrupts the actin filaments, was slower in the patient's fibroblasts. Finally, the patient's fibroblasts bridged a scar gap slower than the control fibroblasts because of slower and indirect movement. This is the first report of a human variation in this PHACTR family member. The knock-out mouse model presented no significant phenotype. Our data underscore the importance of PHACTR2 in regulating the monomeric actin pool, the kinetics of actin polymerization, and cell movement, emphasizing the importance of actin regulation for the normal function of the human heart.

Novel Susceptibility Genes Drive Familial Non-Medullary Thyroid Cancer in a Large Consanguineous Kindred.

Familial non-medullary thyroid cancer (FNMTC) is a well-differentiated thyroid cancer (DTC) of follicular cell origin in two or more first-degree relatives. Patients typically demonstrate an autosomal dominant inheritance pattern with incomplete penetrance. While known genes and chromosomal loci account for some FNMTC, the molecular basis for most FNMTC remains elusive. To identify the variation(s) causing FNMTC in an extended consanguineous family consisting of 16 papillary thyroid carcinoma (PTC) cases, we performed whole exome sequence (WES) analysis of six family patients. We demonstrated an association of ARHGEF28, FBXW10, and SLC47A1 genes with FNMTC. The variations in these genes may affect the structures of their encoded proteins and, thus, their function. The most promising causative gene is ARHGEF28, which has high expression in the thyroid, and its protein-protein interactions (PPIs) suggest predisposition of PTC through ARHGEF28-SQSTM1-TP53 or ARHGEF28-PTCSC2-FOXE1-TP53 associations. Using DNA from a patient's thyroid malignant tissue, we analyzed the possible cooperation of somatic variations with these genes. We revealed two somatic heterozygote variations in XRCC1 and HRAS genes known to implicate thyroid cancer. Thus, the predisposition by the germline variations and a second hit by somatic variations could lead to the progression to PTC.

Distinct Regions of the Haloferax volcanii Dolichol Phosphate-Mannose Synthase AglD Mediate the Assembly and Subsequent Processing of the Lipid-Linked Mannose.

Haloferax volcanii AglD is currently the only archaeal dolichol phosphate (DolP)-mannose synthase shown to participate in N-glycosylation. However, the relation between AglD and Pyrococcus furiosus PF0058, the only archaeal DolP-mannose synthase for which structural information is presently available, was unclear. In this report, similarities between the PF0058 and AglD catalytic domains were revealed. At the same time, AglD includes a transmembrane domain far longer than that of PF0058 or other DolP-mannose synthases. To determine whether this extension affords AglD functions in addition to generating mannose-charged DolP, a series of Hfx. volcanii strains expressing truncated versions of AglD was generated. Mass spectrometry revealed that a version of AglD comprising the catalytic domain and only two of the six to nine predicted membrane-spanning domains could mediate mannose addition to DolP. However, in cells expressing this or other truncated versions of AglD, mannose was not transferred from the lipid to the protein-bound tetrasaccharide precursor of the N-linked pentasaccharide normally decorating Hfx. volcanii glycoproteins. These results thus point to AglD as contributing to additional aspects of Hfx. volcanii N-glycosylation beyond charging DolP with mannose. Accordingly, the possibility that AglD, possibly in coordination with AglR, translocates DolP-mannose across the plasma membrane is discussed.

High-Resolution Cryo-Electron Microscopy Reveals the Unique Striated Hollow Structure of Photocatalytic Macrocyclic Polydiacetylene Nanotubes.

High-resolution structures are crucial for understanding the functional properties of nanomaterials. We applied single-particle cryo-electron microscopy (cryo-EM), a method traditionally used for structure determination of biological macromolecules, to obtain high-resolution structures of synthetic non-biological filaments formed by photopolymerization of macrocyclic diacetylene (MDA) amphiphilic monomers. Tomographic analysis showed that the MDA monomers self-assemble into hollow nanotubes upon dispersion in water. Single-particle analysis revealed tubes consisting of six pairs of covalently bonded filaments held together by hydrophobic interactions, where each filament is composed of macrocyclic rings stacked in parallel "chair" conformations. The hollow MDA nanotube structures we found may account for the efficient scavenging of amphiphilic pollutants in water and subsequent photodegradation of the guest species.

