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

The drug transporter OAT3 (SLC22A8) and endogenous metabolite communication via the gut-liver-kidney axis [Cell Biology]

The organic anion transporters OAT1 (SLC22A6) and OAT3 (SLC22A8) have similar substrate specificity for drugs, but it is far from clear whether this holds for endogenous substrates. By analysis of more than 600 metabolites in the Oat3KO (Oat3 knockout) by LC/MS, we demonstrate OAT3 involvement in the movement of gut microbiome products, key metabolites, and signaling molecules, including those flowing through the gut–liver–kidney axis. Major pathways affected included those involved in metabolism of bile acids, flavonoids, nutrients, amino acids (including tryptophan-derivatives that are uremic toxins), and lipids. OAT3 is also critical in elimination of liver-derived phase II metabolites, particularly those undergoing glucuronidation. Analysis of physicochemical features revealed nine distinct metabolite groups; at least one member of most clusters has been previously validated in transport assays. In contrast to drugs interacting with the OATs, endogenous metabolites accumulating in the Oat1KO (Oat1 knockout) versus Oat3KO have distinct differences in their physicochemical properties; they are very different in size, number of rings, hydrophobicity, and molecular complexity. Consistent with the Remote Sensing and Signaling Hypothesis, the data support the importance of the OAT transporters in inter-organ and inter-organismal remote communication via transporter-mediated movement of key metabolites and signaling molecules (e.g. gut microbiome–to–intestine–to–blood–to–liver–to–kidney–to–urine). We discuss the possibility of an intimate connection between OATs and metabolite sensing and signaling pathways (e.g. bile acids). Furthermore, the metabolomics and pathway analysis support the view that OAT1 plays a greater role in kidney proximal tubule metabolism and OAT3 appears relatively more important in systemic metabolism, modulating levels of metabolites flowing through intestine, liver, and kidney.<img src="" height="1" width="1" alt=""/>
Datum: 22.09.2017

Reconstructed ancestral enzymes reveal that negative selection drove the evolution of substrate specificity in ADP-dependent kinases [Enzymology]

One central goal in molecular evolution is to pinpoint the mechanisms and evolutionary forces that cause an enzyme to change its substrate specificity; however, these processes remain largely unexplored. Using the glycolytic ADP-dependent kinases of archaea, including the orders Thermococcales, Methanosarcinales, and Methanococcales, as a model and employing an approach involving paleoenzymology, evolutionary statistics, and protein structural analysis, we could track changes in substrate specificity during ADP-dependent kinase evolution along with the structural determinants of these changes. To do so, we studied five key resurrected ancestral enzymes as well as their extant counterparts. We found that a major shift in function from a bifunctional ancestor that could phosphorylate either glucose or fructose 6-phosphate (fructose-6-P) as a substrate to a fructose 6-P-specific enzyme was started by a single amino acid substitution resulting in negative selection with a ground-state mode against glucose and a subsequent 1,600-fold change in specificity of the ancestral protein. This change rendered the residual phosphorylation of glucose a promiscuous and physiologically irrelevant activity, highlighting how promiscuity may be an evolutionary vestige of ancestral enzyme activities, which have been eliminated over time. We also could reconstruct the evolutionary history of substrate utilization by using an evolutionary model of discrete binary characters, indicating that substrate uses can be discretely lost or acquired during enzyme evolution. These findings exemplify how negative selection and subtle enzyme changes can lead to major evolutionary shifts in function, which can subsequently generate important adaptive advantages, for example, in improving glycolytic efficiency in Thermococcales.<img src="" height="1" width="1" alt=""/>
Datum: 22.09.2017

The unintended mitochondrial uncoupling effects of the FDA-approved anti-helminth drug nitazoxanide mitigates experimental parkinsonism in mice [Bioenergetics]

Mitochondria play a primary role in the pathophysiology of Parkinson's disease (PD), and small molecules that counteract the initial stages of disease may offer therapeutic benefit. In this regard, we have examined whether the off-target effects of the Food and Drug Administration (FDA)–approved anti-helminth drug nitazoxanide (NTZ) on mitochondrial respiration could possess any therapeutic potential for PD. Results indicate that MPP+-induced loss in oxygen consumption rate (OCR) and ATP production by mitochondria were ameliorated by NTZ in real time by virtue of its mild uncoupling effect. Pretreatment of cells with NTZ mitigated MPP+-induced loss in mitochondrial OCR and reactive oxygen species (ROS). Similarly, addition of NTZ to cells pretreated with MPP+ could reverse block in mitochondrial OCR and reactive oxygen species induced by MPP+ in real time. The observed effects of NTZ were found to be transient and reversible as removal of NTZ from incubation medium restored the mitochondrial respiration to that of controls. Apoptosis induced by MPP+ was ameliorated by NTZ in a dose-dependent manner. In vivo results demonstrated that oral administration of NTZ (50 mg/kg) in an acute MPTP mouse model of PD conferred significant protection against the loss of tyrosine hydroxylase (TH)-positive neurons of substantia nigra. Based on the above observations we believe that repurposing of NTZ for PD may offer therapeutic benefit.<img src="" height="1" width="1" alt=""/>
Datum: 22.09.2017

Kinetic analyses of single-stranded break repair by human DNA ligase III isoforms reveal biochemical differences from DNA ligase I [Enzymology]

Humans have three genes encoding DNA ligases with conserved structural features and activities, but they also have notable differences. The LIG3 gene encodes a ubiquitous isoform in all tissues (LIG3α) and a germ line–specific splicing isoform (LIG3β) that differs in the C-terminal domain. Both isoforms are found in the nucleus and the mitochondria. Here, we determined the kinetics and thermodynamics of single-stranded break ligation by LIG3α and LIG3β and compared this framework to that of LIG1, the nuclear replicative ligase. The kinetic parameters of the LIG3 isoforms are nearly identical under all tested conditions, indicating that the BRCA1 C terminal (BRCT) domain specific to LIG3α does not alter ligation kinetics. Although LIG3 is only 22% identical to LIG1 across their conserved domains, the two enzymes had very similar maximal ligation rates. Comparison of the rate and equilibrium constants for LIG3 and LIG1 nevertheless revealed important differences. The LIG3 isoforms were seven times more efficient than LIG1 at ligating nicked DNA under optimal conditions, mainly because of their lower Km value for the DNA substrate. This could explain why LIG3 is less prone to abortive ligation than LIG1. Surprisingly, the affinity of LIG3 for Mg2+ was ten times weaker than that of LIG1, suggesting that Mg2+ availability regulates DNA ligation in vivo, because Mg2+ levels are higher in the mitochondria than in the nucleus. The biochemical differences between the LIG3 isoforms and LIG1 identified here will guide the understanding of both unique and overlapping biological roles of these critical enzymes.<img src="" height="1" width="1" alt=""/>
Datum: 22.09.2017

The Axl kinase domain in complex with a macrocyclic inhibitor offers first structural insights into an active TAM receptor kinase [Protein Structure and Folding]

