Journal of Biological Chemistry

Current research reports and chronological list of recent articles.


The international scientific Journal of Biological Chemistry (JBC) publishes papers based on original research that are judged to make a novel and important contribution to understanding the molecular and cellular basis of biological processes.

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Journal of Biological Chemistry - Abstracts



Mutant cycle analysis identifies a ligand interaction site in an odorant receptor of the malaria vector Anopheles gambiae [Protein Structure and Folding]

Lack of information about the structure of insect odorant receptors (ORs) hinders the development of more effective repellants to control disease-transmitting insects. Mutagenesis and functional analyses using agonists to map the odorant-binding sites of these receptors have been limited because mutations distant from an agonist-binding site can alter agonist sensitivity. Here we use mutant cycle analysis, an approach for exploring the energetics of protein–protein or protein–ligand interactions, with inhibitors, to identify a component of the odorant-binding site of an OR from the malaria vector, Anopheles gambiae. The closely related odorant-specificity subunits Agam/Or15 and Agam/Or13 were each co-expressed with Agam/Orco (odorant receptor co-receptor subunit) in Xenopus oocytes and assayed by two-electrode voltage clamp electrophysiology. We identified (−)-fenchone as a competitive inhibitor with different potencies at the two receptors and used this difference to screen a panel of 37 Agam/Or15 mutants, surveying all positions that differ between Agam/Or15 and Agam/Or13 in the transmembrane and extracellular regions, identifying position 195 as a determinant of (−)-fenchone sensitivity. Inhibition by (−)-fenchone and six structurally related inhibitors of Agam/Or15 receptors containing each of four different hydrophobic residues at position 195 served as input data for mutant cycle analysis. Several mutant cycles, calculated from the inhibition of two receptors by each of two ligands, yielded coupling energies of ≥1 kcal/mol, indicating a close, physical interaction between the ligand and residue 195 of Agam/Or15. This approach should be useful in further expanding our knowledge of odorant-binding site structures in ORs of disease vector insects.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/dW3xV46eTD4" height="1" width="1" alt=""/>
Datum: 17.11.2017


Plasma membrane phosphatidylinositol 4-phosphate and 4,5-bisphosphate determine the distribution and function of K-Ras4B but not H-Ras proteins [Signal Transduction]

Plasma membrane (PM) localization of Ras proteins is crucial for transmitting signals upon mitogen stimulation. Post-translational lipid modification of Ras proteins plays an important role in their recruitment to the PM. Electrostatic interactions between negatively charged PM phospholipids and basic amino acids found in K-Ras4B (K-Ras) but not in H-Ras are important for permanent K-Ras localization to the PM. Here, we investigated how acute depletion of negatively charged PM polyphosphoinositides (PPIns) from the PM alters the intracellular distribution and activity of K- and H-Ras proteins. PPIns depletion from the PM was achieved either by agonist-induced activation of phospholipase C β or with a rapamycin-inducible system in which various phosphatidylinositol phosphatases were recruited to the PM. Redistribution of the two Ras proteins was monitored with confocal microscopy or with a recently developed bioluminescence resonance energy transfer-based approach involving fusion of the Ras C-terminal targeting sequences or the entire Ras proteins to Venus fluorescent protein. We found that PM PPIns depletion caused rapid translocation of K-Ras but not H-Ras from the PM to the Golgi. PM depletion of either phosphatidylinositol 4-phosphate (PtdIns4P) or PtdIns(4,5)P2 but not PtdIns(3,4,5)P3 was sufficient to evoke K-Ras translocation. This effect was diminished by deltarasin, an inhibitor of the Ras–phosphodiesterase interaction, or by simultaneous depletion of the Golgi PtdIns4P. The PPIns depletion decreased incorporation of [3H]leucine in K-Ras–expressing cells, suggesting that Golgi-localized K-Ras is not as signaling-competent as its PM-bound form. We conclude that PPIns in the PM are important regulators of K-Ras–mediated signals.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/QMKychCrcl4" height="1" width="1" alt=""/>
Datum: 17.11.2017


Bacterial DnaB helicase interacts with the excluded strand to regulate unwinding [Molecular Biophysics]

Replicative hexameric helicases are thought to unwind duplex DNA by steric exclusion (SE) where one DNA strand is encircled by the hexamer and the other is excluded from the central channel. However, interactions with the excluded strand on the exterior surface of hexameric helicases have also been shown to be important for DNA unwinding, giving rise to the steric exclusion and wrapping (SEW) model. For example, the archaeal Sulfolobus solfataricus minichromosome maintenance (SsoMCM) helicase has been shown to unwind DNA via a SEW mode to enhance unwinding efficiency. Using single-molecule FRET, we now show that the analogous Escherichia coli (Ec) DnaB helicase also interacts specifically with the excluded DNA strand during unwinding. Mutation of several conserved and positively charged residues on the exterior surface of EcDnaB resulted in increased interaction dynamics and states compared with wild type. Surprisingly, these mutations also increased the DNA unwinding rate, suggesting that electrostatic contacts with the excluded strand act as a regulator for unwinding activity. In support of this, experiments neutralizing the charge of the excluded strand with a morpholino substrate instead of DNA also dramatically increased the unwinding rate. Of note, although the stability of the excluded strand was nearly identical for EcDnaB and SsoMCM, these enzymes are from different superfamilies and unwind DNA with opposite polarities. These results support the SEW model of unwinding for EcDnaB that expands on the existing SE model of hexameric helicase unwinding to include contributions from the excluded strand to regulate the DNA unwinding rate.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/S0SV8kJjzXA" height="1" width="1" alt=""/>
Datum: 17.11.2017


Structural analyses of the MazEF4 toxin-antitoxin pair in Mycobacterium tuberculosis provide evidence for a unique extracellular death factor [Molecular Biophysics]

The bacterial toxin-antitoxin MazEF system in the tuberculosis (TB)-causing bacterium Mycobacterium tuberculosis is activated under unfavorable conditions, including starvation, antibiotic exposure, and oxidative stress. This system contains the ribonucleolytic enzyme MazF and has emerged as a promising drug target for TB treatments targeting the latent stage of M. tuberculosis infection and reportedly mediates a cell death process via a peptide called extracellular death factor (EDF). Although it is well established that the increase in EDF-mediated toxicity of MazF drives a cell-killing phenomenon, the molecular details are poorly understood. Moreover, the divergence in sequences among reported EDFs suggests that each bacterial species has a unique EDF. To address these open questions, we report here the structures of MazF4 and MazEF4 complexes from M. tuberculosis, representing the first MazEF structures from this organism. We found that MazF4 possesses a negatively charged MazE4-binding pocket in contrast to the positively charged MazE-binding pockets in homologous MazEF complex structures from other bacteria. Moreover, using NMR spectroscopy and biochemical assays, we unraveled the molecular interactions of MazF4 with its RNA substrate and with a new EDF homolog originating from M. tuberculosis. The EDF homolog discovered here possesses a positively charged residue at the C terminus, making this EDF distinct from previously reported EDFs. Overall, our results suggest that M. tuberculosis evolved a unique MazF and EDF and that the distinctive EDF sequence could serve as a starting point for designing new anti-tuberculosis drugs. We therefore conclude that this study might contribute to the development of a new line of anti-tuberculosis agents.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/1lzeX-lIBUo" height="1" width="1" alt=""/>
Datum: 17.11.2017