PSMC1 variant causes a novel neurological syndrome.

Proteasome 26S, the eukaryotic proteasome, serves as the machinery for cellular protein degradation. It is composed of the 20S core particle and one or two 19S regulatory particles, composed of a base and a lid. To date, several human diseases have been associated with mutations within the 26S proteasome subunits; only one of them affects a base subunit. We now delineate an autosomal recessive syndrome of failure to thrive, severe developmental delay and intellectual disability, spastic tetraplegia with central hypotonia, chorea, hearing loss, micropenis and undescended testes, as well as mild elevation of liver enzymes. None of the affected individuals achieved verbal communication or ambulation. Ventriculomegaly was evident on MRI. Homozygosity mapping combined with exome sequencing revealed a disease-associated p.I328T PSMC1 variant. Protein modeling demonstrated that the PSMC1 variant is located at the highly conserved putative ATP binding and hydrolysis domain, and is suggested to interrupt a hydrophobic core within the protein. Fruit flies in which we silenced the Drosophila ortholog Rpt2 specifically in the eye exhibited an apparent phenotype that was highly rescued by the human wild-type PSMC1, yet only partly by the mutant PSMC1, proving the functional effect of the p.I328T disease-causing variant.

Flexible microtubule anchoring modulates the bi-directional motility of the kinesin-5 Cin8.

Two modes of motility have been reported for bi-directional kinesin-5 motors: (a) context-dependent directionality reversal, a mode in which motors undergo persistent minus-end directed motility at the single-molecule level and switch to plus-end directed motility in different assays or under different conditions, such as during MT gliding or antiparallel sliding or as a function of motor clustering; and (b) bi-directional motility, defined as movement in two directions in the same assay, without persistent unidirectional motility. Here, we examine how modulation of motor-microtubule (MT) interactions affects these two modes of motility for the bi-directional kinesin-5, Cin8. We report that the large insert in loop 8 (L8) within the motor domain of Cin8 increases the MT affinity of Cin8 in vivo and in vitro and is required for Cin8 intracellular functions. We consistently found that recombinant purified L8 directly binds MTs and L8 induces single Cin8 motors to behave according to context-dependent directionality reversal and bi-directional motility modes at intermediate ionic strength and according to a bi-directional motility mode in an MT surface-gliding assay under low motor density conditions. We propose that the largely unstructured L8 facilitates flexible anchoring of Cin8 to the MTs. This flexible anchoring enables the direct observation of bi-directional motility in motility assays. Remarkably, although L8-deleted Cin8 variants exhibit a strong minus-end directed bias at the single-molecule level, they also exhibit plus-end directed motility in an MT-gliding assay. Thus, L8-induced flexible MT anchoring is required for bi-directional motility of single Cin8 molecules but is not necessary for context-dependent directionality reversal of Cin8 in an MT-gliding assay.

The cation diffusion facilitator protein MamM's cytoplasmic domain exhibits metal-type dependent binding modes and discriminates against Mn2.

Cation diffusion facilitator (CDF) proteins are a conserved family of divalent transition metal cation transporters. CDF proteins are usually composed of two domains: the transmembrane domain, in which the metal cations are transported through, and a regulatory cytoplasmic C-terminal domain (CTD). Each CDF protein transports either one specific metal or multiple metals from the cytoplasm, and it is not known whether the CTD takes an active regulatory role in metal recognition and discrimination during cation transport. Here, the model CDF protein MamM, an iron transporter from magnetotactic bacteria, was used to probe the role of the CTD in metal recognition and selectivity. Using a combination of biophysical and structural approaches, the binding of different metals to MamM CTD was characterized. Results reveal that different metals bind distinctively to MamM CTD in terms of their binding sites, thermodynamics, and binding-dependent conformations, both in crystal form and in solution, which suggests a varying level of functional discrimination between CDF domains. Furthermore, these results provide the first direct evidence that CDF CTDs play a role in metal selectivity. We demonstrate that MamM's CTD can discriminate against Mn2+, supporting its postulated role in preventing magnetite formation poisoning in magnetotactic bacteria via Mn2+ incorporation.