The receptor tyrosine kinase family consisting of Tyro3, Axl, and Mer (TAM) is one of the most recently identified receptor tyrosine kinase families. TAM receptors are up-regulated postnatally and maintained at high levels in adults. They all play an important role in immunity, but Axl has also been implicated in cancer and therefore is a target in the discovery and development of novel therapeutics. However, of the three members of the TAM family, the Axl kinase domain is the only one that has so far eluded structure determination. To this end, using differential scanning fluorimetry and hydrogen-deuterium exchange mass spectrometry, we show here that a lower stability and greater dynamic nature of the Axl kinase domain may account for its poor crystallizability. We present the first structural characterization of the Axl kinase domain in complex with a small-molecule macrocyclic inhibitor. The Axl crystal structure revealed two distinct conformational states of the enzyme, providing a first glimpse of what an active TAM receptor kinase may look like and suggesting a potential role for the juxtamembrane region in enzyme activity. We noted that the ATP/inhibitor-binding sites of the TAM members closely resemble each other, posing a challenge for the design of a selective inhibitor. We propose that the differences in the conformational dynamics among the TAM family members could potentially be exploited to achieve inhibitor selectivity for targeted receptors.<img src="" height="1" width="1" alt=""/>
Datum: 22.09.2017

The UL8 subunit of the helicase-primase complex of herpes simplex virus promotes DNA annealing and has a high affinity for replication forks [Molecular Biophysics]

During lytic infection, herpes simplex virus (HSV) DNA is replicated by a mechanism involving DNA recombination. For instance, replication of the HSV-1 genome produces X- and Y-branched structures, reminiscent of recombination intermediates. HSV-1's replication machinery includes a trimeric helicase–primase composed of helicase (UL5) and primase (UL52) subunits and a third subunit, UL8. UL8 has been reported to stimulate the helicase and primase activities of the complex in the presence of ICP8, an HSV-1 protein that functions as an annealase, a protein that binds complementary single-stranded DNA (ssDNA) and facilitates its annealing to duplex DNA. UL8 also influences the intracellular localization of the UL5/UL52 subunits, but UL8's catalytic activities are not known. In this study we used a combination of biochemical techniques and transmission electron microscopy. First, we report that UL8 alone forms protein filaments in solution. Moreover, we also found that UL8 binds to ssDNAs >50-nucletides long and promotes the annealing of complementary ssDNA to generate highly branched duplex DNA structures. Finally, UL8 has a very high affinity for replication fork structures containing a gap in the lagging strand as short as 15 nucleotides, suggesting that UL8 may aid in directing or loading the trimeric complex onto a replication fork. The properties of UL8 uncovered here suggest that UL8 may be involved in the generation of the X- and Y-branched structures that are the hallmarks of HSV replication.<img src="" height="1" width="1" alt=""/>
Datum: 22.09.2017

Global proteomic analysis of advanced glycation end products in the Arabidopsis proteome provides evidence for age-related glycation hot spots [Plant Biology]

Glycation is a post-translational modification resulting from the interaction of protein amino and guanidino groups with carbonyl compounds. Initially, amino groups react with reducing carbohydrates, yielding Amadori and Heyns compounds. Their further degradation results in formation of advanced glycation end products (AGEs), also originating from α-dicarbonyl products of monosaccharide autoxidation and primary metabolism. In mammals, AGEs are continuously formed during the life of the organism, accumulate in tissues, are well-known markers of aging, and impact age-related tissue stiffening and atherosclerotic changes. However, the role of AGEs in age-related molecular alterations in plants is still unknown. To fill this gap, we present here a comprehensive study of the age-related changes in the Arabidopsis thaliana glycated proteome, including the proteins affected and specific glycation sites therein. We also consider the qualitative and quantitative changes in glycation patterns in terms of the general metabolic background, pathways of AGE formation, and the status of plant anti-oxidative/anti-glycative defense. Although the patterns of glycated proteins were only minimally influenced by plant age, the abundance of 96 AGE sites in 71 proteins was significantly affected in an age-dependent manner and clearly indicated the existence of age-related glycation hot spots in the plant proteome. Homology modeling revealed glutamyl and aspartyl residues in close proximity (less than 5 Å) to these sites in three aging-specific and eight differentially glycated proteins, four of which were modified in catalytic domains. Thus, the sites of glycation hot spots might be defined by protein structure that indicates, at least partly, site-specific character of glycation.<img src="" height="1" width="1" alt=""/>
Datum: 22.09.2017

Dimerization of the transmembrane domain of amyloid precursor protein is determined by residues around the {gamma}-secretase cleavage sites [Cell Biology]

One of the hallmarks of Alzheimer's disease is the formation of extracellular amyloid plaques that consist mainly of abnormally aggregated forms of amyloid β (Aβ) peptides. These peptides are generated by γ-secretase–catalyzed cleavage of a dimeric membrane-bound C-terminal fragment (C99) of the amyloid precursor protein. Although C99 homodimerization has been linked to Aβ production and changes in the aggregation-determining Aβ42/Aβ40 ratio, the motif through which C99 dimerizes has remained controversial. Here, we have used two independent assays to gain insight into C99 homodimerization in the context of the membrane of live cells: bioluminescence resonance energy transfer and Tango membrane protein–protein interaction assays, which were further confirmed by traditional pull-down assays. Our results indicate a four-amino acid region within the C99 transmembrane helix (43TVIV46) as well as its local secondary structure as critical determinants for homodimerization. These four amino acids are also a hot spot of familial Alzheimer's disease–linked mutations that both decrease C99 homodimerization and γ-secretase cleavage and alter the initial cleavage site to increase the Aβ42/40 ratio.<img src="" height="1" width="1" alt=""/>
Datum: 22.09.2017

Suppressor of Fused restraint of Hedgehog activity level is critical for osteogenic proliferation and differentiation during calvarial bone development [Developmental Biology]

Hedgehog signaling plays crucial roles in the development of calvarial bone, relying on the activation of Gli transcription factors. However, the molecular mechanism of the role of regulated Gli protein level in osteogenic specification of mesenchyme still remains elusive. Here, we show by conditionally inactivating Suppressor of Fused (Sufu), a critical repressor of Hedgehog signaling, in Wnt1-Cre–mediated cranial neural crest (CNC) or Dermo1-Cre–mediated mesodermal lineages that Sufu restraint of Hedgehog activity level is critical for differentiation of preosteogenic mesenchyme. Ablation of Sufu results in failure of calvarial bone formation, including CNC-derived bones and mesoderm-derived bones, depending on the Cre line being used. Although mesenchymal cells populate to frontonasal destinations, where they are then condensed, Sufu deletion significantly inhibits the proliferation of osteoprogenitor cells, and these cells no longer differentiate into osteoblasts. We show that there is suppression of Runx2 and Osterix, the osteogenic regulators, in calvarial mesenchyme in the Sufu mutant. We show that down-regulation of several genes upstream to Runx2 and Osterix is manifested within the calvarial primordia, including Bmp2 and its downstream genes Msx1/2 and Dlx5. By contrast, we find that Gli1, the Hedgehog activity readout gene, is excessively activated in mesenchyme. Deletion of Sufu in CNC leads to a discernible decrease in the repressive Gli3 form and an increase in the full-length Gli2. Finally, we demonstrate that simultaneous deletion of Gli2 and Sufu in CNC completely restores calvarial bone formation, suggesting that a sustained level of Hedgehog activity is critical in specification of the osteogenic mesenchymal cells.<img src="" height="1" width="1" alt=""/>
Datum: 22.09.2017