Prion protein is required for tumor necrosis factor {alpha} (TNF{alpha})-triggered nuclear factor {kappa}B (NF-{kappa}B) signaling and cytokine production [Signal Transduction]

The expression of normal cellular prion protein (PrP) is required for the pathogenesis of prion diseases. However, the physiological functions of PrP remain ambiguous. Here, we identified PrP as being critical for tumor necrosis factor (TNF) α-triggered signaling in a human melanoma cell line, M2, and a pancreatic ductal cell adenocarcinoma cell line, BxPC-3. In M2 cells, TNFα up-regulates the expression of p-IκB-kinase α/β (p-IKKα/β), p-p65, and p-JNK, but down-regulates the IκBα protein, all of which are downstream signaling intermediates in the TNF receptor signaling cascade. When PRNP is deleted in M2 cells, the effects of TNFα are no longer detectable. More importantly, p-p65 and p-JNK responses are restored when PRNP is reintroduced into the PRNP null cells. TNFα also activates NF-κB and increases TNFα production in wild-type M2 cells, but not in PrP-null M2 cells. Similar results are obtained in the BxPC-3 cells. Moreover, TNFα activation of NF-κB requires ubiquitination of receptor-interacting serine/threonine kinase 1 (RIP1) and TNF receptor–associated factor 2 (TRAF2). TNFα treatment increases the binding between PrP and the deubiquitinase tumor suppressor cylindromatosis (CYLD), in these treated cells, binding of CYLD to RIP1 and TRAF2 is reduced. We conclude that PrP traps CYLD, preventing it from binding and deubiquitinating RIP1 and TRAF2. Our findings reveal that PrP enhances the responses to TNFα, promoting proinflammatory cytokine production, which may contribute to inflammation and tumorigenesis.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/jpqgGpZQsgs" height="1" width="1" alt=""/>
Datum: 17.11.2017


Histone arginine demethylase JMJD6 is linked to stress granule assembly through demethylation of the stress granule-nucleating protein G3BP1 [Signal Transduction]

Stress granules (SG) are membrane-less organelles that are condensates of stalled translation initiation complexes and mRNAs. SG formation is a cytoprotective response to environmental stress and results from protein interactions involving regions of low amino acid complexity and poorly defined post-translational modifications of SG components. Many RNA-binding proteins are methylated, and we previously demonstrated that the potent SG–nucleating protein G3BP1 is methylated by protein arginine methyltransferase 1 and 5 (PRMT1 and PRMT5). G3BP1 methylation represses SG formation and is reversible. Here we functionally link JMJD6 (Jumonji C domain-containing protein 6) to G3BP1 demethylation. Our findings reveal that JMJD6 is a novel SG component that interacts with G3BP1 complexes, and its expression reduces G3BP1 monomethylation and asymmetric dimethylation at three Arg residues. Knockdown of JMJD6 repressed SG formation and G3BP1 demethylation, but SG formation and G3BP1 demethylation were rescued with catalytically active but not mutant JMJD6. These results suggest that JMJD6 functions directly or indirectly as an arginine demethylase of G3BP1 that promotes SG formation.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/8rm4Gqzylis" height="1" width="1" alt=""/>
Datum: 17.11.2017


Fluorescence lifetime analyses reveal how the high light-responsive protein LHCSR3 transforms PSII light-harvesting complexes into an energy-dissipative state [Bioenergetics]

In green algae, light-harvesting complex stress-related 3 (LHCSR3) is responsible for the pH-dependent dissipation of absorbed light energy, a function vital for survival under high-light conditions. LHCSR3 binds the photosystem II and light-harvesting complex II (PSII–LHCII) supercomplex and transforms it into an energy-dissipative form under acidic conditions, but the molecular mechanism remains unclear. Here we show that in the green alga Chlamydomonas reinhardtii, LHCSR3 modulates the excitation energy flow and dissipates the excitation energy within the light-harvesting complexes of the PSII supercomplex. Using fluorescence decay–associated spectra analysis, we found that, when the PSII supercomplex is associated with LHCSR3 under high-light conditions, excitation energy transfer from light-harvesting complexes to chlorophyll-binding protein CP43 is selectively inhibited compared with that to CP47, preventing excess excitation energy from overloading the reaction center. By analyzing femtosecond up-conversion fluorescence kinetics, we further found that pH- and LHCSR3-dependent quenching of the PSII—LHCII–LHCSR3 supercomplex is accompanied by a fluorescence emission centered at 684 nm, with a decay time constant of 18.6 ps, which is equivalent to the rise time constant of the lutein radical cation generated within a chlorophyll–lutein heterodimer. These results suggest a mechanism in which LHCSR3 transforms the PSII supercomplex into an energy-dissipative state and provide critical insight into the molecular events and characteristics in LHCSR3-dependent energy quenching.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/2OvmQK5LBi8" height="1" width="1" alt=""/>
Datum: 17.11.2017


Characterization of enhancers and the role of the transcription factor KLF7 in regulating corneal epithelial differentiation [Developmental Biology]

During tissue development, transcription factors bind regulatory DNA regions called enhancers, often located at great distances from the genes they regulate, to control gene expression. The enhancer landscape during embryonic stem cell differentiation has been well characterized. By contrast, little is known about the shared and unique enhancer regulatory mechanisms in different ectodermally derived epithelial cells. Here we use ChIP sequencing (ChIP-seq) to identify domains enriched for the histone marks histone H3 lysine 4 trimethylation, histone H3 lysine 4 monomethylation, and histone H3 lysine 27 acetylation (H3K4me3, H3K4me1, and H3K27ac) and define, for the first time, the super enhancers and typical enhancers active in primary human corneal epithelial cells. We show that regulatory regions are often shared between cell types of the ectodermal lineage and that corneal epithelial super enhancers are already marked as potential regulatory domains in embryonic stem cells. Kruppel-like factor (KLF) motifs were enriched in corneal epithelial enhancers, consistent with the important roles of KLF4 and KLF5 in promoting corneal epithelial differentiation. We now show that the Kruppel family member KLF7 promotes the corneal progenitor cell state; on many genes, KLF7 antagonized the corneal differentiation–promoting KLF4. Furthermore, we found that two SNPs linked previously to corneal diseases, astigmatism, and Stevens-Johnson syndrome fall within corneal epithelial enhancers and alter their activity by disrupting transcription factor motifs that overlap these SNPs. Taken together, our work defines regulatory enhancers in corneal epithelial cells, highlights global gene-regulatory relationships shared among different epithelial cells, identifies a role for KLF7 as a KLF4 antagonist in corneal epithelial cell differentiation, and explains how two SNPs may contribute to corneal diseases.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/-9GjSvrCdeA" height="1" width="1" alt=""/>
Datum: 17.11.2017


Human copper transporter ATP7B (Wilson disease protein) forms stable dimers in vitro and in cells [Membrane Biology]

