Search

Oncogenic mutations in isocitrate dehydrogenase 1 (IDH1) and IDH2 occur in a wide range of cancers, including acute myeloid leukemia (AML) and glioma. Mutant IDH enzymes convert 2-oxoglutarate (2OG) to (R)-2-hydroxyglutarate [(R)-2HG], an oncometabolite that is hypothesized to promote cellular transformation by dysregulating 2OG-dependent enzymes. The only (R)-2HG target that has been convincingly shown to contribute to transformation by mutant IDH is the myeloid tumor suppressor TET2. However, there is ample evidence to suggest that (R)-2HG has other functionally relevant targets in IDH-mutant cancers. Here, we show that (R)-2HG inhibits KDM5 histone lysine demethylases and that this inhibition contributes to cellular transformation in IDH-mutant AML and IDH-mutant glioma. These studies provide the first evidence of a functional link between dysregulation of histone lysine methylation and transformation in IDH-mutant cancers. Significance: Mutant IDH is known to induce histone hypermethylation. However, it is not known if this hypermethylation is functionally significant or is a bystander effect of (R)-2HG accumulation in IDH-mutant cells. Here, we provide evidence that KDM5 inhibition by (R)-2HG contributes to mutant IDH–mediated transformation in AML and glioma.

Characterization of isogenic TF-1 cell lines expressing TET2-targeting shRNAs.

Supplementary Table S9 shows the gene set enrichment analysis gene set that corresponds to Figure S6A.

List of supplementary figures and tables.

Oncogenic mutations in isocitrate dehydrogenase 1 (IDH1) and IDH2 occur in a wide range of cancers, including acute myeloid leukemia (AML) and glioma. Mutant IDH enzymes convert 2-oxoglutarate (2OG) to (R)-2-hydroxyglutarate [(R)-2HG], an oncometabolite that is hypothesized to promote cellular transformation by dysregulating 2OG-dependent enzymes. The only (R)-2HG target that has been convincingly shown to contribute to transformation by mutant IDH is the myeloid tumor suppressor TET2. However, there is ample evidence to suggest that (R)-2HG has other functionally relevant targets in IDH-mutant cancers. Here, we show that (R)-2HG inhibits KDM5 histone lysine demethylases and that this inhibition contributes to cellular transformation in IDH-mutant AML and IDH-mutant glioma. These studies provide the first evidence of a functional link between dysregulation of histone lysine methylation and transformation in IDH-mutant cancers.Significance:Mutant IDH is known to induce histone hypermethylation. However, it is not known if this hypermethylation is functionally significant or is a bystander effect of (R)-2HG accumulation in IDH-mutant cells. Here, we provide evidence that KDM5 inhibition by (R)-2HG contributes to mutant IDH–mediated transformation in AML and glioma.

HIDEA syndrome is caused by biallelic pathogenic variants in P4HTM. The phenotype is characterized by muscular and central hypotonia, hypoventilation including obstructive and central sleep apneas, intellectual disability, dysautonomia, epilepsy, eye abnormalities, and an increased tendency to develop respiratory distress during pneumonia. Here, we report six new patients with HIDEA syndrome caused by five different biallelic P4HTM variants, including three novel variants. We describe two Finnish enriched pathogenic P4HTM variants and demonstrate that these variants are embedded within founder haplotypes. We review the clinical data from all previously published patients with HIDEA and characterize all reported P4HTM pathogenic variants associated with HIDEA in silico. All known pathogenic variants in P4HTM result in either premature stop codons, an intragenic deletion, or amino acid changes that impact the active site or the overall stability of P4H-TM protein. In all cases, normal P4H-TM enzyme function is expected to be lost or severely decreased. This report expands knowledge of the genotypic and phenotypic spectrum of the disease. publishedVersion

Golden open access means publishing scholarly articles in journals that are open without costs for all readers. Green open access means secondary publishing of toll access articles in institutional repositories. Thus, it offers scholars a nice possibility to promote open access without giving up their preference of publishing in high-ranking toll-access journals. But what are the real pros and cons of this path to open access publishing culture?

Kontrolloitu digitaalinen lainaaminen (Controlled Digital Lending, CDL) on lainauskäytäntö, jonka mukaan elektroninen ja Internetissä toimiva kirjasto voi täysin laillisesti digitoida omistamansa kirjan ja lainata digitoitua kappaletta elektronisesti niin, että koko ajan ainoastaan yksi kappale on lainassa tai lainattavissa. Tämän käytännön uranuurtajana on toiminut Internet Archive, mutta se on saanut myös seuraajia, joista tässä artikkelissa esitellään yksi, Digital Theological Library.

