Branched-Chain Amino Acid Deprivation Decreases Lipid Oxidation and Lipogenesis in C2C12 Myotubes

Impaired lipid metabolism is a common risk factor underlying several metabolic diseases such as metabolic syndrome and type 2 diabetes. Branched-chain amino acids (BCAAs) that include valine, leucine and isoleucine have been proven to share a role in lipid metabolism and hence in maintaining metabolic health. We have previously introduced a hypothesis suggesting that BCAA degradation mechanistically connects to lipid oxidation and storage in skeletal muscle. To test our hypothesis, the present study examined the effects of BCAA deprivation and supplementation on lipid oxidation, lipogenesis and lipid droplet characteristics in murine C2C12 myotubes. In addition, the role of myotube contract…

The swinholide biosynthesis gene cluster from a terrestrial cyanobacterium, Nostoc sp. strain UHCC 0450

ABSTRACT Swinholides are 42-carbon ring polyketides with a 2-fold axis of symmetry. They are potent cytotoxins that disrupt the actin cytoskeleton. Swinholides were discovered from the marine sponge Theonella sp. and were long suspected to be produced by symbiotic bacteria. Misakinolide, a structural variant of swinholide, was recently demonstrated to be the product of a symbiotic heterotrophic proteobacterium. Here, we report the production of swinholide A by an axenic strain of the terrestrial cyanobacterium Nostoc sp. strain UHCC 0450. We located the 85-kb trans -AT polyketide synthase (PKS) swinholide biosynthesis gene cluster from a draft genome of Nostoc sp. UHCC 0450. The swinholide …

Arabidopsis RCD1 coordinates chloroplast and mitochondrial functions through interaction with ANAC transcription factors

Reactive oxygen species (ROS)-dependent signaling pathways from chloroplasts and mitochondria merge at the nuclear protein RADICAL-INDUCED CELL DEATH1 (RCD1). RCD1 interacts in vivo and suppresses the activity of the transcription factors ANAC013 and ANAC017, which mediate a ROS-related retrograde signal originating from mitochondrial complex III. Inactivation of RCD1 leads to increased expression of mitochondrial dysfunction stimulon (MDS) genes regulated by ANAC013 and ANAC017. Accumulating MDS gene products, including alternative oxidases (AOXs), affect redox status of the chloroplasts, leading to changes in chloroplast ROS processing and increased protection of photosynthetic apparatus.…

Hydrophobic pocket targeting probes for enteroviruses

Visualization and tracking of viruses without compromising their functionality is crucial in order to understand virus targeting to cells and tissues, and to understand the subsequent subcellular steps leading to virus uncoating and replication. Enteroviruses are important human pathogens causing a vast number of acute infections, and are also suggested to contribute to the development of chronic diseases like type I diabetes. Here, we demonstrate a novel method to target site-specifically the hydrophobic pocket of enteroviruses. A probe, a derivative of Pleconaril, was developed and conjugated to various labels that enabled the visualization of enteroviruses under light and electron micros…

Characterization of the interaction of the antifungal and cytotoxic cyclic glycolipopeptide hassallidin with sterol-containing lipid membranes.

Hassallidins are cyclic glycolipopeptides produced by cyanobacteria and other prokaryotes. The hassallidin structure consists of a peptide ring of eight amino acids where a fatty acid chain, additional amino acids, and sugar moieties are attached. Hassallidins show antifungal activity against several opportunistic human pathogenic fungi, but does not harbor antibacterial effects. However, they have not been studied on mammalian cells, and the mechanism of action is unknown. We purified hassallidin D from cultured cyanobacterium Anabaena sp. UHCC 0258 and characterized its effect on mammalian and fungal cells. Ultrastructural analysis showed that hassallidin D disrupts cell membranes, causin…

Characterization of the interaction between Actinin-Associated LIM Protein (ALP) and the rod domain of α-actinin

Abstract Background The PDZ-LIM proteins are a family of signalling adaptors that interact with the actin cross-linking protein, α-actinin, via their PDZ domains or via internal regions between the PDZ and LIM domains. Three of the PDZ-LIM proteins have a conserved 26-residue ZM motif in the internal region, but the structure of the internal region is unknown. Results In this study, using circular dichroism and nuclear magnetic resonance (NMR), we showed that the ALP internal region (residues 107–273) was largely unfolded in solution, but was able to interact with the α-actinin rod domain in vitro, and to co-localize with α-actinin on stress fibres in vivo. NMR analysis revealed that the ti…