ATP7B is a copper-transporting P1B-type ATPase (Cu-ATPase) with an essential role in human physiology. Mutations in ATP7B cause the potentially fatal Wilson disease, and changes in ATP7B expression are observed in several cancers. Despite its physiologic importance, the biochemical information about ATP7B remains limited because of a complex multidomain organization of the protein. By analogy with the better characterized prokaryotic Cu-ATPases, ATP7B is assumed to be a single-chain monomer. We show that in eukaryotic cells, human ATP7B forms dimers that can be purified following solubilization. Deletion of the four N-terminal metal-binding domains, characteristic for human ATP7B, does not disrupt dimerization, i.e. the dimer interface is formed by the domains that are conserved among Cu-ATPases. Unlike the full-length ATP7B, which is targeted to the trans-Golgi network, 1–4ΔMBD-7B is targeted primarily to vesicles. This result and the analysis of differentially tagged ATP7B variants indicate that the dimeric structure is retained during ATP7B trafficking between the intracellular compartments. Purified dimeric species of 1–4ΔMBD-7B were characterized by a negative stain electron microscopy in the presence of ADP/MgCl2. Single-particle analysis yielded a low-resolution 3D model that provides the first insight into an overall architecture of a human Cu-ATPase, positions of the main domains, and a dimer interface.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/5XiuLV2uC8c" height="1" width="1" alt=""/>
Datum: 17.11.2017


Pleiotrophin, a multifunctional cytokine and growth factor, induces leukocyte responses through the integrin Mac-1 [Protein Structure and Folding]

Pleiotrophin (PTN) is a multifunctional, cationic, glycosaminoglycan-binding cytokine and growth factor involved in numerous physiological and pathological processes, including tissue repair and inflammation-related diseases. PTN has been shown to promote leukocyte responses by inducing their migration and expression of inflammatory cytokines. However, the mechanisms through which PTN mediates these responses remain unclear. Here, we identified the integrin Mac-1 (αMβ2, CD11b/CD18) as the receptor mediating macrophage adhesion and migration to PTN. We also found that expression of Mac-1 on the surface of human embryonic kidney (HEK) 293 cells induced their adhesion and migration to PTN. Accordingly, PTN promoted Mac-1–dependent cell spreading and initiated intracellular signaling manifested in phosphorylation of Erk1/2. While binding to PTN, Mac-1 on Mac-1–expressing HEK293 cells appears to cooperate with cell-surface proteoglycans because both anti-Mac-1 function-blocking mAb and heparin were required to block adhesion. Moreover, biolayer interferometry and NMR indicated a direct interaction between the αMI domain, the major ligand-binding region of Mac-1, and PTN. Using peptide libraries, we found that in PTN the αMI domain bound sequences enriched in basic and hydrophobic residues, indicating that PTN conforms to the general principle of ligand-recognition specificity of the αMI domain toward cationic proteins/peptides. Finally, using recombinant PTN-derived fragments, we show that PTN contains two distinct Mac-1–binding sites in each of its constitutive domains. Collectively, these results identify PTN as a ligand for the integrin Mac-1 on the surface of leukocytes and suggest that this interaction may play a role in inflammatory responses.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/lV-0WAszY-U" height="1" width="1" alt=""/>
Datum: 17.11.2017


A unique cysteine-rich zinc finger domain present in a majority of class II ribonucleotide reductases mediates catalytic turnover [Enzymology]

Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to the corresponding deoxyribonucleotides, used in DNA synthesis and repair. Two different mechanisms help deliver the required electrons to the RNR active site. Formate can be used as reductant directly in the active site, or glutaredoxins or thioredoxins reduce a C-terminal cysteine pair, which then delivers the electrons to the active site. Here, we characterized a novel cysteine-rich C-terminal domain (CRD), which is present in most class II RNRs found in microbes. The NrdJd-type RNR from the bacterium Stackebrandtia nassauensis was used as a model enzyme. We show that the CRD is involved in both higher oligomeric state formation and electron transfer to the active site. The CRD-dependent formation of high oligomers, such as tetramers and hexamers, was induced by addition of dATP or dGTP, but not of dTTP or dCTP. The electron transfer was mediated by an array of six cysteine residues at the very C-terminal end, which also coordinated a zinc atom. The electron transfer can also occur between subunits, depending on the enzyme's oligomeric state. An investigation of the native reductant of the system revealed no interaction with glutaredoxins or thioredoxins, indicating that this class II RNR uses a different electron source. Our results indicate that the CRD has a crucial role in catalytic turnover and a potentially new terminal reduction mechanism and suggest that the CRD is important for the activities of many class II RNRs.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/qDjWJln2axY" height="1" width="1" alt=""/>
Datum: 17.11.2017


Lysine trimethylation regulates 78-kDa glucose-regulated protein proteostasis during endoplasmic reticulum stress [Genomics and Proteomics]

The up-regulation of chaperones such as the 78-kDa glucose-regulated protein (GRP78, also referred to as BiP or HSPA5) is part of the adaptive cellular response to endoplasmic reticulum (ER) stress. GRP78 is widely used as a marker of the unfolded protein response, associated with sustained ER stress. Here we report the discovery of a proteostatic mechanism involving GRP78 trimethylation in the context of ER stress. Using mass spectrometry–based proteomics, we identified two GRP78 fractions, one homeostatic and one induced by ER stress. ER stress leads to de novo biosynthesis of non-trimethylated GRP78, whereas homeostatic, METTL21A-dependent lysine 585–trimethylated GRP78 is reduced. This proteostatic mechanism, dependent on the posttranslational modification of GRP78, allows cells to differentially regulate specific protein abundance during cellular stress.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/mb7Z5S__9ko" height="1" width="1" alt=""/>
Datum: 17.11.2017


Discovery of leucokinin-like neuropeptides that modulate a specific parameter of feeding motor programs in the molluscan model, Aplysia [Protein Synthesis and Degradation]

A better understanding of neuromodulation in a behavioral system requires identification of active modulatory transmitters. Here, we used identifiable neurons in a neurobiological model system, the mollusc Aplysia, to study neuropeptides, a diverse class of neuromodulators. We took advantage of two types of feeding neurons, B48 and B1/B2, in the Aplysia buccal ganglion that might contain different neuropeptides. We performed a representational difference analysis (RDA) by subtraction of mRNAs in B48 versus mRNAs in B1/B2. The RDA identified an unusually long (2025 amino acids) peptide precursor encoding Aplysia leucokinin-like peptides (ALKs; e.g. ALK-1 and ALK-2). Northern blot analysis revealed that, compared with other ganglia (e.g. the pedal-pleural ganglion), ALK mRNA is predominantly present in the buccal ganglion, which controls feeding behavior. We then used in situ hybridization and immunohistochemistry to localize ALKs to specific neurons, including B48. MALDI-TOF MS on single buccal neurons revealed expression of 40 ALK precursor–derived peptides. Among these, ALK-1 and ALK-2 are active in the feeding network; they shortened the radula protraction phase of feeding motor programs triggered by a command-like neuron. We also found that this effect may be mediated by the ALK-stimulated enhancement of activity of an interneuron, which has previously been shown to terminate protraction. We conclude that our multipronged approach is effective for determining the structure and defining the diverse functions of leucokinin-like peptides. Notably, the ALK precursor is the first verified nonarthropod precursor for leucokinin-like peptides with a novel, marked modulatory effect on a specific parameter (protraction duration) of feeding motor programs.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/f28Fd9WEUuM" height="1" width="1" alt=""/>
Datum: 17.11.2017