A new syndrome with hypotonia, intellectual disability, and eye abnormalities (HIDEA) was previously described in a large consanguineous family. Linkage analysis identified the recessive disease locus, and genome sequencing yielded three candidate genes with potentially pathogenic biallelic variants: transketolase (TKT), transmembrane prolyl 4-hydroxylase (P4HTM), and ubiquitin specific peptidase 4 (USP4). However, the causative gene remained elusive. International collaboration and exome sequencing were used to identify new patients with HIDEA and biallelic, potentially pathogenic, P4HTM variants. Segregation analysis was performed using Sanger sequencing. P4H-TM wild-type and variant constructs without the transmembrane region were overexpressed in insect cells and analyzed using sodium dodecyl sulfate–polyacrylamide gel electrophoresis and western blot. Five different homozygous or compound heterozygous pathogenic P4HTM gene variants were identified in six new and six previously published patients presenting with HIDEA. Hypoventilation, obstructive and central sleep apnea, and dysautonomia were identified as novel features associated with the phenotype. Characterization of three of the P4H-TM variants demonstrated yielding insoluble protein products and, thus, loss-of-function. Biallelic loss-of-function P4HTM variants were shown to cause HIDEA syndrome. Our findings enable diagnosis of the condition, and highlight the importance of assessing the need for noninvasive ventilatory support in patients.

Non

Gain-of-function mutations in isocitrate dehydrogenase enzymes IDH1 and IDH2 occur in ∼10% of acute myeloid leukemias (AML) and >80% of gliomas. The mutant enzymes convert 2-oxoglutarate (2OG) to the oncometabolite R-2-hydroxyglutarate (R-2HG). R-2HG promotes cellular transformation by modulating the activities of 2OG-dependent dioxygenases (2OGDDs). The only functionally validated direct target of R-2HG is TET2, a 2OGDD myeloid tumor suppressor that catalyzes the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Interestingly, in clonal myeloid disorders the patterns of IDH and TET2 mutations are vastly different. TET2 mutations occur at similar frequencies in both low- and high-grade disease. IDH mutations, on the other hand, are associated with higher-grade and blast-phase MPN and MDS and with de novo AML and are rare in CHIP and low-grade MDS. This suggests that mutant IDH promotes a more aggressive disease phenotype and that R-2HG has additional targets other than TET2 that contribute to its leukemogenic activity. To identify other 2OGDD myeloid tumor suppressors that could be contributing to R-2HG-mediated transformation, we performed a positive-selection CRISPR-Cas9 screen under cytokine-poor conditions in TF-1 cells. We identified three H3K4 histone lysine demethylases, KDM5A, KDM5C and KDM5D, as genes whose sgRNAs were enriched upon cytokine withdrawal. Triple knockout of KDM5A, KDM5C and KDM5D (TKO) in TF-1 cells induces robust cytokine independence as does treatment of TF-1 cells with KDM5c70, a KDM5 inhibitor. We further found that R-2HG is a more potent inhibitor of KDM5A, KDM5C and KDM5D than of TET2. We then assessed the effect of mutant IDH1 expression, TKO, R-2HG treatment and KDM5c70 treatment on H3K4 trimethylation by ChIP-seq and found that each of these perturbations results in a significant enrichment in H3K4me3 peaks relative to controls, with the same sites being enriched. We then asked if mutant IDH positivity is associated with increased levels of H3K4 methylation in glioma. We performed ChIP-seq on a panel of IDH wild-type and IDH mutant glioma MCTS lines and found H3K4me peaks to be highly enriched in the IDH mutant lines when compared to IDH wild-type lines. Trimethyl-H3K4 levels were likewise increased in isogenic normal human astrocyte (NHA) cells ectopically expressing mutant IDH1. Expression of mutant IDH or treatment KDM5 inhibitor was sufficient to cause increased colony formation in NHA cells relative to controls. Collectively, these data suggest that R-2HG inhibits KDM5 histone lysine demethylases to promote mutant IDH-mediated transformation in AML and glioma. These studies identify a novel direct target of R-2HG in IDH mutant tumors and provide a functional link between IDH mutations and dysregulated histone lysine methylation in cancer. Citation Format: Kathryn E. Gunn, Matti Myllykoski, John Cao, Bofu Huang, Betty Rouaisnel, Bill Diplas, Michael M. Levitt, Ryan Looper, John G. Doench, Keith L. Ligon, Harley I. Kornblum, Hai Yan, Samuel McBrayer, Lucy A. Godley, Peppi Koivunen, Julie-Aurore Losman. (R)-2-hydroxyglutarate inhibits KDM5 histone lysine demethylases to drive tumorigenesis in IDH-mutant cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5853.

By far most macromolecular crystallographic data collection and experimental phasing is nowadays carried out using synchrotron radiation. Here, we present two crystallographic datasets collected on a home-source X-ray diffractometer, which can per se be use to experimentally solve the atomic-resolution crystal structure of the Src homology 3(SH3)-like domain from the postsynaptic protein Shank3. The refined structure was described in the article “Structure of an unconventional SH3 domain from the postsynaptic density protein Shank3 at ultrahigh resolution” (Ponna et al., 2017) [1]. Crystals of the Shank3 SH3 domain were derivatized through soaking in 1 M sodium iodide prior to diffraction data collection at a wavelength of 1.54 Å. High-resolution data are reported for a native crystal to 1.01 Å and an iodide-derivatized one to 1.60 Å. The crystals suffered from several anomalies affecting experimental phasing: a high fraction (34–40%) of pseudomerohedral twinning, significant pseudotranslational symmetry (> 15%) with the operator 0.5,0,0.5, and a low solvent content. Twinning with the operator h,-k,-l is made possible by the space group P21 coupled with a unit cell β angle of 90.0°. The data can be used to repeat and optimize derivatization and phasing procedures, to understand halide interactions with protein surfaces, to promote the use of home X-ray sources for protein structure determination, as well as for educational purposes and protocol development. Keywords: Protein crystallography, Diffraction, Experimental phasing, Data collection, Home-source X-ray diffractometer