Discovery of a microbial transglutaminase enabling highly site-specific labeling of proteins [Protein Structure and Folding]

Microbial transglutaminases (MTGs) catalyze the formation of Gln–Lys isopeptide bonds and are widely used for the cross-linking of proteins and peptides in food and biotechnological applications (e.g. to improve the texture of protein-rich foods or in generating antibody-drug conjugates). Currently used MTGs have low substrate specificity, impeding their biotechnological use as enzymes that do not cross-react with nontarget substrates (i.e. as bio-orthogonal labeling systems). Here, we report the discovery of an MTG from Kutzneria albida (KalbTG), which exhibited no cross-reactivity with known MTG substrates or commonly used target proteins, such as antibodies. KalbTG was produced in Escherichia coli as soluble and active enzyme in the presence of its natural inhibitor ammonium to prevent potentially toxic cross-linking activity. The crystal structure of KalbTG revealed a conserved core similar to other MTGs but very short surface loops, making it the smallest MTG characterized to date. Ultra-dense peptide array technology involving a pool of 1.4 million unique peptides identified specific recognition motifs for KalbTG in these peptides. We determined that the motifs YRYRQ and RYESK are the best Gln and Lys substrates of KalbTG, respectively. By first reacting a bifunctionalized peptide with the more specific KalbTG and in a second step with the less specific MTG from Streptomyces mobaraensis, a successful bio-orthogonal labeling system was demonstrated. Fusing the KalbTG recognition motif to an antibody allowed for site-specific and ratio-controlled labeling using low label excess. Its site specificity, favorable kinetics, ease of use, and cost-effective production render KalbTG an attractive tool for a broad range of applications, including production of therapeutic antibody-drug conjugates.<img src="" height="1" width="1" alt=""/>
Datum: 22.09.2017

Crystal structure of the human Polε B-subunit in complex with the C-terminal domain of the catalytic subunit [Protein Structure and Folding]

The eukaryotic B-family DNA polymerases include four members: Polα, Polδ, Polϵ, and Polζ, which share common architectural features, such as the exonuclease/polymerase and C-terminal domains (CTDs) of catalytic subunits bound to indispensable B-subunits, which serve as scaffolds that mediate interactions with other components of the replication machinery. Crystal structures for the B-subunits of Polα and Polδ/Polζ have been reported: the former within the primosome and separately with CTD and the latter with the N-terminal domain of the C-subunit. Here we present the crystal structure of the human Polϵ B-subunit (p59) in complex with CTD of the catalytic subunit (p261C). The structure revealed a well defined electron density for p261C and the phosphodiesterase and oligonucleotide/oligosaccharide-binding domains of p59. However, electron density was missing for the p59 N-terminal domain and for the linker connecting it to the phosphodiesterase domain. Similar to Polα, p261C of Polϵ contains a three-helix bundle in the middle and zinc-binding modules on each side. Intersubunit interactions involving 11 hydrogen bonds and numerous hydrophobic contacts account for stable complex formation with a buried surface area of 3094 Å2. Comparative structural analysis of p59–p261C with the corresponding Polα complex revealed significant differences between the B-subunits and CTDs, as well as their interaction interfaces. The B-subunit of Polδ/Polζ also substantially differs from B-subunits of either Polα or Polϵ. This work provides a structural basis to explain biochemical and genetic data on the importance of B-subunit integrity in replisome function in vivo.<img src="" height="1" width="1" alt=""/>
Datum: 22.09.2017

MicroRNA-125b is a key epigenetic regulatory factor that promotes nuclear transfer reprogramming [Cell Biology]

Somatic cell nuclear transfer (SCNT)-mediated reprogramming is a rapid, efficient, and sophisticated process that reprograms differentiated somatic cells to a pluripotent state. However, many factors in this elaborate reprogramming process remain largely unknown. Here, we report that the microRNA (miR) miR-125b is an important component of SCNT-mediated reprogramming. Luciferase reporter assay, quantitative PCR, and Western blotting demonstrated that miR-125b directly binds the 3′-untranslated region of SUV39H1, encoding the histone-lysine N-methyltransferase SUV39H1, to down-regulate histone H3 lysine-9 tri-methylation (H3K9me3) in SCNT embryos. Furthermore, the miR-125b/SUV39H1 interaction induced loss of SUV39H1-mediated H3K9me3, caused heterochromatin relaxation, and promoted the development of SCNT embryos. Transcriptome analyses of SCNT blastomeres indicated that HNF1 homeobox B (HNF1B), a gene encoding a transcription factor downstream of and controlled by the miR-125b/SUV39H1 axis, is important for conferring developmental competence on preimplantation embryos. We conclude that miR-125b promotes SCNT-mediated nuclear reprogramming by targeting SUV39H1 to decrease the deposition of repressive H3K9me3 modifications.<img src="" height="1" width="1" alt=""/>
Datum: 22.09.2017

Structural analyses of the bacterial primosomal protein DnaB reveal that it is a tetramer and forms a complex with a primosomal re-initiation protein [Protein Structure and Folding]

The DnaB primosomal protein from Gram-positive bacteria plays a key role in DNA replication and restart as a loader protein for the recruitment of replisome cascade proteins. Previous investigations have established that DnaB is composed of an N-terminal domain, a middle domain, and a C-terminal domain. However, structural evidence for how DnaB functions at the atomic level is lacking. Here, we report the crystal structure of DnaB, encompassing the N-terminal and middle domains (residues 1–300), from Geobacillus stearothermophilus (GstDnaB1–300) at 2.8 Å resolution. Our structure revealed that GstDnaB1–300 forms a tetramer with two basket-like architectures, a finding consistent with those from solution studies using analytical ultracentrifugation. Furthermore, our results from both GST pulldown assays and analytical ultracentrifugation show that GstDnaB1–300 is sufficient to form a complex with PriA, the primosomal reinitiation protein. Moreover, with the aid of small angle X-ray scattering experiments, we also determined the structural envelope of full-length DnaB (GstDnaBFL) in solution. These small angle X-ray scattering studies indicated that GstDnaBFL has an elongated conformation and that the protruding density envelopes originating from GstDnaB1–300 could completely accommodate the GstDnaB C-terminal domain (residues 301–461). Taken together with biochemical assays, our results suggest that GstDnaB uses different domains to distinguish the PriA interaction and single-stranded DNA binding. These findings can further extend our understanding of primosomal assembly in replication restart.<img src="" height="1" width="1" alt=""/>
Datum: 22.09.2017

An in vitro tag-and-modify protein sample generation method for single-molecule fluorescence resonance energy transfer [Protein Structure and Folding]