Psychedelics promote plasticity by directly binding to BDNF receptor TrkB

10 paginas, 15 fguras

Structure of SNX9 SH3 in complex with a viral ligand reveals the molecular basis of its unique specificity for alanine-containing class I SH3 motifs

Class I SH3 domain-binding motifs generally comply with the consensus sequence [R/K]x0PxxP, the hydrophobic residue 0 being proline or leucine. We have studied the unusual 0 = Ala-specificity of SNX9 SH3 by determining its complex structure with a peptide present in eastern equine encephalitis virus (EEEV) nsP3. The structure revealed the length and composition of the n-Src loop as important factors determining specificity. We also compared the affinities of EEEV nsP3 peptide, its mutants, and cellular ligands to SNX9 SH3. These data suggest that nsP3 has evolved to minimize reduction of conformational entropy upon binding, hence acquiring stronger affinity, enabling takeover of SNX9. The R…

The structure and biosynthesis of heinamides A1-A3 and B1-B5, antifungal members of the laxaphycin lipopeptide family.

Laxaphycins are a family of cyclic lipopeptides with synergistic antifungal and antiproliferative activities. They are produced by multiple cyanobacterial genera and comprise two sets of structurally unrelated 11- and 12-residue macrocyclic lipopeptides. Here, we report the discovery of new antifungal laxaphycins from Nostoc sp. UHCC 0702, which we name heinamides, through antimicrobial bioactivity screening. We characterized the chemical structures of eight heinamide structural variants A1-A3 and B1-B5. These variants contain the rare non-proteinogenic amino acids 3-hydroxy-4-methylproline, 4-hydroxyproline, 3-hydroxy-d-leucine, dehydrobutyrine, 5-hydroxyl beta-amino octanoic acid, and O-c…

Decreased temperature increases the expression of a disordered bacterial late embryogenesis abundant (LEA) protein that enhances natural transformation

Late embryogenesis abundant (LEA) proteins are important players in the management of responses to stressful conditions, such as drought, high salinity, and changes in temperature. Many LEA proteins do not have defined three-dimensional structures, so they are intrinsically disordered proteins (IDPs) and are often highly hydrophilic. Although LEA-like sequences have been identified in bacterial genomes, the functions of bacterial LEA proteins have been studied only recently. Sequence analysis of outer membrane interleukin receptor I (BilRI) from the oral pathogen Aggregatibacter actinomycetemcomitans indicated that it shared sequence similarity with group 3/3b/4 LEA proteins. Comprehensive …

1H, 13C, and 15N NMR chemical shift assignment of the complex formed by the first EPEC EspF repeat and N-WASP GTPase binding domain

AbstractLEE-encoded effector EspF (EspF) is an effector protein part of enteropathogenic Escherichia coli’s (EPEC’s) arsenal for intestinal infection. This intrinsically disordered protein contains three highly conserved repeats which together compose over half of the protein’s complete amino acid sequence. EPEC uses EspF to hijack host proteins in order to promote infection. In the attack EspF is translocated, together with other effector proteins, to host cell via type III secretion system. Inside host EspF stimulates actin polymerization by interacting with Neural Wiskott-Aldrich syndrome protein (N-WASP), a regulator in actin polymerization machinery. It is presumed that EspF acts by di…

Structural and functional insights into lysostaphin–substrate interaction

Lysostaphin from Staphylococcus simulans and its family enzymes rapidly acquire prominence as the next generation agents in treatment of S. aureus infections. The specificity of lysostaphin is promoted by its C-terminal cell wall targeting domain selectivity towards pentaglycine bridges in S. aureus cell wall. Scission of these cross-links is carried out by its N-terminal catalytic domain, a zinc-dependent endopeptidase. Understanding the determinants affecting the efficiency of catalysis and strength and specificity of interactions lies at the heart of all lysostaphin family enzyme applications. To this end, we have used NMR, SAXS and molecular dynamics simulations to characterize lysostap…

Flexible Structure of Peptide-Bound Filamin A Mechanosensor Domain Pair 20-21.