The Shank proteins are crucial scaffolding elements of the post-synaptic density (PSD). One of the best-characterized domains in Shank is the PDZ domain, which binds to C-terminal segments of several other PSD proteins. We carried out a detailed structural analysis of Shank3 PDZ domain-peptide complexes, to understand determinants of binding affinity towards different ligand proteins. Ligand peptides from four different proteins were cocrystallized with the Shank3 PDZ domain, and binding affinities were determined calorimetrically. In addition to conserved class I interactions between the first and third C-terminal peptide residue and Shank3, side chain interactions of other residues in the peptide with the PDZ domain are important factors in defining affinity. Structural conservation suggests that the binding specificities of the PDZ domains from different Shanks are similar. Two conserved buried water molecules in PDZ domains may affect correct local folding of ligand recognition determinants. The solution structure of a tandem Shank3 construct containing the SH3 and PDZ domains showed that the two domains are close to each other, which could be of relevance, when recognizing and binding full target proteins. The SH3 domain did not affect the affinity of the PDZ domain towards short target peptides, and the schizophrenia-linked Shank3 mutation R536W in the linker between the domains had no effect on the structure or peptide interactions of the Shank3 SH3-PDZ unit. Our data show the spatial arrangement of two adjacent Shank domains and pinpoint affinity determinants for short PDZ domain ligands with limited sequence homology.

Gain-of-function mutations in isocitrate dehydrogenase enzymes IDH1 and IDH2 occur in ∼10% of acute myeloid leukemias (AML) and >80% of gliomas. The mutant enzymes convert 2-oxoglutarate (2OG) to the oncometabolite R-2-hydroxyglutarate (R-2HG). R-2HG promotes cellular transformation by modulating the activities of 2OG-dependent dioxygenases (2OGDDs). The only functionally validated direct target of R-2HG is TET2, a 2OGDD myeloid tumor suppressor that catalyzes the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Interestingly, in clonal myeloid disorders the patterns of IDH and TET2 mutations are vastly different. TET2 mutations occur at similar frequencies in clonal hematopoiesis of unknown significance (CHIP), lower- and higher-grade myeloproliferative (MPN) and myelodysplastic (MDS) disorders, and primary and secondary AML. IDH mutations, on the other hand, are associated with higher-grade and blast-phase MPN and MDS and with de novo AML and are rare in CHIP and low-grade MDS. This suggests that mutant IDH promotes a more aggressive disease phenotype and that R-2HG has additional targets other than TET2 that contribute to its leukemogenic activity. To ask if the in vitro transforming activity of R-2HG directly correlates with TET2 inhibition, we treated TF-1 cells, a cytokine-dependent human AML cell line, with a dose range of cell-permeable esterified R-2HG. We found that R-2HG induces cytokine independence at concentrations that have no effect on 5hmC levels. To identify other 2OGDD myeloid tumor suppressors that could be contributing to R-2HG-mediated transformation, we performed a positive-selection CRISPR-Cas9 screen under cytokine-poor conditions in TF-1 cells. We identified three H3K4 histone lysine demethylases, KDM5A, KDM5C and KDM5D, as genes whose sgRNAs were enriched upon cytokine withdrawal. Triple knockout of KDM5A, KDM5C and KDM5D (TKO) in TF-1 cells induces robust cytokine independence. Likewise, treatment of TF-1 cells with KDM5c70, a specific inhibitor of KDM5 enzymes, strongly induces TF-1 cytokine independence. Of note, KDM5 inhibition has no effect on TET2 expression or 5hmC levels. We further found that R-2HG is a more potent inhibitor of KDM5A, KDM5C and KDM5D than of TET2. We then assessed the effect of mutant IDH1 expression, TKO, R-2HG treatment and KDM5c70 treatment on H3K4 trimethylation by ChIP-seq and found that each of these perturbations results in a significant enrichment in H3K4me3 peaks relative to controls. TET enzymes are not recurrently mutated in glioma and although there is a strong correlation between mutant IDH status and the CpG island methylator phenotype (CIMP), direct inhibition of TET2 by R-2HG has not been reproducibly demonstrated in glioma. To ask if TET2 activity is suppressed in IDH mutant glioma, we quantified 5hmC levels in a panel of primary IDH wild-type and IDH mutant glioma and AML samples by mass spectrometry. We found that, unlike in AML, in glioma there is no correlation between IDH1 mutation status and loss of 5hmC. We likewise saw no correlation between 5hmC levels and either IDH mutation status or intracellular R-2HG levels in patient derived xenograft (PDX) models of glioma. Given the lack of evidence that TET enzymes are tumor suppressor targets of R-2HG in IDH mutant glioma, we asked if mutant IDH positivity is associated with increased levels of H3K4 methylation in glioma. We performed ChIP-seq on a panel of IDH wild-type and IDH mutant glioma PDX lines and found H3K4me peaks to be highly enriched in the IDH mutant lines when compared to IDH wild-type lines. Trimethyl-H3K4 levels were likewise increased in isogenic normal human astrocyte (NHA) cells ectopically expressing mutant IDH1. Collectively, these data suggest that R-2HG inhibits KDM5 histone lysine demethylases to promote mutant IDH-mediated transformation in AML and glioma. These studies identify a novel direct target of R-2HG in IDH mutant tumors and provide a functional link between IDH mutations and dysregulated histone lysine methylation in cancer. Disclosures No relevant conflicts of interest to declare.