Biomolecular systems exhibit many dynamic and biologically relevant properties, such as conformational fluctuations, multistep catalysis, transient interactions, folding, and allosteric structural transitions. These properties are challenging to detect and engineer using standard ensemble-based techniques. To address this drawback, single-molecule methods offer a way to access conformational distributions, transient states, and asynchronous dynamics inaccessible to these standard techniques. Fluorescence-based single-molecule approaches are parallelizable and compatible with multiplexed detection; to date, however, they have remained limited to serial screens of small protein libraries. This stems from the current absence of methods for generating either individual dual-labeled protein samples at high throughputs or protein libraries compatible with multiplexed screening platforms. Here, we demonstrate that by combining purified and reconstituted in vitro translation, quantitative unnatural amino acid incorporation via AUG codon reassignment, and copper-catalyzed azide-alkyne cycloaddition, we can overcome these challenges for target proteins that are, or can be, methionine-depleted. We present an in vitro parallelizable approach that does not require laborious target-specific purification to generate dual-labeled proteins and ribosome-nascent chain libraries suitable for single-molecule FRET-based conformational phenotyping. We demonstrate the power of this approach by tracking the effects of mutations, C-terminal extensions, and ribosomal tethering on the structure and stability of three protein model systems: barnase, spectrin, and T4 lysozyme. Importantly, dual-labeled ribosome-nascent chain libraries enable single-molecule co-localization of genotypes with phenotypes, are well suited for multiplexed single-molecule screening of protein libraries, and should enable the in vitro directed evolution of proteins with designer single-molecule conformational phenotypes of interest.<img src="" height="1" width="1" alt=""/>
Datum: 22.09.2017

A computational combinatorial approach identifies a protein inhibitor of superoxide dismutase 1 misfolding, aggregation, and cytotoxicity [Molecular Bases of Disease]

Molecular agents that specifically bind and neutralize misfolded and toxic superoxide dismutase 1 (SOD1) mutant proteins may find application in attenuating the disease progression of familial amyotrophic lateral sclerosis. However, high structural similarities between the wild-type and mutant SOD1 proteins limit the utility of this approach. Here we addressed this challenge by converting a promiscuous natural human IgG-binding domain, the hyperthermophilic variant of protein G (HTB1), into a highly specific aggregation inhibitor (designated HTB1M) of two familial amyotrophic lateral sclerosis–linked SOD1 mutants, SOD1G93A and SOD1G85R. We utilized a computational algorithm for mapping protein surfaces predisposed to HTB1 intermolecular interactions to construct a focused HTB1 library, complemented with an experimental platform based on yeast surface display for affinity and specificity screening. HTB1M displayed high binding specificity toward SOD1 mutants, inhibited their amyloid aggregation in vitro, prevented the accumulation of misfolded proteins in living cells, and reduced the cytotoxicity of SOD1G93A expressed in motor neuron–like cells. Competition assays and molecular docking simulations suggested that HTB1M binds to SOD1 via both its α-helical and β-sheet domains at the native dimer interface that becomes exposed upon mutated SOD1 misfolding and monomerization. Our results demonstrate the utility of computational mapping of the protein–protein interaction potential for designing focused protein libraries to be used in directed evolution. They also provide new insight into the mechanism of conversion of broad-spectrum immunoglobulin-binding proteins, such as HTB1, into target-specific proteins, thereby paving the way for the development of new selective drugs targeting the amyloidogenic proteins implicated in a variety of human diseases.<img src="" height="1" width="1" alt=""/>
Datum: 22.09.2017

How glycosaminoglycans promote fibrillation of salmon calcitonin. [Additions and Corrections]

VOLUME 291 (2016) PAGES 16849–16862PAGE 16850:The structures shown for chondroitin sulfate A, dermatan sulfate, and heparan sulfate in Fig. 1 were incorrect. This error has now been corrected.jbc;292/38/15992/FU1F1FU1<img src="" height="1" width="1" alt=""/>
Datum: 22.09.2017

Crystal structures of TdsC, a dibenzothiophene monooxygenase from the thermophile Paenibacillus sp. A11-2, reveal potential for expanding its substrate selectivity [Enzymology]

Sulfur compounds in fossil fuels are a major source of environmental pollution, and microbial desulfurization has emerged as a promising technology for removing sulfur under mild conditions. The enzyme TdsC from the thermophile Paenibacillus sp. A11-2 is a two-component flavin-dependent monooxygenase that catalyzes the oxygenation of dibenzothiophene (DBT) to its sulfoxide (DBTO) and sulfone (DBTO2) during microbial desulfurization. The crystal structures of the apo and flavin mononucleotide (FMN)-bound forms of DszC, an ortholog of TdsC, were previously determined, although the structure of the ternary substrate–FMN–enzyme complex remains unknown. Herein, we report the crystal structures of the DBT–FMN–TdsC and DBTO–FMN–TdsC complexes. These ternary structures revealed many hydrophobic and hydrogen-bonding interactions with the substrate, and the position of the substrate could reasonably explain the two-step oxygenation of DBT by TdsC. We also determined the crystal structure of the indole-bound enzyme because TdsC, but not DszC, can also oxidize indole, and we observed that indole binding did not induce global conformational changes in TdsC with or without bound FMN. We also found that the two loop regions close to the FMN-binding site are disordered in apo-TdsC and become structured upon FMN binding. Alanine substitutions of Tyr-93 and His-388, which are located close to the substrate and FMN bound to TdsC, significantly decreased benzothiophene oxygenation activity, suggesting their involvement in supplying protons to the active site. Interestingly, these substitutions increased DBT oxygenation activity by TdsC, indicating that expanding the substrate-binding site can increase the oxygenation activity of TdsC on larger sulfur-containing substrates, a property that should prove useful for future microbial desulfurization applications.<img src="" height="1" width="1" alt=""/>
Datum: 22.09.2017

Endoplasmic reticulum oxidase 1{alpha} is critical for collagen secretion from and membrane type 1-matrix metalloproteinase levels in hepatic stellate cells [Signal Transduction]

Upon liver injury, excessive deposition of collagen from activated hepatic stellate cells (HSCs) is a leading cause of liver fibrosis. An understanding of the mechanism by which collagen biosynthesis is regulated in HSCs will provide important clues for practical anti-fibrotic therapy. Endoplasmic reticulum oxidase 1α (ERO1α) functions as an oxidative enzyme of protein disulfide isomerase, which forms intramolecular disulfide bonds of membrane and secreted proteins. However, the role of ERO1α in HSCs remains unclear. Here, we show that ERO1α is expressed and mainly localized in the endoplasmic reticulum in human HSCs. When HSCs were transfected with ERO1α siRNA or an ERO1α shRNA-expressing plasmid, expression of ERO1α was completely silenced. Silencing of ERO1α expression in HSCs markedly suppressed their proliferation but did not induce apoptosis, which was accompanied by impaired secretion of collagen type 1. Silencing of ERO1α expression induced impaired disulfide bond formation and inhibited autophagy via activation of the Akt/mammalian target of rapamycin signaling pathway, resulting in intracellular accumulation of collagen type 1 in HSCs. Furthermore, silencing of ERO1α expression also promoted proteasome-dependent degradation of membrane type 1-matrix metalloproteinase (MT1-MMP), which stimulates cell proliferation through cleavage of secreted collagens. The inhibition of HSC proliferation was reversed by treatment with MT1-MMP–cleaved collagen type 1. The results suggest that ERO1α plays a crucial role in HSC proliferation via posttranslational modification of collagen and MT1-MMP and, therefore, may be a suitable therapeutic target for managing liver fibrosis.<img src="" height="1" width="1" alt=""/>
Datum: 22.09.2017