Filamins (FLNs) are large, multidomain actin cross-linking proteins with diverse functions. Besides regulating the actin cytoskeleton, they serve as important links between the extracellular matrix and the cytoskeleton by binding cell surface receptors, functioning as scaffolds for signaling proteins, and binding several other cytoskeletal proteins that regulate cell adhesion dynamics. Structurally, FLNs are formed of an amino terminal actin-binding domain followed by 24 immunoglobulin-like domains (IgFLNs). Recent studies have demonstrated that myosin-mediated contractile forces can reveal hidden protein binding sites in the domain pairs IgFLNa18-19 and 20-21, enabling FLNs to transduce me…

Dispersion from Cα or NH: 4D experiments for backbone resonance assignment of intrinsically disordered proteins

AbstractResonance assignment of intrinsically disordered proteins is remarkably challenging due to scant chemical shift dispersion arising from conformational heterogeneity. The challenge is even greater if repeating segments are present in the amino acid sequence. To forward unambiguous resonance assignment of intrinsically disordered proteins, we present iHACANCO, HACACON and (HACA)CONCAHA, three Hα-detected 4D experiments with Cα as an additional dimension. In addition, we present (HACA)CON(CA)NH and (HACA)N(CA)CONH, new 4D Hα-start, HN-detect experiments which have two NH dimensions to enhance peak dispersion in a sequential walk through C′, NH and HN, and provide more accurate NH/HN ch…

Aerobic exercise training and gut microbiome-associated metabolic shifts in women with overweight : a multi-omic study

Physical activity is essential in weight management, improves overall health, and mitigates obesity-related risk markers. Besides inducing changes in systemic metabolism, habitual exercise may improve gut’s microbial diversity and increase the abundance of beneficial taxa in a correlated fashion. Since there is a lack of integrative omics studies on exercise and overweight populations, we studied the metabolomes and gut microbiota associated with programmed exercise in obese individuals. We measured the serum and fecal metabolites of 17 adult women with overweight during a 6-week endurance exercise program. Further, we integrated the exercise-responsive metabolites with variations in the gu…

Characterization of sulfhydryl oxidase from Aspergillus tubingensis

Background Despite of the presence of sulfhydryl oxidases (SOXs) in the secretomes of industrially relevant organisms and their many potential applications, only few of these enzymes have been biochemically characterized. In addition, basic functions of most of the SOX enzymes reported so far are not fully understood. In particular, the physiological role of secreted fungal SOXs is unclear. Results The recently identified SOX from Aspergillus tubingensis (AtSOX) was produced, purified and characterized in the present work. AtSOX had a pH optimum of 6.5, and showed a good pH stability retaining more than 80% of the initial activity in a pH range 4-8.5 within 20 h. More than 70% of the initia…

Antifungal activity improved by coproduction of cyclodextrins and anabaenolysins in Cyanobacteria

Department of Chemistry, Nanoscience Center, University of Jyväskylä, FI-40014, Jyväskylä, Finland Cyclodextrins are cyclic oligosaccharides widely used in the pharmaceutical industry to improve drug delivery and to increase the solubility of hydrophobic compounds. Anabaenolysins are lipopeptides produced by cyanobacteria with potent lytic activity in cholesterolcontaining membranes. Here, we identified the 23- To 24-kb gene clusters responsible for the production of the lipopeptide anabaenolysin. The hybrid nonribosomal peptide synthetase and polyketide synthase biosynthetic gene cluster is encoded in the genomes of three anabaenolysin-producing strains of Anabaena.We detected previously u…

Potent Inhibitor of Human Trypsins from the Aeruginosin Family of Natural Products

Funding Information: We would like to thank A. Löfhjelm and L. Saari for excellent technical assistance. This work was supported by a Sigrid Jusélius Foundation grant to H.K. and the Academy of Finland funding (321809) to T.S. We would also like to thank the Erkko Foundation and Nordforsk Nordic center of Excellency NordAqua (project number #82845) and University of Helsinki’s Doctoral Programme in Microbiology and Biotechnology funding to M.N.A. D.O.A. was supported by a postdoctoral research fellowship from the São Paulo Research Foundation (FAPESP #2018/01563-2). We thank Biocenter Kuopio for the use of their facilities for molecular modeling and MD simulations. We thank the DNA Sequenci…