O2 sensing-associated glycosylation exposes the F-box-combining site of the Dictyostelium Skp1 subunit in E3 ubiquitin ligases [Molecular Biophysics]

Skp1 is a conserved protein linking cullin-1 to F-box proteins in SCF (Skp1/Cullin-1/F-box protein) E3 ubiquitin ligases, which modify protein substrates with polyubiquitin chains that typically target them for 26S proteasome-mediated degradation. In Dictyostelium (a social amoeba), Toxoplasma gondii (the agent for human toxoplasmosis), and other protists, Skp1 is regulated by a unique pentasaccharide attached to hydroxylated Pro-143 within its C-terminal F-box–binding domain. Prolyl hydroxylation of Skp1 contributes to O2-dependent Dictyostelium development, but full glycosylation at that position is required for optimal O2 sensing. Previous studies have shown that the glycan promotes organization of the F-box–binding region in Skp1 and aids in Skp1's association with F-box proteins. Here, NMR and MS approaches were used to determine the glycan structure, and then a combination of NMR and molecular dynamics simulations were employed to characterize the impact of the glycan on the conformation and motions of the intrinsically flexible F-box–binding domain of Skp1. Molecular dynamics trajectories of glycosylated Skp1 whose calculated monosaccharide relaxation kinetics and rotational correlation times agreed with the NMR data indicated that the glycan interacts with the loop connecting two α-helices of the F-box–combining site. In these trajectories, the helices separated from one another to create a more accessible and dynamic F-box interface. These results offer an unprecedented view of how a glycan modification influences a disordered region of a full-length protein. The increased sampling of an open Skp1 conformation can explain how glycosylation enhances interactions with F-box proteins in cells.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/w6kr7on56DY" height="1" width="1" alt=""/>
Datum: 17.11.2017


Chemical reprogramming of mouse embryonic and adult fibroblast into endoderm lineage [Developmental Biology]

We report here an approach to redirecting somatic cell fate under chemically defined conditions without transcription factors. We start by converting mouse embryonic fibroblasts to epithelial-like cells with chemicals and growth factors. Subsequent cell fate mapping reveals a robust induction of SOX17 in the resulting epithelial-like cells that can be further reprogrammed to endodermal progenitor cells. Interestingly, these cells can self-renew in vitro and further differentiate into albumin-producing hepatocytes that can rescue mice from acute liver injury. Our results demonstrate a rational approach to convert mouse embryonic fibroblasts to hepatocytes and suggest that this mechanism-driven approach may be generalized for other cells.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/XwgyPXr7DZA" height="1" width="1" alt=""/>
Datum: 17.11.2017


ZNF143 protein is an important regulator of the myeloid transcription factor C/EBP{alpha} [Gene Regulation]

The transcription factor C/EBPα is essential for myeloid differentiation and is frequently dysregulated in acute myeloid leukemia. Although studied extensively, the precise regulation of its gene by upstream factors has remained largely elusive. Here, we investigated its transcriptional activation during myeloid differentiation. We identified an evolutionarily conserved octameric sequence, CCCAGCAG, ∼100 bases upstream of the CEBPA transcription start site, and demonstrated through mutational analysis that this sequence is crucial for C/EBPα expression. This sequence is present in the genes encoding C/EBPα in humans, rodents, chickens, and frogs and is also present in the promoters of other C/EBP family members. We identified that ZNF143, the human homolog of the Xenopus transcriptional activator STAF, specifically binds to this 8-bp sequence to activate C/EBPα expression in myeloid cells through a mechanism that is distinct from that observed in liver cells and adipocytes. Altogether, our data suggest that ZNF143 plays an important role in the expression of C/EBPα in myeloid cells.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/nyrWeVjEHyA" height="1" width="1" alt=""/>
Datum: 17.11.2017


Mutagenic potential of nitrogen mustard-induced formamidopyrimidine DNA adduct: Contribution of the non-canonical {alpha}-anomer [Cell Biology]

Nitrogen mustards (NMs) are DNA-alkylating compounds that represent the earliest anticancer drugs. However, clinical use of NMs is limited because of their own mutagenic properties. The mechanisms of NM-induced mutagenesis remain unclear. The major product of DNA alkylation by NMs is a cationic NM-N7-dG adduct that can yield the imidazole ring-fragmented lesion, N5-NM-substituted formamidopyrimidine (NM-Fapy-dG). Characterization of this adduct is complicated because it adopts different conformations, including both a canonical β- and an unnatural α-anomeric configuration. Although formation of NM-Fapy-dG in cellular DNA has been demonstrated, its potential role in NM-induced mutagenesis is unknown. Here, we created site-specifically modified single-stranded vectors for replication in primate (COS7) or Escherichia coli cells. In COS7 cells, NM-Fapy-dG caused targeted mutations, predominantly G → T transversions, with overall frequencies of ∼11–12%. These frequencies were ∼2-fold higher than that induced by 8-oxo-dG adduct. Replication in E. coli was essentially error-free. To elucidate the mechanisms of bypass of NM-Fapy-dG, we performed replication assays in vitro with a high-fidelity DNA polymerase, Saccharomyces cerevisiae polymerase (pol) δ. It was found that pol δ could catalyze high-fidelity synthesis past NM-Fapy-dG, but only on a template subpopulation, presumably containing the β-anomeric adduct. Consistent with the low mutagenic potential of the β-anomer in vitro, the mutation frequency was significantly reduced when conditions for vector preparation were modified to favor this configuration. Collectively, these data implicate the α-anomer as a major contributor to NM-Fapy-dG-induced mutagenesis in primate cells.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/v8Xsygc-LNE" height="1" width="1" alt=""/>
Datum: 17.11.2017


The phospholipase PNPLA7 functions as a lysophosphatidylcholine hydrolase and interacts with lipid droplets through its catalytic domain [Enzymology]

Mammalian patatin-like phospholipase domain–containing proteins (PNPLAs) are lipid-metabolizing enzymes with essential roles in energy metabolism, skin barrier development, and brain function. A detailed annotation of enzymatic activities and structure–function relationships remains an important prerequisite to understand PNPLA functions in (patho-)physiology, for example, in disorders such as neutral lipid storage disease, non-alcoholic fatty liver disease, and neurodegenerative syndromes. In this study, we characterized the structural features controlling the subcellular localization and enzymatic activity of PNPLA7, a poorly annotated phospholipase linked to insulin signaling and energy metabolism. We show that PNPLA7 is an endoplasmic reticulum (ER) transmembrane protein that specifically promotes hydrolysis of lysophosphatidylcholine in mammalian cells. We found that transmembrane and regulatory domains in the PNPLA7 N-terminal region cooperate to regulate ER targeting but are dispensable for substrate hydrolysis. Enzymatic activity is instead mediated by the C-terminal domain, which maintains full catalytic competence even in the absence of N-terminal regions. Upon elevated fatty acid flux, the catalytic domain targets cellular lipid droplets and promotes interactions of PNPLA7 with these organelles in response to increased cAMP levels. We conclude that PNPLA7 acts as an ER-anchored lysophosphatidylcholine hydrolase that is composed of specific functional domains mediating catalytic activity, subcellular positioning, and interactions with cellular organelles. Our study provides critical structural insights into an evolutionarily conserved class of phospholipid-metabolizing enzymes.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/CQMykXrAO1Q" height="1" width="1" alt=""/>
Datum: 17.11.2017