O-Glycosylation modulates the stability of epidermal growth factor-like repeats and thereby regulates Notch trafficking [Protein Structure and Folding]

Glycosylation in the endoplasmic reticulum (ER) is closely associated with protein folding and quality control. We recently described a non-canonical ER quality control mechanism for folding of thrombospondin type 1 repeats by protein O-fucosyltransferase 2 (POFUT2). Epidermal growth factor-like (EGF) repeats are also small cysteine-rich protein motifs that can be O-glycosylated by several ER-localized enzymes, including protein O-glucosyltransferase 1 (POGLUT1) and POFUT1. Both POGLUT1 and POFUT1 modify the Notch receptor on multiple EGF repeats and are essential for full Notch function. The fact that POGLUT1 and POFUT1 can distinguish between folded and unfolded EGF repeats raised the possibility that they participate in a quality control pathway for folding of EGF repeats in proteins such as Notch. Here, we demonstrate that cell-surface expression of endogenous Notch1 in HEK293T cells is dependent on the presence of POGLUT1 and POFUT1 in an additive manner. In vitro unfolding assays reveal that addition of O-glucose or O-fucose stabilizes a single EGF repeat and that addition of both O-glucose and O-fucose enhances stability in an additive manner. Finally, we solved the crystal structure of a single EGF repeat covalently modified by a full O-glucose trisaccharide at 2.2 Å resolution. The structure reveals that the glycan fills up a surface groove of the EGF with multiple contacts with the protein, providing a chemical basis for the stabilizing effects of the glycans. Taken together, this work suggests that O-fucose and O-glucose glycans cooperatively stabilize individual EGF repeats through intramolecular interactions, thereby regulating Notch trafficking in cells.<img src="" height="1" width="1" alt=""/>
Datum: 22.09.2017

Characterization of a stable HIV-1 B/C recombinant, soluble, and trimeric envelope glycoprotein (Env) highly resistant to CD4-induced conformational changes [Microbiology]

The HIV-1 envelope (Env) is a glycoprotein consisting of a trimer of heterodimers containing gp120 and gp41 subunits that mediates virus entry and is a major target of broadly neutralizing antibodies (bnAbs) developed during infection in some individuals. The engagement of the HIV-1 gp120 glycoprotein to the host CD4 protein triggers conformational changes in gp120 that allow its binding to co-receptors and is necessary for virus entry to establish infection. Native-like HIV-1 Env immunogens representing distinct clades have been proposed to improve immunogenicity. In the present study, we examined the basis of resistance of an HIV-1 B/C recombinant Env (LT5.J4b12C) to non-neutralizing antibodies targeting CD4-induced Env epitopes in the presence of soluble CD4 (sCD4). Using native polyacrylamide gel shift assay and negative-stain EM, we found that the prefusion conformational state of LT5.J4b12C trimeric Env was largely unaffected in the presence of excess sCD4 with most Env trimers appearing to be in a ligand-free state. This resistance to CD4-induced conformational changes was associated with a lower affinity for CD4. Moreover, the LT5.J4b12C trimeric Env preferentially bound to the neutralizing antibodies compared with non-neutralizing antibodies. Taken together, we report on an HIV-1 B/C recombinant, native-like trimeric Env protein that is highly resistant to CD4-induced conformational changes but displays epitopes recognized by a diverse array of bnAbs. Such features make this B/C recombinant trimeric Env a useful addition to the pool of other recently identified native-like HIV-1 Env trimers suitable for use as antigenic bait for bnAb isolation, structural studies, and use as potential immunogens.<img src="" height="1" width="1" alt=""/>
Datum: 22.09.2017

Quantifying enzyme activity in living cells [Enzymology]

For over a century, enzymatic activity has been studied in vitro, assuming similar activity in the crowded cellular milieu. Here, we determined in real time the catalytic activity of TEM1-β-lactamase inside living cells and compared the values to those obtained in vitro. We found the apparent in vivo catalytic efficiency, kcat/Km, to be lower than in vitro, with significant cell-to-cell variability. Surprisingly, the results show that inside the cell the apparent catalytic efficiency decreases, and Km increases with increasing enzyme concentration. To rationalize these findings, we measured enzyme and substrate diffusion rates in the cell and found the latter to be slower than expected. Simulations showed that for attenuated diffusion the substrate flux becomes rate-limiting, explaining why reaction rates in vivo can be independent on enzyme concentrations. The octanol/water partition of the substrate is 4.5, which is in the range of Food and Drug Administration–approved drugs. This suggests substrate-limited reaction rates to be common. These findings indicate that in vitro data cannot be simply extrapolated to the crowded in vivo environment.<img src="" height="1" width="1" alt=""/>
Datum: 22.09.2017

Unraveling the interactions of the physiological reductant flavodoxin with the different conformations of the Fe protein in the nitrogenase cycle [Protein Structure and Folding]

Nitrogenase reduces dinitrogen (N2) to ammonia in biological nitrogen fixation. The nitrogenase Fe protein cycle involves a transient association between the reduced, MgATP-bound Fe protein and the MoFe protein and includes electron transfer, ATP hydrolysis, release of Pi, and dissociation of the oxidized, MgADP-bound Fe protein from the MoFe protein. The cycle is completed by reduction of oxidized Fe protein and nucleotide exchange. Recently, a kinetic study of the nitrogenase Fe protein cycle involving the physiological reductant flavodoxin reported a major revision of the rate-limiting step from MoFe protein and Fe protein dissociation to release of Pi. Because the Fe protein cannot interact with flavodoxin and the MoFe protein simultaneously, knowledge of the interactions between flavodoxin and the different nucleotide states of the Fe protein is critically important for understanding the Fe protein cycle. Here we used time-resolved limited proteolysis and chemical cross-linking to examine nucleotide-induced structural changes in the Fe protein and their effects on interactions with flavodoxin. Differences in proteolytic cleavage patterns and chemical cross-linking patterns were consistent with known nucleotide-induced structural differences in the Fe protein and indicated that MgATP-bound Fe protein resembles the structure of the Fe protein in the stabilized nitrogenase complex structures. Docking models and cross-linking patterns between the Fe protein and flavodoxin revealed that the MgADP-bound state of the Fe protein has the most complementary docking interface with flavodoxin compared with the MgATP-bound state. Together, these findings provide new insights into the control mechanisms in protein–protein interactions during the Fe protein cycle.<img src="" height="1" width="1" alt=""/>
Datum: 22.09.2017

Identification and characterization of a bacterial cytochrome P450 monooxygenase catalyzing the 3-nitration of tyrosine in rufomycin biosynthesis [Microbiology]