The Shank family comprises three large multi-domain proteins playing central roles as protein scaffolds in the neuronal postsynaptic density. The Shank proteins are closely linked to neuropsychiatric diseases, such as autism spectrum disorders. One characteristic domain in the Shank family is the SH3 domain, assumed to play a role in protein-protein interactions; however, no specific ligand binding to any Shank SH3 domain has been described. We solved the crystal structure of the SH3 domain from Shank3 at sub-atomic resolution. While the structure presents the canonical SH3 domain fold, the binding site for proline-rich peptides is not conserved. In line with this, no binding of Pro-rich sequences by the Shank3 SH3 domain was observed. Sequence comparisons indicate that all Shank isoforms have similarly lost the classical Pro-rich peptide binding site from the SH3 domain. Whether the corresponding site in the Shank SH3 domains has evolved to bind a non-poly-Pro target sequence is currently not known. Our work provides an intriguing example of the evolution of a well-characterized protein-protein interaction domain within the context of multi-domain protein scaffolds, allowing the conservation of structural features, but losing canonical functional sites. The data are further discussed in light of known mutations in the SH3 domain or its vicinity in the different Shank isoforms.

Collapsin response mediator protein 2 (CRMP-2) is a neuronal protein involved in axonal pathfinding. Intense research is focusing on its role in various neurological diseases. Despite a wealth of studies, not much is known about the molecular mechanisms of CRMP-2 function in vivo. The detailed structure-function relationships of CRMP-2 have also largely remained unknown, in part due to the fact that the available crystal structures lack the C-terminal tail, which is known to be a target for many post-translational modifications and protein interactions. Although CRMP-2, and other CRMPs, belong to the dihydropyrimidinase family, they have lost the enzymatic active site. Drug candidates for CRMP-2-related processes have come up during the recent years, but no reports of CRMP-2 complexes with small molecules have emerged. Here, CRMP-2 was studied at 1.25-Å resolution using X-ray crystallography. In addition, ligands were docked into the homotetrameric structure, and the C-terminal tail of CRMP-2 was produced recombinantly and analyzed. We have obtained the human CRMP-2 crystal structure at atomic resolution and could identify small-molecule binding pockets in the protein. Structures obtained in different crystal forms highlight flexible regions near possible ligand-binding pockets. We also used the CRMP-2 structure to analyze known or suggested post-translational modifications at the 3D structural level. The high-resolution CRMP-2 structure was also used for docking experiments with the sulfur amino acid metabolite lanthionine ketimine and its ester. We show that the C-terminal tail is intrinsically disordered, but it has conserved segments that may act as interaction sites. Our data provide the most accurate structural data on CRMPs to date and will be useful in further computational and experimental studies on CRMP-2, its function, and its binding to small-molecule ligands.

Oxygen sensing revisited The cellular response to hypoxia (oxygen deficiency) is a contributing factor in many human diseases. Previous studies examining the way in which hypoxia alters gene expression have focused on oxygen-sensing enzymes that regulate the activity of a transcription factor called hypoxia-inducible factor (see the Perspective by Gallipoli and Huntly). Chakraborty et al. and Batie et al. now show that hypoxia can also affect gene expression through direct effects on chromatin regulators. Certain histone demethylases, such as KDM6A and KDM5A, were found to be direct sensors of oxygen. In cell-culture models, hypoxia diminished the activity of these enzymes and caused changes in the expression of genes that govern cell fate. Science , this issue p. 1217 , p. 1222 ; see also p. 1148

Mammalian cells express multiple 2-oxoglutarate (OG)-dependent dioxygenases, including many chromatin regulators. The oxygen affinities, and hence oxygen sensing capabilities, of the 2-oxoglutarate (OG)-dependent dioxygenases reported to date vary widely. Hypoxia can affect chromatin, but whether this reflects a direct effect on chromatin-modifying dioxygenases, or indirect effects caused by the hypoxic-induction of the HIF transcription factor or the endogenous 2-OG competitor 2-hydroxyglutarate (2-HG), is unclear. Here we report that hypoxia induces a HIF- and 2-HG-independent histone modification signature consistent with KDM inactivation. We also show that the H3K27 histone demethylase KDM6A (also called UTX), but not its paralog KDM6B, is oxygen-sensitive. KDM6A loss, like hypoxia, prevented H3K27me3 erasure and blocked differentiation. Conversely, restoring H3K27me3 homeostasis in hypoxic cells reversed these effects. Therefore, oxygen directly affects chromatin regulators to control cell fate.One Sentence SummaryKDM6A demethylase activity is diminished under hypoxic conditions and causes changes in gene expression programs that govern cell fate.