Enteroviruses and coronaviruses: similarities and therapeutic targets

ABSTRACT Introduction: Enteroviruses are common viruses causing a huge number of acute and chronic infections and producing towering economic costs. Similarly, coronaviruses cause seasonal mild infections, epidemics, and even pandemics and can lead to severe respiratory symptoms. It is important to develop broadly acting antiviral molecules to efficiently tackle the infections caused by thes. Areas covered: This review illuminates the differences and similarities between enteroviruses and coronaviruses and examines the most appealing therapeutic targets to combat both virus groups. Publications of both virus groups and deposited structures discovered through PubMed to March 2021 for viral p…

A novel intrinsically disordered outer membrane lipoprotein ofAggregatibacter actinomycetemcomitansbinds various cytokines and plays a role in biofilm response to interleukin-1β and interleukin-8

Intrinsically disordered proteins (IDPs) do not have a well-defined and stable 3-dimensional fold. Some IDPs can function as either transient or permanent binders of other proteins and may interact with an array of ligands by adopting different conformations. A novel outer membrane lipoprotein, bacterial interleukin receptor I (BilRI) of the opportunistic oral pathogen Aggregatibacter actinomycetemcomitans binds a key gatekeeper proinflammatory cytokine interleukin (IL)-1b. Because the amino acid sequence of the novel lipoprotein resembles that of fibrinogen binder A of Haemophilus ducreyi, BilRI could have the potential to bind other proteins, such as host matrix proteins. However, from th…

Direct pathway cloning and expression of the radiosumin biosynthetic gene cluster

Radiosumins are a structurally diverse family of low molecular weight natural products that are produced by cyanobacteria and exhibit potent serine protease inhibition. Members of this family are dipeptides characterized by the presence of two similar non-proteinogenic amino acids. Here we used a comparative bioinformatic analysis to identify radiosumin biosynthetic gene clusters from the genomes of 13 filamentous cyanobacteria. We used direct pathway cloning to capture and express the entire 16.8 kb radiosumin biosynthetic gene cluster from Dolichospermum planctonicum UHCC 0167 in Escherichia coli. Bioinformatic analysis demonstrates that radiosumins represent a new group of chorismate-der…

The swinholide biosynthetic gene cluster from a terrestrial cyanobacterium Nostoc sp. UHCC 0450

Swinholides are 42-carbon ring polyketides with a 2-fold axis of symmetry. They are potent cytotoxins that disrupt the actin cytoskeleton. Swinholides were discovered from the marine sponge Theonella sp. and were long suspected to be produced by symbiotic bacteria. Misakinolide, a structural variant of swinholide, was recently demonstrated to be the product of a symbiotic heterotrophic proteobacterium. Here, we report the production of swinholide A by an axenic strain of the terrestrial cyanobacterium Nostoc sp. strain UHCC 0450. We located the 85-kb trans-AT polyketide synthase (PKS) swinholide biosynthesis gene cluster from a draft genome of Nostoc sp. UHCC 0450. The swinholide and misaki…

Additional file 7: of Characterization of sulfhydryl oxidase from Aspergillus tubingensis

Part of the alignment of 25 sequences from the same protein family (InterPro IPR000103). On the first line is shown AtSOX retrieved from A. tubingensis genome. The C-X-X-C motifs are marked with a box. The sequences: AtSOX, secreted SOX from A. tubingensis; AnSOX, secreted SOX from A. niger; AoSOX, secreted SOX from A. oryzae; DepH, enzyme from C. violaceum; GliT, enzyme from A. fumigatus; HlmI, enzyme from S. clavuligerus. The other abbreviations are shown in the legend of Fig. 7. (PPTX 95Â kb)

Additional file 3: of Characterization of sulfhydryl oxidase from Aspergillus tubingensis

AtSOX activity measured by HVA-peroxidase coupled assay using reduced GSH (5 mM) as a substrate according to [33]. The enzyme reaction is at the enzymatic rate (linear area ca. 0 - 150 s). The production of the fluorescent HVA dimer was followed at excitation wavelength 320 nm and emission wavelength 420 nm. The reduced AtSOX activity with the inhibitor zinc sulphate (10 mM) is also shown (dashed line). The triplicate measurements were done. (PPTX 121 kb)