Rufomycin is a circular heptapeptide with anti-mycobacterial activity and is produced by Streptomyces atratus ATCC 14046. Its structure contains three non-proteinogenic amino acids, N-dimethylallyltryptophan, trans-2-crotylglycine, and 3-nitrotyrosine (3NTyr). Although the rufomycin structure was already reported in the 1960s, its biosynthesis, including 3NTyr generation, remains unclear. To elucidate the rufomycin biosynthetic pathway, we assembled a draft genome sequence of S. atratus and identified the rufomycin biosynthetic gene cluster (ruf cluster), consisting of 20 ORFs (rufA–rufT). We found a putative heptamodular nonribosomal peptide synthetase encoded by rufT, a putative tryptophan N-dimethylallyltransferase encoded by rufP, and a putative trimodular type I polyketide synthase encoded by rufEF. Moreover, the ruf cluster contains an apparent operon harboring putative cytochrome P450 (rufO) and nitric oxide synthase (rufN) genes. A similar operon, txtDE, is responsible for the formation of 4-nitrotryptophan in thaxtomin biosynthesis; the cytochrome P450 TxtE catalyzes the 4-nitration of Trp. Therefore, we hypothesized that RufO should catalyze the Tyr 3-nitration. Disruption of rufO abolished rufomycin production by S. atratus, which was restored when 3NTyr was added to the culture medium of the disruptant. Recombinant RufO protein exhibited Tyr 3-nitration activity both in vitro and in vivo. Spectroscopic analysis further revealed that RufO recognizes Tyr as the substrate with a dissociation constant of ∼0.1 μm. These results indicate that RufO is an unprecedented cytochrome P450 that catalyzes Tyr nitration. Taken together with the results of an in silico analysis of the ruf cluster, we propose a rufomycin biosynthetic pathway in S. atratus.<img src="" height="1" width="1" alt=""/>
Datum: 22.09.2017

Linchpin DNA-binding residues serve as go/no-go controls in the replication factor C-catalyzed clamp-loading mechanism [Enzymology]

DNA polymerases depend on circular sliding clamps for processive replication. Clamps must be loaded onto primer–template DNA (ptDNA) by clamp loaders that open and close clamps around ptDNA in an ATP-fueled reaction. All clamp loaders share a core structure in which five subunits form a spiral chamber that binds the clamp at its base in a twisted open form and encloses ptDNA within, while binding and hydrolyzing ATP to topologically link the clamp and ptDNA. To understand how clamp loaders perform this complex task, here we focused on conserved arginines that might play a central coordinating role in the mechanism because they can alternately contact ptDNA or Walker B glutamate in the ATPase site and lie close to the clamp loader–clamp-binding interface. We mutated Arg-84, Arg-88, and Arg-101 in the ATPase-active B, C, and D subunits of Saccharomyces cerevisiae replication factor C (RFC) clamp loader, respectively, and assessed the impact on multiple transient events in the reaction: proliferating cell nuclear antigen (PCNA) clamp binding/opening/closure/release, ptDNA binding/release, and ATP hydrolysis/product release. The results show that these arginines relay critical information between the PCNA-binding, DNA-binding, and ATPase sites at all steps of the reaction, particularly at a checkpoint before RFC commits to ATP hydrolysis. Moreover, their actions are subunit-specific with RFC-C Arg-88 serving as an accelerator that enables rapid ATP hydrolysis upon contact with ptDNA and RFC-D Arg-101 serving as a brake that confers specificity for ptDNA as the correct substrate for loading PCNA.<img src="" height="1" width="1" alt=""/>
Datum: 22.09.2017

In vitro reconstitution of the yeast spore wall dityrosine layer discloses the mechanism of its assembly [Cell Biology]

In response to nutrient starvation, diploid cells of the budding yeast Saccharomyces cerevisiae differentiate into a dormant form of haploid cell termed a spore. The dityrosine layer forms the outermost layer of the wall of S. cerevisiae spores and endows them with resistance to environmental stresses. ll-Bisformyl dityrosine is the main constituent of the dityrosine layer, but the mechanism of its assembly remains elusive. Here, we found that ll-bisformyl dityrosine, but not ll-dityrosine, stably associated in vitro with dit1Δ spores, which lack the dityrosine layer. No other soluble cytosolic materials were required for this incorporation. In several aspects, the dityrosine incorporated in trans resembled the dityrosine layer. For example, dityrosine incorporation obscured access of the dye calcofluor white to the underlying chitosan layer, and ll-bisformyl dityrosine molecules bound to dit1Δ spores were partly isomerized to the dl-form. Mutational analyses revealed several spore wall components required for this binding. One was the chitosan layer located immediately below the dityrosine layer in the spore wall. However, ll-bisformyl dityrosine did not stably bind to chitosan particles, indicating that chitosan is not sufficient for this association. Several lines of evidence demonstrated that spore-resident proteins are involved in the incorporation, including the Lds proteins, which are localized to lipid droplets attached to the developing spore wall. In conclusion, our results provide insight into the mechanism of dityrosine layer formation, and the in vitro assay described here may be used to investigate additional mechanisms in spore wall assembly.<img src="" height="1" width="1" alt=""/>
Datum: 22.09.2017

Ticks from diverse genera encode chemokine-inhibitory evasin proteins [Immunology]

To prolong residence on their hosts, ticks secrete many salivary factors that target host defense molecules. In particular, the tick Rhipicephalus sanguineus has been shown to produce three salivary glycoproteins named “evasins,” which bind to host chemokines, thereby inhibiting the recruitment of leukocytes to the location of the tick bite. Using sequence similarity searches, we have identified 257 new putative evasin sequences encoded by the genomes or salivary or visceral transcriptomes of numerous hard ticks, spanning the genera Rhipicephalus, Amblyomma, and Ixodes of the Ixodidae family. Nine representative sequences were successfully expressed in Escherichia coli, and eight of the nine candidates exhibited high-affinity binding to human chemokines. Sequence alignments enabled classification of the evasins into two subfamilies: C8 evasins share a conserved set of eight Cys residues (four disulfide bonds), whereas C6 evasins have only three of these disulfide bonds. Most of the identified sequences contain predicted secretion leader sequences, N-linked glycosylation sites, and a putative site of tyrosine sulfation. We conclude that chemokine-binding evasin proteins are widely expressed among tick species of the Ixodidae family, are likely to play important roles in subverting host defenses, and constitute a valuable pool of anti-inflammatory proteins for potential future therapeutic applications.<img src="" height="1" width="1" alt=""/>
Datum: 22.09.2017

Intracellular rebinding of transition-state analogues provides extended in vivo inhibition lifetimes on human purine nucleoside phosphorylase [Enzymology]