The journal of biological chemistry 296, 100197 (2021). doi:10.1074/jbc.RA120.016542, Prolyl 4-hydroxylases (P4Hs) catalyze post-translational hydroxylation of peptidyl proline residues. In addition to collagen P4Hs and hypoxia-inducible factor P4Hs, a third P4H – the poorly characterized endoplasmic reticulum (ER)-localized transmembrane prolyl 4-hydroxylase (P4H-TM) – is found in animals. P4H-TM variants are associated with the familiar neurological HIDEA syndrome, but how these variants might contribute to disease is unknown. Here, we explored this question in a structural and functional analysis of soluble human P4H-TM. The crystal structure revealed an EF-domain with two Ca$^{2+}$-binding motifs inserted within the catalytic domain. A substrate-binding groove was formed between the EF-domain and the conserved core of the catalytic domain. The proximity of the EF-domain to the active site suggests that Ca$^{2+}$-binding is relevant to the catalytic activity. Functional analysis demonstrated that Ca$^{2+}$-binding affinity of P4H-TM is within the range of physiological Ca$^{2+}$ concentration in the ER. P4H-TM was found both as a monomer and a dimer in solution, but the monomer-dimer equilibrium was not regulated by Ca$^{2+}$. The catalytic site contained bound Fe$^{2+}$ and N-oxalylglycine, which is an analogue of the cosubstrate 2-oxoglutarate. Comparison to homologous P4H structures complexed with peptide substrates showed that the substrate interacting residues and the lid structure that folds over the substrate are conserved in P4H-TM, whereas the extensive loop structures that surround the substrate-binding groove, generating a negative surface potential, are different. Analysis of the structure suggests that the HIDEA variants cause loss of P4H-TM function. In conclusion, P4H-TM shares key structural elements with other P4Hs while having an unique EF-domain., Published by Soc., Bethesda, Md.

2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) is an abundant membrane-associated enzyme within the vertebrate myelin sheath. While the physiological function of CNPase still remains to be characterized in detail, it is known - in addition to its in vitro enzymatic activity - to interact with other proteins, small molecules, and membrane surfaces. From an evolutionary point of view, it can be deduced that CNPase is not restricted to myelin-forming cells or vertebrate tissues. Its evolution has involved gene fusion, addition of other small segments with distinct functions, such as membrane attachment, and possibly loss of function at the polynucleotide kinase-like domain. Currently, it is unclear whether the enzymatic function of the conserved phosphodiesterase domain in vertebrate myelin has a physiological role, or if CNPase could actually function - like many other classical myelin proteins - in a more structural role. This article is part of a Special Issue entitled SI: Myelin Evolution.

Compact myelin forms the basis of nerve insulation essential for higher vertebrates. Dozens of myelin membrane bilayers undergo tight stacking, and in the peripheral nervous system, this is partially enabled by myelin protein zero (P0). Consisting of an immunoglobulin (Ig)-like extracellular domain, a single transmembrane helix, and a cytoplasmic extension (P0ct), P0 harbours an important task in ensuring the integrity of compact myelin in the extracellular compartment, referred to as the intraperiod line. Several disease mutations resulting in peripheral neuropathies have been identified for P0, reflecting its physiological importance, but the arrangement of P0 within the myelin ultrastructure remains obscure. We performed a biophysical characterization of recombinant P0ct. P0ct contributes to the binding affinity between apposed cytoplasmic myelin membrane leaflets, which not only results in changes of the bilayer properties, but also potentially involves the arrangement of the Ig-like domains in a manner that stabilizes the intraperiod line. Transmission electron cryomicroscopy of native full-length P0 showed that P0 stacks lipid membranes by forming antiparallel dimers between the extracellular Ig-like domains. The zipper-like arrangement of the P0 extracellular domains between two membranes explains the double structure of the myelin intraperiod line. Our results contribute to the understanding of PNS myelin, the role of P0 therein, and the underlying molecular foundation of compact myelin stability in health and disease., Scientific Reports, 9, ISSN:2045-2322

The close association of myelinated axons and their myelin sheaths involves numerous intercellular molecular interactions. For example, myelin-associated glycoprotein (MAG) mediates myelin-to-axon adhesion and signalling via molecules on the axonal surface. However, knowledge about intracellular binding partners of myelin proteins, including MAG, has remained limited. The two splice isoforms of MAG, S- and L-MAG, display distinct cytoplasmic domains and spatiotemporal expression profiles. We used yeast two-hybrid screening to identify interaction partners of L-MAG and found the dynein light chain DYNLL1 (also termed dynein light chain 8). DYNLL1 homodimers are known to facilitate dimerization of target proteins. L-MAG and DYNLL1 associate with high affinity, as confirmed with recombinant proteins in vitro. Structural analyses of the purified complex indicate that the DYNLL1-binding segment is localized close to the L-MAG C terminus, next to the Fyn kinase Tyr phosphorylation site. The crystal structure of the complex between DYNLL1 and its binding segment on L-MAG shows 2 : 2 binding in a parallel arrangement, indicating a heterotetrameric complex. The homology between L-MAG and previously characterized DYNLL1-ligands is limited, and some details of binding site interactions are unique for L-MAG. The structure of the complex between the entire L-MAG cytoplasmic domain and DYNLL1, as well as that of the extracellular domain of MAG, were modelled based on small-angle X-ray scattering data, allowing structural insights into L-MAG interactions on both membrane surfaces. Our data imply that DYNLL1 dimerizes L-MAG, but not S-MAG, through the formation of a specific 2 : 2 heterotetramer. This arrangement is likely to affect, in an isoform-specific manner, the functions of MAG in adhesion and myelin-to-axon signalling. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/. Read the Editorial Highlight for this article on page 712.