Ligand-dependent corepressor (LCoR) represses the transcription factor C/EBP{beta} during early adipocyte differentiation [Gene Regulation]

Nuclear receptors (NRs) regulate gene transcription by recruiting coregulators, involved in chromatin remodeling and assembly of the basal transcription machinery. The NR-associated protein ligand-dependent corepressor (LCoR) has previously been shown to suppress hepatic lipogenesis by decreasing the binding of steroid receptor coactivators to thyroid hormone receptor. However, the role of LCoR in adipogenesis has not been established. Here, we show that LCoR expression is reduced in the early stage of adipogenesis in vitro. LCoR overexpression inhibited 3T3-L1 adipocyte differentiation, whereas LCoR knockdown promoted it. Using an unbiased affinity purification approach, we identified CCAAT/enhancer-binding protein β (C/EBPβ), a key transcriptional regulator in early adipogenesis, and corepressor C-terminal binding proteins as potential components of an LCoR-containing complex in 3T3-L1 adipocytes. We found that LCoR directly interacts with C/EBPβ through its C-terminal helix-turn-helix domain, required for LCoR's inhibitory effects on adipogenesis. LCoR overexpression also inhibited C/EBPβ transcriptional activity, leading to inhibition of mitotic clonal expansion and transcriptional repression of C/EBPα and peroxisome proliferator-activated receptor γ2 (PPARγ2). However, LCoR overexpression did not affect the recruitment of C/EBPβ to the promoters of C/EBPα and PPARγ2 in 3T3-L1 adipocytes. Of note, restoration of PPARγ2 or C/EBPα expression attenuated the inhibitory effect of LCoR on adipogenesis. Mechanistically, LCoR suppressed C/EBPβ-mediated transcription by recruiting C-terminal binding proteins to the C/EBPα and PPARγ2 promoters and by modulating histone modifications. Taken together, our results indicate that LCoR negatively regulates early adipogenesis by repressing C/EBPβ transcriptional activity and add LCoR to the growing list of transcriptional corepressors of adipogenesis.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/mB0O-vd8Adc" height="1" width="1" alt=""/>
Datum: 17.11.2017


Fibroblast growth factor 2 (FGF2) regulates cytoglobin expression and activation of human hepatic stellate cells via JNK signaling [Signal Transduction]

Cytoglobin (CYGB) belongs to the mammalian globin family and is exclusively expressed in hepatic stellate cells (HSCs) in the liver. In addition to its gas-binding ability, CYGB is relevant to hepatic inflammation, fibrosis, and cancer because of its anti-oxidative properties; however, the regulation of CYGB gene expression remains unknown. Here, we sought to identify factors that induce CYGB expression in HSCs and to clarify the molecular mechanism involved. We used the human HSC cell line HHSteC and primary human HSCs isolated from intact human liver tissues. In HHSteC cells, treatment with a culture supplement solution that included fibroblast growth factor 2 (FGF2) increased CYGB expression with concomitant and time-dependent α-smooth muscle actin (αSMA) down-regulation. We found that FGF2 is a key factor in inducing the alteration in both CYGB and αSMA expression in HHSteCs and primary HSCs and that FGF2 triggered the rapid phosphorylation of both c-Jun N-terminal kinase (JNK) and c-JUN. Both the JNK inhibitor PS600125 and transfection of c-JUN–targeting siRNA abrogated FGF2-mediated CYGB induction, and conversely, c-JUN overexpression induced CYGB and reduced αSMA expression. Chromatin immunoprecipitation analyses revealed that upon FGF2 stimulation, phospho-c-JUN bound to its consensus motif (5′-TGA(C/G)TCA), located −218 to −222 bases from the transcription initiation site in the CYGB promoter. Of note, in bile duct–ligated mice, FGF2 administration ameliorated liver fibrosis and significantly reduced HSC activation. In conclusion, FGF2 triggers CYGB gene expression and deactivation of myofibroblastic human HSCs, indicating that FGF2 has therapeutic potential for managing liver fibrosis.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/34A22mzvorA" height="1" width="1" alt=""/>
Datum: 17.11.2017


Transient alkalinization of the leaf apoplast stiffens the cell wall during onset of chloride salinity in corn leaves [Protein Synthesis and Degradation]

During chloride salinity, the pH of the leaf apoplast (pHapo) transiently alkalizes. There is an ongoing debate about the physiological relevance of these stress-induced pHapo changes. Using proteomic analyses of expanding leaves of corn (Zea mays L.), we show that this transition in pHapo conveys functionality by (i) adjusting protein abundances and (ii) affecting the rheological properties of the cell wall. pHapo was monitored in planta via microscopy-based ratio imaging, and the leaf-proteomic response to the transient leaf apoplastic alkalinization was analyzed via ultra-high performance liquid chromatography–MS. This analysis identified 1459 proteins, of which 44 exhibited increased abundance specifically through the chloride-induced transient rise in pHapo. These elevated protein abundances did not directly arise from high tissue concentrations of Cl− or Na+ but were due to changes in the pHapo. Most of these proteins functioned in growth-relevant processes and in the synthesis of cell wall–building components such as arabinose. Measurements with a linear-variable differential transducer revealed that the transient alkalinization rigidified (i.e. stiffened) the cell wall during the onset of chloride salinity. A decrease in t-coumaric and t-ferulic acids indicates that the wall stiffening arises from cross-linkage to cell wall polymers. We conclude that the pH of the apoplast represents a dynamic factor that is mechanistically coupled to cellular responses to chloride stress. By hardening the wall, the increased pH abrogates wall loosening required for cell expansion and growth. We conclude that the transient alkalinization of the leaf apoplast is related to salinity-induced growth reduction.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/BHLB2Jm8jQI" height="1" width="1" alt=""/>
Datum: 17.11.2017


Thioredoxin-1 maintains mechanistic target of rapamycin (mTOR) function during oxidative stress in cardiomyocytes [Signal Transduction]