Aldosterone synthase (CYP11B2) deficiency among Palestinian infants: Three novel variants and genetic heterogeneity.

Aldosterone synthase deficiency (ASD) is a rare potentially life-threatening genetic disorder that usually presents during infancy due to pathogenic variants in the CYP11B2 gene. Knowledge about CYP11B2 variants in the Arab population is scarce. Here, we present and analyze five Palestinian patients and their different novel pathogenic variants. Data on clinical presentation, electrolytes, plasma renin activity, and steroid hormone levels of five patients diagnosed with ASD were summarized. Sequencing of the CYP11B2 gene exons was followed by evolutionary conservation analysis and structural modeling of the variants. All patients were from highly consanguineous Palestinian families. The patients presented at 1-4 months of age with recurrent vomiting, poor weight gain, hyponatremia, hyperkalemia, and low aldosterone levels. Genetic analysis of the CYP11B2 gene revealed three homozygous pathogenic variants: p.Ser344Profs*9, p.G452W in two patients from an extended family, and p.Q338stop. A previously described pathogenic variant was found in one patient: p.G288S. We described four different CYP11B2 gene pathogenic variants in a relatively small population. Our findings may contribute to the future early diagnosis and therapy for patients with ASD among Arab patients who present with failure to thrive and compatible electrolyte disturbances.

Specificity of protein-DNA interactions in hypersaline environment: structural studies on complexes of Halobacterium salinarum oxidative stress-dependent protein hsRosR.

Interactions between proteins and DNA are crucial for all biological systems. Many studies have shown the dependence of protein-DNA interactions on the surrounding salt concentration. How these interactions are maintained in the hypersaline environments that halophiles inhabit remains puzzling. Towards solving this enigma, we identified the DNA motif recognized by the Halobactrium salinarum ROS-dependent transcription factor (hsRosR), determined the structure of several hsRosR-DNA complexes and investigated the DNA-binding process under extreme high-salt conditions. The picture that emerges from this work contributes to our understanding of the principles underlying the interplay between electrostatic interactions and salt-mediated protein-DNA interactions in an ionic environment characterized by molar salt concentrations.

A common genetic variant in zinc transporter ZnT2 (Thr288Ser) is present in women with low milk volume and alters lysosome function and cell energetics.

Suboptimal lactation is a common, yet underappreciated cause for early cessation of breastfeeding. Molecular regulation of mammary gland function is critical to the process lactation; however, physiological factors underlying insufficient milk production are poorly understood. The zinc (Zn) transporter ZnT2 is critical for regulation of mammary gland development and maturation during puberty, lactation, and postlactation gland remodeling. Numerous genetic variants in the gene encoding ZnT2 (SLC30A2) are associated with low milk Zn concentration and result in severe Zn deficiency in exclusively breastfed infants. However, the functional impacts of genetic variation in ZnT2 on key mammary epithelial cell functions have not yet been systematically explored at the cellular level. Here we determined a common mutation in SLC30A2/ZnT2 substituting serine for threonine at amino acid 288 (Thr288Ser) was found in 20% of women producing low milk volume (n = 2/10) but was not identified in women producing normal volume. Exploration of cellular consequences in vitro using phosphomimetics showed the serine substitution promoted preferential phosphorylation of ZnT2, driving localization to the lysosome and increasing lysosome biogenesis and acidification. While the substitution did not initiate lysosome-mediated cell death, cellular ATP levels were significantly reduced. Our findings demonstrate the Thr288Ser mutation in SLC30A2/ZnT2 impairs critical functions of mammary epithelial cells and suggest a role for genetic variation in the regulation of milk production and lactation performance.

Zinc transporter 10 (ZnT10)-dependent extrusion of cellular Mn2+ is driven by an active Ca2+-coupled exchange.