Histone lysine demethylases (KDMs) are 2-oxoglutarate-dependent dioxygenases (2-OGDDs) that regulate gene expression by altering chromatin structure. Their dysregulation has been associated with many cancers. We set out to study the catalytic and inhibitory properties of human KDM4A, KDM4B, KDM5B, KDM6A and KDM6B, aiming in particular to reveal which of these enzymes are targeted by cancer-associated 2-oxoglutarate (2-OG) analogues. We used affinity-purified insect cell-produced enzymes and synthetic peptides with trimethylated lysines as substrates for the in vitro enzyme activity assays. In addition, we treated breast cancer cell lines with cell-permeable forms of 2-OG analogues and studied their effects on the global histone methylation state. Our data show that KDMs have substrate specificity. Among the enzymes studied, KDM5B had the highest affinity for the peptide substrate but the lowest affinity for the 2-OG and the Fe2+ cosubstrate/cofactors. R-2-hydroxyglutarate (R-2HG) was the most efficient inhibitor of KDM6A, KDM4A and KDM4B, followed by S-2HG. This finding was supported by accumulations of the histone H3K9me3 and H3K27me3 marks in cells treated with the cell-permeable forms of these compounds. KDM5B was especially resistant to inhibition by R-2HG, while citrate was the most efficient inhibitor of KDM6B. We conclude that KDM catalytic activity is susceptible to inhibition by tumorigenic 2-OG analogues and suggest that the inhibition of KDMs is involved in the disease mechanism of cancers in which these compounds accumulate, such as the isocitrate dehydrogenase mutations.

Summary The myelin sheath is a multilamellar plasma membrane extension of highly specialized glial cells laid down in regularly spaced segments along axons. Recent studies indicate that myelin is metabolically active and capable of communicating with the underlying axon. To be functionally connected to the neuron, oligodendrocytes maintain non-compacted myelin as cytoplasmic nanochannels. Here, we used high-pressure freezing for electron microscopy to study these cytoplasmic regions within myelin close to their native state. We identified 2,′3′-cyclic nucleotide 3′-phosphodiesterase (CNP), an oligodendrocyte-specific protein previously implicated in the maintenance of axonal integrity, as an essential factor in generating and maintaining cytoplasm within the myelin compartment. We provide evidence that CNP directly associates with and organizes the actin cytoskeleton, thereby providing an intracellular strut that counteracts membrane compaction by myelin basic protein (MBP). Our study provides a molecular and structural framework for understanding how myelin maintains its cytoplasm to function as an active axon-glial unit., Graphical Abstract, Highlights • Characterization of “cytoplasmic channels” in myelin close to their native state • Antagonistic functions of MBP and CNP in generating cytoplasmic channels • CNP interacts with and bundles actin • Reducing MBP levels rescues axonal pathology in CNP-deficient mice, Snaidero et al. provide evidence that a system of cytoplasmic-rich channels is generated in myelin sheaths by the antagonist function of MBP and CNP. The authors suggest that these channels are required to provide trophic support to neurons and maintain functional axon-glial units over a long period of time.

Compact myelin comprises most of the dry weight of myelin, and its insulative nature is the basis for saltatory conduction of nerve impulses. The major dense line (MDL) is a 3-nm compartment between two cytoplasmic leaflets of stacked myelin membranes, mostly occupied by a myelin basic protein (MBP) phase. MBP is an abundant myelin protein involved in demyelinating diseases, such as multiple sclerosis. The association of MBP with lipid membranes has been studied for decades, but the MBP-driven formation of the MDL remains elusive at the biomolecular level. We employed complementary biophysical methods, including atomic force microscopy, cryo-electron microscopy, and neutron scattering, to investigate the formation of membrane stacks all the way from MBP binding onto a single membrane leaflet to the organisation of a stable MDL. Our results support the formation of an amorphous protein phase of MBP between two membrane bilayers and provide a molecular model for MDL formation during myelination, which is of importance when understanding myelin assembly and demyelinating conditions., Scientific Reports, 7 (1), ISSN:2045-2322

2H phosphoesterases catalyze reactions on nucleotide substrates and contain two conserved histidine residues in the active site. Very limited information is currently available on the details of the active site and substrate/product binding during the catalytic cycle of these enzymes. We performed a comprehensive X-ray crystallographic study of mouse 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase), a membrane-associated enzyme present at high levels in the tetrapod myelin sheath. We determined crystal structures of the CNPase phosphodiesterase domain complexed with substrate, product, and phosphorothioate analogues. The data provide detailed information on the CNPase reaction mechanism, including substrate binding mode and coordination of the nucleophilic water molecule. Linked to the reaction, an open/close motion of the β5–α7 loop is observed. The role of the N terminus of helix α7—unique for CNPase in the 2H family—during the reaction indicates that 2H phosphoesterases differ in their respective reaction mechanisms despite the conserved catalytic residues. Furthermore, based on small-angle X-ray scattering, we present a model for the full-length enzyme, indicating that the two domains of CNPase form an elongated molecule. Finally, based on our structural data and a comprehensive bioinformatics study, we discuss the conservation of CNPase in various organisms., Graphical Abstract, Highlights • A detailed structural analysis of the CNPase catalytic cycle was carried out. • Complexes with substrates, products, and analogues highlight roles for a nearby helix and loop in the reaction mechanism. • The full-length CNPase adopts an elongated conformation in solution. • CNPase is a unique member of the 2H family, and the results will help understand its physiological significance.