Thioredoxin 1 (Trx1) is a 12-kDa oxidoreductase that catalyzes thiol-disulfide exchange reactions to reduce proteins with disulfide bonds. As such, Trx1 helps protect the heart against stresses, such as ischemia and pressure overload. Mechanistic target of rapamycin (mTOR) is a serine/threonine kinase that regulates cell growth, metabolism, and survival. We have shown previously that mTOR activity is increased in response to myocardial ischemia–reperfusion injury. However, whether Trx1 interacts with mTOR to preserve heart function remains unknown. Using a substrate-trapping mutant of Trx1 (Trx1C35S), we show here that mTOR is a direct interacting partner of Trx1 in the heart. In response to H2O2 treatment in cardiomyocytes, mTOR exhibited a high molecular weight shift in non-reducing SDS-PAGE in a 2-mercaptoethanol-sensitive manner, suggesting that mTOR is oxidized and forms disulfide bonds with itself or other proteins. The mTOR oxidation was accompanied by reduced phosphorylation of endogenous substrates, such as S6 kinase (S6K) and 4E-binding protein 1 (4E-BP1) in cardiomyocytes. Immune complex kinase assays disclosed that H2O2 treatment diminished mTOR kinase activity, indicating that mTOR is inhibited by oxidation. Of note, Trx1 overexpression attenuated both H2O2-mediated mTOR oxidation and inhibition, whereas Trx1 knockdown increased mTOR oxidation and inhibition. Moreover, Trx1 normalized H2O2-induced down-regulation of metabolic genes and stimulation of cell death, and an mTOR inhibitor abolished Trx1-mediated rescue of gene expression. H2O2-induced oxidation and inhibition of mTOR were attenuated when Cys-1483 of mTOR was mutated to phenylalanine. These results suggest that Trx1 protects cardiomyocytes against stress by reducing mTOR at Cys-1483, thereby preserving the activity of mTOR and inhibiting cell death.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/DYBEzkKLTgM" height="1" width="1" alt=""/>
Datum: 17.11.2017


Intramolecular autoinhibition of checkpoint kinase 1 is mediated by conserved basic motifs of the C-terminal kinase-associated 1 domain [Molecular Bases of Disease]

Precise control of the cell cycle allows for timely repair of genetic material prior to replication. One factor intimately involved in this process is checkpoint kinase 1 (Chk1), a DNA damage repair inducing Ser/Thr protein kinase that contains an N-terminal kinase domain and a C-terminal regulatory region consisting of a ∼100-residue linker followed by a putative kinase-associated 1 (KA1) domain. We report the crystal structure of the human Chk1 KA1 domain, demonstrating striking structural homology with other sequentially diverse KA1 domains. Separately purified Chk1 kinase and KA1 domains are intimately associated in solution, which results in inhibition of Chk1 kinase activity. Using truncation mutants and site-directed mutagenesis, we define the inhibitory face of the KA1 domain as a series of basic residues residing on two conserved regions of the primary structure. These findings point to KA1-mediated intramolecular autoinhibition as a key regulatory mechanism of human Chk1, and provide new therapeutic possibilities with which to attack this validated oncology target with small molecules.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/tP4C64uEZ54" height="1" width="1" alt=""/>
Datum: 17.11.2017


Role of the endothelial caveolae microdomain in shear stress-mediated coronary vasorelaxation [Molecular Biophysics]

In this study, we determined the role of caveolae and the ionic mechanisms that mediate shear stress–mediated vasodilation (SSD). We found that both TRPV4 and SK channels are targeted to caveolae in freshly isolated bovine coronary endothelial cells (BCECs) and that TRPV4 and KCa2.3 (SK3) channels are co-immunoprecipitated by anti-caveolin-1 antibodies. Acute exposure of BCECs seeded in a capillary tube to 10 dynes/cm2 of shear stress (SS) resulted in activation of TRPV4 and SK currents. However, after incubation with HC067047 (TRPV4 inhibitor), SK currents could no longer be activated by SS, suggesting SK channel activation by SS was mediated through TRPV4. SK currents in BCECs were also activated by isoproterenol or by GSK1016790A (TRPV4 activator). In addition, preincubation of isolated coronary arterioles with apamin (SK inhibitor) resulted in a significant diminution of SSD whereas preincubation with HC067047 produced vasoconstriction by SS. Exposure of BCECs to SS (15 dynes/cm2 16 h) enhanced the production of nitric oxide and prostacyclin (PGI2) and facilitated the translocation of TRPV4 to the caveolae. Inhibition of TRPV4 abolished the SS-mediated intracellular Ca2+ ([Ca2+]i) increase in BCECs. These results indicate a dynamic interaction in the vascular endothelium among caveolae TRPV4 and SK3 channels. This caveolae–TRPV4–SK3 channel complex underlies the molecular and ionic mechanisms that modulate SSD in the coronary circulation.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/fgNjBL6znr8" height="1" width="1" alt=""/>
Datum: 17.11.2017


How activating mutations affect MEK1 regulation and function [Developmental Biology]

The MEK1 kinase directly phosphorylates ERK2, after the activation loop of MEK1 is itself phosphorylated by Raf. Studies over the past decade have revealed a large number of disease-related mutations in the MEK1 gene that lead to tumorigenesis and abnormal development. Several of these mutations result in MEK1 constitutive activity, but how they affect MEK1 regulation and function remains largely unknown. Here, we address these questions focusing on two pathogenic variants of the Phe-53 residue, which maps to the well-characterized negative regulatory region of MEK1. We found that these variants are phosphorylated by Raf faster than the wild-type enzyme, and this phosphorylation further increases their enzymatic activity. However, the maximal activities of fully phosphorylated wild-type and mutant enzymes are indistinguishable. On the basis of available structural information, we propose that the activating substitutions destabilize the inactive conformation of MEK1, resulting in its constitutive activity and making it more prone to Raf-mediated phosphorylation. Experiments in zebrafish revealed that the effects of activating variants on embryonic development reflect the joint control of the negative regulatory region and activating phosphorylation. Our results underscore the complexity of the effects of activating mutations on signaling systems, even at the level of a single protein.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/7BsQlP3OP94" height="1" width="1" alt=""/>
Datum: 17.11.2017


The actin-related p41ARC subunit contributes to p21-activated kinase-1 (PAK1)-mediated glucose uptake into skeletal muscle cells [Signal Transduction]

Defects in translocation of the glucose transporter GLUT4 are associated with peripheral insulin resistance, preclinical diabetes, and progression to type 2 diabetes. GLUT4 recruitment to the plasma membrane of skeletal muscle cells requires F-actin remodeling. Insulin signaling in muscle requires p21-activated kinase-1 (PAK1), whose downstream signaling triggers actin remodeling, which promotes GLUT4 vesicle translocation and glucose uptake into skeletal muscle cells. Actin remodeling is a cyclic process, and although PAK1 is known to initiate changes to the cortical actin-binding protein cofilin to stimulate the depolymerizing arm of the cycle, how PAK1 might trigger the polymerizing arm of the cycle remains unresolved. Toward this, we investigated whether PAK1 contributes to the mechanisms involving the actin-binding and -polymerizing proteins neural Wiskott-Aldrich syndrome protein (N-WASP), cortactin, and ARP2/3 subunits. We found that the actin-polymerizing ARP2/3 subunit p41ARC is a PAK1 substrate in skeletal muscle cells. Moreover, co-immunoprecipitation experiments revealed that insulin stimulates p41ARC phosphorylation and increases its association with N-WASP coordinately with the associations of N-WASP with cortactin and actin. Importantly, all of these associations were ablated by the PAK inhibitor IPA3, suggesting that PAK1 activation lies upstream of these actin-polymerizing complexes. Using the N-WASP inhibitor wiskostatin, we further demonstrated that N-WASP is required for localized F-actin polymerization, GLUT4 vesicle translocation, and glucose uptake. These results expand the model of insulin-stimulated glucose uptake in skeletal muscle cells by implicating p41ARC as a new component of the insulin-signaling cascade and connecting PAK1 signaling to N-WASP-cortactin–mediated actin polymerization and GLUT4 vesicle translocation.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/_lVwBEIhY4A" height="1" width="1" alt=""/>
Datum: 17.11.2017