Purine nucleoside phosphorylase (PNP) is part of the human purine salvage pathway. Its deficiency triggers apoptosis of activated T-cells, making it a target for T-cell proliferative disorders. Transition-state analogues of PNP bind with picomolar (pm) dissociation constants. Tight-binding PNP inhibitors show exceptionally long lifetimes on the target enzyme. We solve the mechanism of the target residence time by comparing functional off-rates in vitro and in vivo. We report in vitro PNP-inhibitor dissociation rates (t½) from 3 to 31 min for seven Immucillins with dissociation constants of 115 to 6 pm. Treatment of human erythrocytes with DADMe-Immucillin-H (DADMe-ImmH, 22 pm) causes complete inhibition of PNP. Loss of [14C]DADMe-ImmH from erythrocytes during multiple washes is slow and biphasic, resulting from inhibitor release and rebinding to PNP catalytic sites. The slow phase gave a t½ of 84 h. Loss of [14C]DADMe-ImmH from erythrocytes in the presence of excess unlabeled DADMe-ImmH increased to a t½ of 1.6 h by preventing rebinding. Thus, in human erythrocytes, rebinding of DADMe-ImmH is 50-fold more likely than diffusional loss of the inhibitor from the erythrocyte. Humans treated with a single oral dose of DADMe-ImmH in phase 1 clinical trials exhibit regain of PNP activity with a t½ of 59 days, corresponding to the erythropoiesis rate in humans. Thus, the PNP catalytic site recapture of DADMe-ImmH is highly favored in vivo. We conclude that transition-state analogues with picomolar dissociation constants exhibit long lifetimes on their targets in vivo because the probability of the target enzyme recapturing inhibitor molecules is greater than diffusional loss to the extracellular space.<img src="" height="1" width="1" alt=""/>
Datum: 22.09.2017

The Ror1 receptor tyrosine kinase plays a critical role in regulating satellite cell proliferation during regeneration of injured muscle [Cell Biology]

The Ror family receptor tyrosine kinases, Ror1 and Ror2, play important roles in regulating developmental morphogenesis and tissue- and organogenesis, but their roles in tissue regeneration in adult animals remain largely unknown. In this study, we examined the expression and function of Ror1 and Ror2 during skeletal muscle regeneration. Using an in vivo skeletal muscle injury model, we show that expression of Ror1 and Ror2 in skeletal muscles is induced transiently by the inflammatory cytokines, TNF-α and IL-1β, after injury and that inhibition of TNF-α and IL-1β by neutralizing antibodies suppresses expression of Ror1 and Ror2 in injured muscles. Importantly, expression of Ror1, but not Ror2, was induced primarily in Pax7-positive satellite cells (SCs) after muscle injury, and administration of neutralizing antibodies decreased the proportion of Pax7-positive proliferative SCs after muscle injury. We also found that stimulation of a mouse myogenic cell line, C2C12 cells, with TNF-α or IL-1β induced expression of Ror1 via NF-κB activation and that suppressed expression of Ror1 inhibited their proliferative responses in SCs. Intriguingly, SC-specific depletion of Ror1 decreased the number of Pax7-positive SCs after muscle injury. Collectively, these findings indicate for the first time that Ror1 has a critical role in regulating SC proliferation during skeletal muscle regeneration. We conclude that Ror1 might be a suitable target in the development of diagnostic and therapeutic approaches to manage muscular disorders.<img src="" height="1" width="1" alt=""/>
Datum: 22.09.2017

Regulation of the epithelial Na+ channel by paraoxonase-2 [Cell Biology]

Paraoxonase-2 (PON-2) is a membrane-bound lactonase with unique anti-oxidative and anti-atherosclerotic properties. PON-2 shares key structural elements with MEC-6, an endoplasmic reticulum–resident molecular chaperone in Caenorhabditis elegans. MEC-6 modulates the expression of a mechanotransductive ion channel comprising MEC-4 and MEC-10 in touch-receptor neurons. Because pon-2 mRNA resides in multiple rat nephron segments, including the aldosterone-sensitive distal nephron where the epithelial Na+ channel (ENaC) is expressed, we hypothesized that PON-2 would similarly regulate ENaC expression. We observed PON-2 expression in aquaporin 2–positive principal cells of the distal nephron of adult human kidney. PON-2 also co-immunoprecipitated with ENaC when co-expressed in HEK293 cells. When PON-2 was co-expressed with ENaC in Xenopus oocytes, ENaC activity was reduced, reflecting a reduction in ENaC surface expression. MEC-6 also reduced ENaC activity when co-expressed in Xenopus oocytes. The PON-2 inhibitory effect was ENaC-specific, as PON-2 had no effect on functional expression of the renal outer medullary potassium channel. PON-2 did not alter the response of ENaC to extracellular Na+, mechanical shear stress, or α-chymotrypsin–mediated proteolysis, suggesting that PON-2 did not alter the regulation of ENaC by these factors. Together, our data suggest that PON-2 regulates ENaC activity by modulating its intracellular trafficking and surface expression.<img src="" height="1" width="1" alt=""/>
Datum: 22.09.2017

A solute-binding protein in the closed conformation induces ATP hydrolysis in a bacterial ATP-binding cassette transporter involved in the import of alginate [Protein Structure and Folding]

The Gram-negative bacterium Sphingomonas sp. A1 incorporates alginate into cells via the cell-surface pit without prior depolymerization by extracellular enzymes. Alginate import across cytoplasmic membranes thereby depends on the ATP-binding cassette transporter AlgM1M2SS (a heterotetramer of AlgM1, AlgM2, and AlgS), which cooperates with the periplasmic solute-binding protein AlgQ1 or AlgQ2; however, several details of AlgM1M2SS-mediated alginate import are not well-understood. Herein, we analyzed ATPase and transport activities of AlgM1M2SS after reconstitution into liposomes with AlgQ2 and alginate oligosaccharide substrates having different polymerization degrees (PDs). Longer alginate oligosaccharides (PD ≥ 5) stimulated the ATPase activity of AlgM1M2SS but were inert as substrates of AlgM1M2SS-mediated transport, indicating that AlgM1M2SS-mediated ATP hydrolysis can be stimulated independently of substrate transport. Using X-ray crystallography in the presence of AlgQ2 and long alginate oligosaccharides (PD 6–8) and with the humid air and glue-coating method, we determined the crystal structure of AlgM1M2SS in complex with oligosaccharide-bound AlgQ2 at 3.6 Å resolution. The structure of the ATP-binding cassette transporter in complex with non-transport ligand-bound periplasmic solute-binding protein revealed that AlgM1M2SS and AlgQ2 adopt inward-facing and closed conformations, respectively. These in vitro assays and structural analyses indicated that interactions between AlgM1M2SS in the inward-facing conformation and periplasmic ligand-bound AlgQ2 in the closed conformation induce ATP hydrolysis by the ATP-binding protein AlgS. We conclude that substrate-bound AlgQ2 in the closed conformation initially interacts with AlgM1M2SS, the AlgM1M2SS–AlgQ2 complex then forms, and this formation is followed by ATP hydrolysis.<img src="" height="1" width="1" alt=""/>
Datum: 22.09.2017

p63{alpha} protein up-regulates heat shock protein 70 expression via E2F1 transcription factor 1, promoting Wasf3/Wave3/MMP9 signaling and bladder cancer invasion [Gene Regulation]