The post-synaptic density multidomain scaffolding proteins of the Shank family are structurally poorly characterised. The Shank family consists of three members, and domain-specific interactions of Shank are involved in forming a network of proteins at the post-synaptic region for intracellular signalling and cellular scaffolding. While X-ray crystallography has provided some information on individual Shank domains, the structural basis of Shank interactions is still largely unknown. In this study, the production and crystallisation of the previously uncharacterised Shank3 SH3 domain is presented. The highly twinned crystals diffracted synchrotron X-rays to a resolution higher than 0.9 Å, and these crystals will eventually have the potential to provide an ultrahigh-resolution view into the Shank family SH3 domains and their interactions.

Most protein preparations require purification steps prior to biophysical analysis assessing protein stability, secondary structure and degree of folding. It was, therefore, the aim of this study to develop a system to separate and purify a protein from a commercially available medicinal product, recombinant human growth hormone (rhGH) and show preservation of conformation and function following the gel-based procedure. The rhGH was run on clear native (CN) gels and recovered from the gels by electroelution using D-Tube Dialyzer Midi under rigorous cooling. Melting point studies indicated preservation of the structural integrity. This finding was confirmed by synchrotron radiation circular dichroism spectroscopy (SRCD) revealing an identical folding pattern for the sample before and after electrophoretic separation and purification. Synchrotron small-angle X-ray scattering (SAXS) indicated that the sample was folded and monomeric, both before and after separation and purification, and that its shape corresponded well to the known crystal structure of GH. Binding properties of rhGH to a receptor-model system before and after clear native electrophoresis were comparable. This analytical and preparative approach to purify and concentrate a protein preserving conformation and function may be helpful for many applications in analytical, protein and stereochemistry.

Myelin basic protein (MBP) is present between the cytoplasmic leaflets of the compact myelin membrane in both the peripheral and central nervous systems, and characterized to be intrinsically disordered in solution. One of the best-characterized protein ligands for MBP is calmodulin (CaM), a highly acidic calcium sensor. We pulled down MBP from human brain white matter as the major calcium-dependent CaM-binding protein. We then used full-length brain MBP, and a peptide from rodent MBP, to structurally characterize the MBP-CaM complex in solution by small-angle X-ray scattering, NMR spectroscopy, synchrotron radiation circular dichroism spectroscopy, and size exclusion chromatography. We determined 3D structures for the full-length protein-protein complex at different stoichiometries and detect ligand-induced folding of MBP. We also obtained thermodynamic data for the two CaM-binding sites of MBP, indicating that CaM does not collapse upon binding to MBP, and show that CaM and MBP colocalize in myelin sheaths. In addition, we analyzed the post-translational modifications of rat brain MBP, identifying a novel MBP modification, glucosylation. Our results provide a detailed picture of the MBP-CaM interaction, including a 3D model of the complex between full-length proteins.

In an article published earlier this year (NTS 55.1: 104–15), a full reconstruction of the less intelligible side of P.Oxy. 4009 (lines 1–13) was presented, and it was argued that this text belongs to the Gospel of Peter. These 13 lines parallel the Lukan pericope of the sinful woman (Luke 7.45–49) and demonstrate that the Gospel of Peter used manuscripts that represent the Western text of the earlier Gospels. The most notable Western feature, the omission in P.Oxy. 4009 of Luke 7.47b–48, is no coincidence. There are weighty arguments for the omission of these verses in the Lukan original as well.

In 1993, Dieter Lührmann published a reconstruction of the more intelligible side of P.Oxy. 4009. He demonstrated that this side, which he called the recto, consists of passages parallel to Matt 10.16 par., Luke 10.3 and 2 Clem. 5.2–4. He also argued that the passage stems from the Gospel of Peter. However, Lührmann considered it impossible (‘ausgeschlossen’) to reconstruct the other side of the fragment. The aim of the present article is to demonstrate that a full reconstruction of this less intelligible side of P.Oxy. 4009, lines 1–13, is possible and that it enriches our knowledge of the Gospel of Peter with a new pericope which is an interesting parallel of Luke 7.36–50. The reconstruction also demonstrates that the side reconstructed by Lührmann is actually the verso, and that both sides together point towards the well-known anti-Jewish redactional tendencies of the author of the Gospel of Peter.

Part I of this article, published in CBR 5.1 (pp. 73-122), includes a general introduction to the scholarship on James the Just, discussion on James and the family of Jesus in the Gospels, Jesus' resurrection and the leadership of James, and a detailed survey on scholarly opinions concerning the so-called apostolic council and the conflict at Antioch (Acts 15 and Gal. 2). Part II of the article focuses on the study of traditions concerning James's ritual purity, his martyrdom, and succession, as well as his role in the Gnostic writings and later Christian evidence. The article concludes with reflections on the role of James the Just in present scholarship.