Dopamine transporter phosphorylation site threonine 53 is stimulated by amphetamines and regulates dopamine transport, efflux, and cocaine analog binding [Neurobiology]

The dopamine transporter (DAT) controls the spatial and temporal dynamics of dopamine neurotransmission through reuptake of extracellular transmitter and is a target for addictive compounds such as cocaine, amphetamine (AMPH), and methamphetamine (METH). Reuptake is regulated by kinase pathways and drug exposure, allowing for fine-tuning of clearance in response to specific conditions, and here we examine the impact of transporter ligands on DAT residue Thr-53, a proline-directed phosphorylation site previously implicated in AMPH-stimulated efflux mechanisms. Our findings show that Thr-53 phosphorylation is stimulated in a transporter-dependent manner by AMPH and METH in model cells and rat striatal synaptosomes, and in striatum of rats given subcutaneous injection of METH. Rotating disc electrode voltammetry revealed that initial rates of uptake and AMPH-induced efflux were elevated in phosphorylation-null T53A DAT relative to WT and charge-substituted T53D DATs, consistent with functions related to charge or polarity. These effects occurred without alterations of surface transporter levels, and mutants also showed reduced cocaine analog binding affinity that was not rescued by Zn2+. Together these findings support a role for Thr-53 phosphorylation in regulation of transporter kinetic properties that could impact DAT responses to amphetamines and cocaine.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/-dQi4SFcDJA" height="1" width="1" alt=""/>
Datum: 17.11.2017


Preferential association with ClC-3 permits sorting of ClC-4 into endosomal compartments [Molecular Biophysics]

ClC-4 is an intracellular Cl−/H+ exchanger that is highly expressed in the brain and whose dysfunction has been linked to intellectual disability and epilepsy. Here we studied the subcellular localization of human ClC-4 in heterologous expression systems. ClC-4 is retained in the endoplasmic reticulum (ER) upon overexpression in HEK293T cells. Co-expression with distinct ClC-3 splice variants targets ClC-4 to late endosome/lysosomes (ClC-3a and ClC-3b) or recycling endosome (ClC-3c). When expressed in cultured astrocytes, ClC-4 sorted to endocytic compartments in WT cells but was retained in the ER in Clcn3−/− cells. To understand the virtual absence of ER-localized ClC-4 in WT astrocytes, we performed association studies by high-resolution clear native gel electrophoresis. Although other CLC channels and transporters form stable dimers, ClC-4 was mostly observed as monomer, with ClC-3–ClC-4 heterodimers being more stable than ClC-4 homodimers. We conclude that unique oligomerization properties of ClC-4 permit regulated targeting of ClC-4 to various endosomal compartment systems via expression of different ClC-3 splice variants.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/VOnCI2aOQm0" height="1" width="1" alt=""/>
Datum: 17.11.2017


DNA-binding affinity and transcriptional activity of the RelA homodimer of nuclear factor {kappa}B are not correlated [Gene Regulation]

The nuclear factor κB (NF-κB) transcription factor family regulates genes involved in cell proliferation and inflammation. The promoters of these genes often contain NF-κB-binding sites (κB sites) arranged in tandem. How NF-κB activates transcription through these multiple sites is incompletely understood. We report here an X-ray crystal structure of homodimers comprising the RelA DNA-binding domain containing the Rel homology region (RHR) in NF-κB bound to an E-selectin promoter fragment with tandem κB sites. This structure revealed that two dimers bind asymmetrically to the symmetrically arranged κB sites at which multiple cognate contacts between one dimer to the corresponding DNA are broken. Because simultaneous RelA-RHR dimer binding to tandem sites in solution was anti-cooperative, we inferred that asymmetric RelA-RHR binding with fewer contacts likely indicates a dissociative binding mode. We found that both κB sites are essential for reporter gene activation by full-length RelA homodimer, suggesting that dimers facilitate DNA binding to each other even though their stable co-occupation is not promoted. Promoter variants with altered spacing and orientation of tandem κB sites displayed unexpected reporter activities that were not explained by the solution-binding pattern of RelA-RHR. Remarkably, full-length RelA bound all DNAs with a weaker affinity and specificity. Moreover, the transactivation domain played a negative role in DNA binding. These observations suggest that other nuclear factors influence full-length RelA binding to DNA by neutralizing the transactivation domain negative effect. We propose that DNA binding by NF-κB dimers is highly complex and modulated by facilitated association–dissociation processes.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/D6-3E-aUX7E" height="1" width="1" alt=""/>
Datum: 17.11.2017


Asparagine and glutamine ladders promote cross-species prion conversion [Neurobiology]

Prion transmission between species is governed in part by primary sequence similarity between the infectious prion aggregate, PrPSc, and the cellular prion protein of the host, PrPC. A puzzling feature of prion formation is that certain PrPC sequences, such as that of bank vole, can be converted by a remarkably broad array of different mammalian prions, whereas others, such as rabbit, show robust resistance to cross-species prion conversion. To examine the structural determinants that confer susceptibility or resistance to prion conversion, we systematically tested over 40 PrPC variants of susceptible and resistant PrPC sequences in a prion conversion assay. Five key residue positions markedly impacted prion conversion, four of which were in steric zipper segments where side chains from amino acids tightly interdigitate in a dry interface. Strikingly, all five residue substitutions modulating prion conversion involved the gain or loss of an asparagine or glutamine residue. For two of the four positions, Asn and Gln residues were not interchangeable, revealing a strict requirement for either an Asn or Gln residue. Bank voles have a high number of Asn and Gln residues and a high Asn:Gln ratio. These findings suggest that a high number of Asn and Gln residues at specific positions may stabilize β-sheets and lower the energy barrier for cross-species prion transmission, potentially because of hydrogen bond networks from side chain amides forming extended Asn/Gln ladders. These data also suggest that multiple PrPC segments containing Asn/Gln residues may act in concert along a replicative interface to promote prion conversion.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/0Y2onpC29jY" height="1" width="1" alt=""/>
Datum: 17.11.2017


Intrinsically disordered sequences enable modulation of protein phase separation through distributed tyrosine motifs [Cell Biology]

Liquid–liquid phase separation (LLPS) is thought to contribute to the establishment of many biomolecular condensates, eukaryotic cell structures that concentrate diverse macromolecules but lack a bounding membrane. RNA granules control RNA metabolism and comprise a large class of condensates that are enriched in RNA-binding proteins and RNA molecules. Many RNA granule proteins are composed of both modular domains and intrinsically disordered regions (IDRs) having low amino acid sequence complexity. Phase separation of these molecules likely plays an important role in the generation and stability of RNA granules. To understand how folded domains and IDRs can cooperate to modulate LLPS, we generated a series of engineered proteins. These were based on fusions of an IDR derived from the RNA granule protein FUS (fused in sarcoma) to a multivalent poly-Src homology 3 (SH3) domain protein that phase-separates when mixed with a poly-proline–rich-motif (polyPRM) ligand. We found that the wild-type IDR promotes LLPS of the polySH3–polyPRM system, decreasing the phase separation threshold concentration by 8-fold. Systematic mutation of tyrosine residues in Gly/Ser-Tyr-Gly/Ser motifs of the IDR reduced this effect, depending on the number but not on the position of these substitutions. Mutating all tyrosines to non-aromatic residues or phosphorylating the IDR raised the phase separation threshold above that of the unmodified polySH3–polyPRM pair. These results show that low-complexity IDRs can modulate LLPS both positively and negatively, depending on the degree of aromaticity and phosphorylation status. Our findings provide plausible mechanisms by which these sequences could alter RNA granule properties on evolutionary and cellular timescales.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/1-zKKSaNaYg" height="1" width="1" alt=""/>
Datum: 17.11.2017