Manganese (Mn2+) is extruded from the cell by the zinc transporter 10 (ZnT10). Loss of ZnT10 expression caused by autosomal mutations in the ZnT10 gene leads to hypermanganesemia in multiple organs. Here, combining fluorescent monitoring of cation influx in HEK293-T cells expressing human ZnT10 with molecular modeling of ZnT10 cation selectivity, we show that ZnT10 is exploiting the transmembrane Ca2+ inward gradient for active cellular exchange of Mn2+ In analyzing ZnT10 activity we used the ability of Fura-2 to spectrally distinguish between Mn2+ and Ca2+ fluxes. We found that (a) application of Mn2+-containing Ca2+-free solution to ZnT10-expressing cells triggers an influx of Mn2+, (b) reintroduction of Ca2+ leads to cellular Mn2+ extrusion against an inward Mn2+ gradient, and (c) the cellular transport of Mn2+ by ZnT10 is coupled to a reciprocal movement of Ca2+ Remarkably, replacing a single asparagine residue in ZnT10 (Asp-43) with threonine (ZnT10 N43T) converted the Mn2+/Ca2+ exchange to an uncoupled channel mode, permeable to both Ca2+ and Mn2+ The findings in our study identify the first ion transporter that uses the Ca2+ gradient for active counter-ion exchange. They highlight a remarkable versatility in metal selectivity and mode of transport controlled by the tetrahedral metal transport site of ZnT proteins.

Folding of an Intrinsically Disordered Iron-Binding Peptide in Response to Sedimentation Revealed by Cryo-EM.

Biomineralization is mediated by specialized proteins that guide and control mineral sedimentation. In many cases, the active regions of these biomineralization proteins are intrinsically disordered. High-resolution structures of these proteins while they interact with minerals are essential for understanding biomineralization processes and the function of intrinsically disordered proteins (IDPs). Here we used the cavity of ferritin as a nanoreactor where the interaction between M6A, an intrinsically disordered iron-binding domain, and an iron oxide particle was visualized at high resolution by cryo-EM. Taking advantage of the differences in the electron-dose sensitivity of the protein and the iron oxide particles, we developed a method to determine the irregular shape of the particles found in our density maps. We found that the folding of M6A correlates with the detection of mineral particles in its vicinity. M6A interacts with the iron oxide particles through its C-terminal side, resulting in the stabilization of a helix at its N-terminal side. The stabilization of the helix at a region that is not in direct contact with the iron oxide particle demonstrates the ability of IDPs to respond to signals from their surroundings by conformational changes. These findings provide the first glimpse toward the long-suspected mechanism for biomineralization protein control over mineral microstructure, where unstructured regions of these proteins become more ordered in response to their interaction with the nascent mineral particles.

Correction: Mitochondrial 16S rRNA Is Methylated by tRNA Methyltransferase TRMT61B in All Vertebrates.

[This corrects the article DOI: 10.1371/journal.pbio.1002557.].

CAP2 mutation leads to impaired actin dynamics and associates with supraventricular tachycardia and dilated cardiomyopathy.

BACKGROUND: Dilated cardiomyopathy (DCM) is a primary myocardial disease leading to contractile dysfunction, progressive heart failure and excessive risk of sudden cardiac death. Around half of DCM cases are idiopathic, and genetic factors seem to play an important role. AIM: We investigated a possible genetic cause of DCM in two consanguineous children from a Bedouin family. METHODS AND RESULTS: Using exome sequencing and searching for rare homozygous variations, we identified a nucleotide change in the donor splice consensus sequence of exon 7 in CAP2 as the causative mutation. Using patient-derived fibroblasts, we demonstrated that the mutation causes skipping of exons 6 and 7. The resulting protein is missing 64 amino acids in its N-CAP domain that should prevent its correct folding. CAP2 protein level was markedly reduced without notable compensation by the homolog CAP1. However, ß-actin mRNA was elevated as demonstrated by real-time qPCR. In agreement with the essential role of CAP2 in actin filament polymerization, we demonstrate that the mutation affects the kinetics of repolymerization of actin in patient fibroblasts. CONCLUSIONS: This is the first report of a recessive deleterious mutation in CAP2 and its association with DCM in humans. The clinical phenotype recapitulates the damaging effects on the heart observed in Cap2 knockout mice including DCM and cardiac conduction disease, but not the other effects on growth, viability, wound healing and eye development. Our data underscore the importance of the proper kinetics of actin polymerization for normal function of the human heart.