Bladder cancer (BC) is the sixth most common cancer in the United States and is the number one cause of death among patients with urinary system malignancies. This makes the identification of invasive regulator(s)/effector(s) as the potential therapeutic targets for managing BC a high priority. p63 is a member of the p53 family of tumor suppressor genes/proteins, plays a role in the differentiation of epithelial tissues, and is believed to function as a tumor suppressor. However, it remains unclear whether and how p63 functions in BC cell invasion after tumorigenesis. Here, we show that p63α protein levels were much higher in mouse high-invasive BC tissues than in normal tissues. Our results also revealed that p63α is crucial for heat shock protein 70 (Hsp70) expression and subsequently increases the ability of BC invasion. Mechanistic experiments demonstrated that p63α can transcriptionally up-regulate Hsp70 expression, thereby promoting BC cell invasion via the Hsp70/Wasf3/Wave3/MMP-9 axis. We further show that E2F transcription factor 1 (E2F1) mediates p63α overexpression-induced Hsp70 transcription. We also found that p63α overexpression activates E2F1 transcription, which appears to be stimulated by p63α together with E2F1. Collectively, our results demonstrate that p63α is a positive regulator of BC cell invasion after tumorigenesis, providing significant insights into the biological function of p63α in BC and supporting the notion that p63α might be a potential target for invasive BC therapy.<img src="" height="1" width="1" alt=""/>
Datum: 22.09.2017

A synergistic role of IRP1 and FBXL5 proteins in coordinating iron metabolism during cell proliferation [Metabolism]

Iron-regulatory protein 1 (IRP1) belongs to a family of RNA-binding proteins that modulate metazoan iron metabolism. Multiple mechanisms are employed to control the action of IRP1 in dictating changes in the uptake and metabolic fate of iron. Inactivation of IRP1 RNA binding by iron primarily involves insertion of a [4Fe-4S] cluster by the cytosolic iron–sulfur cluster assembly (CIA) system, converting it into cytosolic aconitase (c-acon), but can also involve iron-mediated degradation of IRP1 by the E3 ligase FBXL5 that also targets IRP2. How CIA and FBXL5 collaborate to maintain cellular iron homeostasis through IRP1 and other pathways is poorly understood. Because impaired Fe-S cluster biogenesis associates with human disease, we determined the importance of FBXL5 for regulating IRP1 when CIA is impaired. Suppression of FBXL5 expression coupled with induction of an IRP1 mutant (IRP13C>3S) that cannot insert the Fe-S cluster, or along with knockdown of the CIA factors NUBP2 or FAM96A, reduced cell viability. Iron supplementation reversed this growth defect and was associated with FBXL5-dependent polyubiquitination of IRP1. Phosphorylation of IRP1 at Ser-138 increased when CIA was inhibited and was required for iron rescue. Impaired CIA activity, as noted by reduced c-acon activity, was associated with enhanced FBXL5 expression and a concomitant reduction in IRP1 and IRP2 protein level and RNA-binding activity. Conversely, expression of either IRP induced FBXL5 protein level, demonstrating a negative feedback loop limiting excessive accumulation of iron-response element RNA-binding activity, whose disruption reduces cell growth. We conclude that a regulatory circuit involving FBXL5 and CIA acts through both IRPs to control iron metabolism and promote optimal cell growth.<img src="" height="1" width="1" alt=""/>
Datum: 22.09.2017

A sweet development in Notch regulation [Protein Structure and Folding]

The transmembrane signaling protein Notch, which is crucial for embryonic cell fate decisions, has 36 extracellular EGF domains that are glycosylated in variable and complex ways. A new study shows that O-fucose and O-glucose stabilize the repeats but that extension of glucose by xylose weakens stability, explained by the binding of the glycan to a protein groove. This work shows how different types of glycosylation can distinctly influence protein stability and structure.<img src="" height="1" width="1" alt=""/>
Datum: 22.09.2017

Copper homeostasis networks in the bacterium Pseudomonas aeruginosa [Gene Regulation]

Bacterial copper (Cu+) homeostasis enables both precise metallation of diverse cuproproteins and control of variable metal levels. To this end, protein networks mobilize Cu+ to cellular targets with remarkable specificity. However, the understanding of these processes is rather fragmented. Here, we use genome-wide transcriptomic analysis by RNA-Seq to characterize the response of Pseudomonas aeruginosa to external 0.5 mm CuSO4, a condition that did not generate pleiotropic effects. Pre-steady-state (5-min) and steady-state (2-h) Cu+ fluxes resulted in distinct transcriptome landscapes. Cells quickly responded to Cu2+ stress by slowing down metabolism. This was restored once steady state was reached. Specific Cu+ homeostasis genes were strongly regulated in both conditions. Our system-wide analysis revealed induction of three Cu+ efflux systems (a P1B-ATPase, a porin, and a resistance-nodulation-division (RND) system) and of a putative Cu+-binding periplasmic chaperone and the unusual presence of two cytoplasmic CopZ proteins. Both CopZ chaperones could bind Cu+ with high affinity. Importantly, novel transmembrane transporters probably mediating Cu+ influx were among those largely repressed upon Cu+ stress. Compartmental Cu+ levels appear independently controlled; the cytoplasmic Cu+ sensor CueR controls cytoplasmic chaperones and plasma membrane transporters, whereas CopR/S responds to periplasmic Cu+. Analysis of ΔcopR and ΔcueR mutant strains revealed a CopR regulon composed of genes involved in periplasmic Cu+ homeostasis and its putative DNA recognition sequence. In conclusion, our study establishes a system-wide model of a network of sensors/regulators, soluble chaperones, and influx/efflux transporters that control the Cu+ levels in P. aeruginosa compartments.<img src="" height="1" width="1" alt=""/>
Datum: 22.09.2017

Checks and balances for the iron bank [Metabolism]

The RNA-binding iron regulatory proteins IRP1 and IRP2 are inactivated by either Fe-S cluster insertion or protein degradation mediated by the E3 ligase component FBXL5. However, the mechanisms for coordination between Fe-S cluster assembly, FBXL5, and IRP1/IRP2 activity are poorly defined. A new study reveals that FBXL5 plays a critical role in limiting IRP1 and IRP2 overaccumulation when cytosolic Fe-S cluster assembly is impaired in order to maintain optimal iron levels for cell viability.<img src="" height="1" width="1" alt=""/>
Datum: 22.09.2017

Molecular mechanisms and genomic maps of DNA excision repair in Escherichia coli and humans [DNA and Chromosomes]

Nucleotide excision repair is a major DNA repair mechanism in all cellular organisms. In this repair system, the DNA damage is removed by concerted dual incisions bracketing the damage and at a precise distance from the damage. Here, we review the basic mechanisms of excision repair in Escherichia coli and humans and the recent genome-wide mapping of DNA damage and repair in these organisms at single-nucleotide resolution.<img src="" height="1" width="1" alt=""/>
Datum: 22.09.2017

Effects of hypoxia on monocyte inflammatory mediator production: Dissociation between changes in cyclooxygenase-2 expression and eicosanoid synthesis. [Expressions of Concern]

VOLUME 278 (2003) PAGES 38607–38616The publisher of the Journal of Biological Chemistry is issuing an Expression of Concern to inform readers that credible concerns have been raised regarding some of the data and conclusions in the article listed above. The Journal of Biological Chemistry will provide additional information as it becomes available.<img src="" height="1" width="1" alt=""/>
Datum: 22.09.2017

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