High-resolution structure of a lytic polysaccharide monooxygenase from Hypocrea jecorina reveals a predicted linker as an integral part of the catalytic domain [Molecular Biophysics]

For decades, the enzymes of the fungus Hypocrea jecorina have served as a model system for the breakdown of cellulose. Three-dimensional structures for almost all H. jecorina cellulose-degrading enzymes are available, except for HjLPMO9A, belonging to the AA9 family of lytic polysaccharide monooxygenases (LPMOs). These enzymes enhance the hydrolytic activity of cellulases and are essential for cost-efficient conversion of lignocellulosic biomass. Here, using structural and spectroscopic analyses, we found that native HjLPMO9A contains a catalytic domain and a family-1 carbohydrate-binding module (CBM1) connected via a linker sequence. A C terminally truncated variant of HjLPMO9A containing 21 residues of the predicted linker was expressed at levels sufficient for analysis. Here, using structural, spectroscopic, and biochemical analyses, we found that this truncated variant exhibited reduced binding to and activity on cellulose compared with the full-length enzyme. Importantly, a 0.95-Å resolution X-ray structure of truncated HjLPMO9A revealed that the linker forms an integral part of the catalytic domain structure, covering a hydrophobic patch on the catalytic AA9 module. We noted that the oxidized catalytic center contains a Cu(II) coordinated by two His ligands, one of which has a His-brace in which the His-1 terminal amine group also coordinates to a copper. The final equatorial position of the Cu(II) is occupied by a water-derived ligand. The spectroscopic characteristics of the truncated variant were not measurably different from those of full-length HjLPMO9A, indicating that the presence of the CBM1 module increases the affinity of HjLPMO9A for cellulose binding, but does not affect the active site.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/gT64_e9C-N8" height="1" width="1" alt=""/>
Datum: 17.11.2017


Sulfur denitrosylation by an engineered Trx-like DsbG enzyme identifies nucleophilic cysteine hydrogen bonds as key functional determinant. [Additions and Corrections]

VOLUME 291 (2016) PAGES 15020–15028Table 2 was inadvertently omitted in the version of this article that was published on July 15, 2016. This error has now been corrected.jbc;292/46/18831/T2T1T2Table 2X-ray data collection, processing, and refinement statisticsDsbGCGPCDsbGCGPC-T200MData collection    BeamlineProxima IProxima I    Wavelength (Å)0.980110.98011    Space groupC2C2    Unit cella = 116.65, b = 57.03, and c = 85.55 Å; β = 95.14 and α = γ = 90°a = 116.92, b = 57.22, and c =86.04; β = 94.75 and α = γ = 90°    Resolution limits (Å)a46.12–1.70 (1.80–1.70)46.44–1.96 (2.00–1.96)    Total no. of reflections201,432 (26,100)150,037 (8488)    No. of unique reflections61,470 (9506)40,644 (2344)    Completeness99.5 (98.2)99.4 (99.9)    Rmerge0.051 (0.425)0.063 (0.442)    〈I/σ(I)〉16.73 (2.80)15.58 (3.53)    Rcrystb16.8219.16    Rfreec19.2324.08Ramachandran profile    Most favored (%)98.2598.57    Additionally allowed (%)1.000.71    Disallowed (%)0.750.72R.m.s. deviations    Bond lengths (Å)0.0070.007    Bond angles0.972°0.900°    PDB entry5G1L5G1Ka Data in brackets are for the highest resolution shell.b Rcryst = S||Fobs| − |Fcalc||/S|/Fobs|, Fobs and Fcalc are observed and calculated structure factor amplitudes.c Rfree as for Rcryst using a random subset of the data excluded from the refinement.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/bQGMQxejIdw" height="1" width="1" alt=""/>
Datum: 17.11.2017


The ATAD2 bromodomain binds different acetylation marks on the histone H4 in similar fuzzy complexes. [Additions and Corrections]

VOLUME 292 (2017) PAGES 16734–16745Fig. 6 and supplemental Fig. S12 were inadvertently duplicated. The correct Fig. 6 is shown here. This error does not affect the results or conclusions of this work.jbc;292/46/19121/FU1F1FU1<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/q-1boqD-URw" height="1" width="1" alt=""/>
Datum: 17.11.2017


A B-Z ȷunction induced by an A … A mismatch in GAC repeats in the gene for cartilage oligomeric matrix protein promotes binding with the hZ{alpha}ADAR1 protein [Computational Biology]

GAC repeat expansion from five to seven in the exonic region of the gene for cartilage oligomeric matrix protein (COMP) leads to pseudoachondroplasia, a skeletal abnormality. However, the molecular mechanism by which GAC expansions in the COMP gene lead to skeletal dysplasias is poorly understood. Here we used molecular dynamics simulations, which indicate that an A … A mismatch in a d(GAC)6·d(GAC)6 duplex induces negative supercoiling, leading to a local B-to-Z DNA transition. This transition facilitates the binding of d(GAC)7·d(GAC)7 with the Zα-binding domain of human adenosine deaminase acting on RNA 1 (ADAR1, hZαADAR1), as confirmed by CD, NMR, and microscale thermophoresis studies. The CD results indicated that hZαADAR1 recognizes the zigzag backbone of d(GAC)7·d(GAC)7 at the B–Z junction and subsequently converts it into Z-DNA via the so-called passive mechanism. Molecular dynamics simulations carried out for the modeled hZαADAR1–d(GAC)6.d(GAC)6 complex confirmed the retention of previously reported important interactions between the two molecules. These findings suggest that hZαADAR1 binding with the GAC hairpin stem in COMP can lead to a non-genetic, RNA editing–mediated substitution in COMP that may then play a crucial role in the development of pseudoachondroplasia.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/x0jt5OlyAyo" height="1" width="1" alt=""/>
Datum: 17.11.2017


An intermediate cell state allows rerouting of cell fate [Developmental Biology]

Somatic cell reprogramming into pluripotent stem cells using transcriptional factors or chemical compounds has been shown to include an intermediate cell state with epithelial features. Two recent papers show that this intermediate state can be redirected to create other differentiated cell types—specifically hepatocytes and neurons—using chemical cocktails. These results shed new light on a critical intermediate in cell fate conversion with mechanistic and practical implications.<img src="http://feeds.feedburner.com/~r/jbc/SUcv/~4/IbqoJuvOIMo" height="1" width="1" alt=""/>
Datum: 17.11.2017






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