The dual role of MamB in magnetosome membrane assembly and magnetite biomineralization.

Magnetospirillum gryphiswaldense MSR-1 synthesizes membrane-enclosed magnetite (Fe3 O4 ) nanoparticles, magnetosomes, for magnetotaxis. Formation of these organelles involves a complex process comprising key steps which are governed by specific magnetosome-associated proteins. MamB, a cation diffusion facilitator (CDF) family member has been implicated in magnetosome-directed iron transport. However, deletion mutagenesis studies revealed that MamB is essential for the formation of magnetosome membrane vesicles, but its precise role remains elusive. In this study, we employed a multi-disciplinary approach to define the role of MamB during magnetosome formation. Using site-directed mutagenesis complemented by structural analyses, fluorescence microscopy and cryo-electron tomography, we show that MamB is most likely an active magnetosome-directed transporter serving two distinct, yet essential functions. First, MamB initiates magnetosome vesicle formation in a transport-independent process, probably by serving as a landmark protein. Second, MamB transport activity is required for magnetite nucleation. Furthermore, by determining the crystal structure of the MamB cytosolic C-terminal domain, we also provide mechanistic insight into transport regulation. Additionally, we present evidence that magnetosome vesicle growth and chain formation are independent of magnetite nucleation and magnetic interactions respectively. Together, our data provide novel insight into the role of the key bifunctional magnetosome protein MamB, and the early steps of magnetosome formation.

Mitochondrial 16S rRNA Is Methylated by tRNA Methyltransferase TRMT61B in All Vertebrates.

The mitochondrial ribosome, which translates all mitochondrial DNA (mtDNA)-encoded proteins, should be tightly regulated pre- and post-transcriptionally. Recently, we found RNA-DNA differences (RDDs) at human mitochondrial 16S (large) rRNA position 947 that were indicative of post-transcriptional modification. Here, we show that these 16S rRNA RDDs result from a 1-methyladenosine (m1A) modification introduced by TRMT61B, thus being the first vertebrate methyltransferase that modifies both tRNA and rRNAs. m1A947 is conserved in humans and all vertebrates having adenine at the corresponding mtDNA position (90% of vertebrates). However, this mtDNA base is a thymine in 10% of the vertebrates and a guanine in the 23S rRNA of 95% of bacteria, suggesting alternative evolutionary solutions. m1A, uridine, or guanine may stabilize the local structure of mitochondrial and bacterial ribosomes. Experimental assessment of genome-edited Escherichia coli showed that unmodified adenine caused impaired protein synthesis and growth. Our findings revealed a conserved mechanism of rRNA modification that has been selected instead of DNA mutations to enable proper mitochondrial ribosome function.

The 3-D structure of VNG0258H/RosR - A haloarchaeal DNA-binding protein in its ionic shell.

Protein-DNA interactions are highly dependent on salt concentration. To gain insight into how such interactions are maintained in the highly saline cytoplasm of halophilic archaea, we determined the 3-D structure of VNG0258H/RosR, the first haloarchaeal DNA-binding protein from the extreme halophilic archaeon Halobactrium salinarum. It is a dimeric winged-helix-turn-helix (wHTH) protein with unique features due to adaptation to the halophilic environment. As ions are major players in DNA binding processes, particularly in halophilic environments, we investigated the solution structure of the ionic envelope and located anions in the first shell around the protein in the crystal using anomalous scattering. Anions that were found to be tightly bound to residues in the positively charged DNA-binding site would probably be released upon DNA binding and will thus make significant contribution to the driving force of the binding process. Unexpectedly, ions were also found in a buried internal cavity connected to the external medium by a tunnel. Our structure lays a solid groundwork for future structural, computational and biochemical studies on complexes of the protein with cognate DNA sequences, with implications to protein-DNA interactions in hyper-saline environments.