James the Just, the brother of Jesus, is known from the New Testament as the chief apostle of the Torah-obedient Christians. Up to the last quarter of the twentieth century, Jewish Christianity was regarded as an unimportant branch of the early Christian movement. Correspondingly, there was remarkably little interest in James. However, in the past two decades, while early Christianity has been studied as a form of Judaism, the literature on James has grown considerably. Now some scholars tend to assume that James was a loyal follower of his brother right from the beginning, and that his leadership in the church was stronger than traditionally has been assumed. Fresh studies on Acts 15 and Galatians 2 have opened new questions about the Christian Judaism of James and social formation of the community which he led. Part II of this article, to be published in a later issue of Currents, will treat the rest of the James tradition—James's ritual purity, martyrdom and succession, and his role in the Gnostic writings and later Christian evidence. It will conclude with reflections concerning James and earliest Jewish-Christian theology.

The 2H phosphoesterase family contains enzymes with two His-X-Ser/Thr motifs in the active site. 2H enzymes are found in all kingdoms of life, sharing little sequence identity despite the conserved overall fold and active site. For many 2H enzymes, the physiological function is unknown. Here, we studied the structure of the 2H family member LigT from Escherichia coli both in the apo form and complexed with different active-site ligands, including ATP, 2′-AMP, 3′-AMP, phosphate, and NADP+. Comparisons to the well-characterized vertebrate myelin enzyme 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) highlight specific features of the catalytic cycle and substrate recognition in both enzymes. The role played by the helix α7, unique to CNPases within the 2H family, is apparently taken over by Arg130 in the bacterial enzyme. Other residues and loops lining the active site groove are likely to be important for RNA substrate binding. We visualized conformational changes related to ligand binding, as well as the position of the nucleophilic water molecule. We also present a low-resolution model of E. coli LigT bound to tRNA in solution, and provide a model for RNA binding by LigT, involving flexible loops lining the active site cavity. Taken together, our results both aid in understanding the common features of 2H family enzymes and help highlight the distinct features in the 2H family members, which must result in different reaction mechanisms. Unique aspects in different 2H family members can be observed in ligand recognition and binding, and in the coordination of the nucleophilic water molecule and the reactive phosphate moiety. publishedVersion

Valistuksen aikana alkaneessa Jeesus-tutkimuksessa Jeesusta pidettiin juutalaiseen lakiin sitoutuneena oman kansansa messiaana. Tübingenin koulussa tämän tulkinnan rinnalle nousi ajatus Mooseksen laista irroittautuvasta Jeesuksesta.

The myelin sheath is a unique membrane, tightly wrapped around selected axons in the vertebrate nervous system. The multilayered myelin proteolipid membrane contains a specific set of proteins, many of which share no homology with other known proteins. We are interested in the structure, function, interactions, and dynamics of myelin proteins and use multidisciplinary methods to study these relationships. We have solved crystal structures of myelin-specific proteins, characterized their membrane-binding properties, and initiated experiments to pinpoint details of protein dynamics. Among our most recent results are a comprehensive X-ray crystal structure-based characterization of the reaction cycle of the myelin enzyme CNPase, the identification of several myelin-specific proteins as intrinsically disordered molecules, and the analysis of molecular dynamics in myelin proteins and the lipid bilayers they interact with by neutron scattering. All the results will be important in understanding the formation of the myelin membrane multilayer, as well as the roles the myelin proteins play in myelination and myelin-related diseases.

The myelin sheath is a multilayered membrane in the nervous system, which has unique biochemical properties. Myelin carries a set of specific high-abundance proteins, the structure and function of which are still poorly understood. The proteins of the myelin sheath are involved in a number of neurological diseases, including autoimmune diseases and inherited neuropathies. In this review, we briefly discuss the structural properties and functions of selected myelin-specific proteins (P0, myelin oligodendrocyte glycoprotein, myelin-associated glycoprotein, myelin basic protein, myelin-associated oligodendrocytic basic protein, P2, proteolipid protein, peripheral myelin protein of 22 kDa, 2′,3′-cyclic nucleotide 3′-phosphodiesterase, and periaxin); such properties include, for example, interactions with lipid bilayers and the presence of large intrinsically disordered regions in some myelin proteins. A detailed understanding of myelin protein structure and function at the molecular level will be required to fully grasp their physiological roles in the myelin sheath. © 2013 BioFactors, 2013

2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) is a quantitatively major enzyme in myelin, where it localizes to the non-compact regions and is bound to the membrane surface. Although its catalytic activity in vitro has been characterized, the physiological function and in vivo substrate of CNPase remain unknown. Especially the N-terminal domain has been poorly characterized; previously, we have shown it is involved in CNPase dimerization and RNA binding. Here, we show that purified CNPase binds to the calcium sensor protein calmodulin (CaM) in a calcium-dependent manner; the binding site is in the N-terminal domain of CNPase. CaM does not affect the phosphodiesterase activity of CNPase in vitro, nor does it influence polyadenylic acid binding. The colocalization of CNPase and CaM during Schwann cell myelination in culture was observed, and CaM antagonists induced the colocalization of CNPase with microtubules in differentiated CG-4 oligodendrocytes. An analysis of post-translational modifications of CNPase from rat brain revealed the presence of two novel phosphorylation sites on Tyr110 and Ser169 within the N-terminal domain. The results indicate a role for the N-terminal domain of CNPase in mediating multiple molecular interactions and provide a starting point for detailed structure-function studies on CNPase and its N-terminal domain.