Additional file 2: of Characterization of sulfhydryl oxidase from Aspergillus tubingensis

AtSOX activity measured by oxygen consumption assay using 3 mM reduced GSH as a substrate (continuous line). The reaction occurred at the enzymatic rate (the linear area ca. 0.5 - 3.5 min). The amount of dissolved oxygen in the reduced GSH solution prior addition of enzyme is shown with a dashed line. The triplicate measurements were done. (PPTX 6691 kb)

The Interaction Mechanism of Intrinsically Disordered PP2A Inhibitor Proteins ARPP-16 and ARPP-19 With PP2A

Protein phosphatase 2A (PP2A) activity is critical for maintaining normal physiological cellular functions. PP2A is inhibited by endogenous inhibitor proteins in several pathological conditions including cancer. A PP2A inhibitor protein, ARPP-19, has recently been connected to several human cancer types. Accordingly, the knowledge about ARPP-19—PP2A inhibition mechanism is crucial for the understanding the disease development and the therapeutic targeting of ARPP-19—PP2A. Here, we show the first structural characterization of ARPP-19, and its splice variant ARPP-16 using NMR spectroscopy, and SAXS. The results reveal that both ARPP proteins are intrinsically disordered but contain transient…

The cytoprotective protein MANF promotes neuronal survival independently from its role as a GRP78 cofactor

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an endoplasmic reticulum (ER)-stress-regulated protein exhibiting cytoprotective properties through a poorly understood mechanism in various in vitro and in vivo models of neuronal and non-neuronal damage. Although initially characterized as a secreted neurotrophic factor for midbrain dopamine neurons, MANF has recently gained more interest for its intracellular role in regulating the ER homeostasis, including serving as a cofactor of the chaperone glucose-regulated protein 78 (GRP78). We aimed for a better understanding of the neuroprotective mechanisms of MANF. Here we show for the first time that MANF promotes the survival of …

Higher glucose availability augments the metabolic responses of the C2C12 myotubes to exercise-like electrical pulse stimulation

The application of exercise-like electrical pulse simulation (EL-EPS) has become a widely used exercise mimetic in vitro. EL-EPS produces similar physiological responses as in vivo exercise, while less is known about the detailed metabolic effects. Routinely, the C2C12 myotubes are cultured in high-glucose medium (4.5 g/L), which may alter EL-EPS responses. In this study, we evaluate the metabolic effects of EL-EPS under the high- and low-glucose (1.0 g/L) conditions to understand how substrate availability affects the myotube response to EL-EPS. The C2C12 myotube, media, and cell-free media metabolites were analyzed using untargeted nuclear magnetic resonance (NMR)-based metabolomics. Furt…

Molecular basis of filamin a-filGAP interaction and its impairment in congenital disorders associated with filamin a mutations

Background Mutations in filamin A (FLNa), an essential cytoskeletal protein with multiple binding partners, cause developmental anomalies in humans. Methodology/Principal Findings We determined the structure of the 23rd Ig repeat of FLNa (IgFLNa23) that interacts with FilGAP, a Rac-specific GTPase-activating protein and regulator of cell polarity and movement, and the effect of the three disease-related mutations on this interaction. A combination of NMR structural analysis and in silico modeling revealed the structural interface details between the C and D β-strands of the IgFLNa23 and the C-terminal 32 residues of FilGAP. Mutagenesis of the predicted key interface residues confirmed the b…

Maternal Inheritance of a Recessive RBP4 Defect in Canine Congenital Eye Disease

SUMMARY Maternally skewed transmission of traits has been associated with genomic imprinting and oocyte-derived mRNA. We report canine congenital eye malformations, caused by an amino acid deletion (K12del) near the N terminus of retinol-binding protein (RBP4). The disease is only expressed when both dam and offspring are deletion homozygotes. RBP carries vitamin A (retinol) from hepatic stores to peripheral tissues, including the placenta and developing eye, where it is required to synthesize retinoic acid. Gestational vitamin A deficiency is a known risk factor for ocular birth defects. The K12del mutation disrupts RBP folding in vivo, decreasing its secretion from hepatocytes to serum. T…

Discovery of a Pederin Family Compound in a Nonsymbiotic Bloom-Forming Cyanobacterium

The pederin family includes a number of bioactive compounds isolated from symbiotic organisms of diverse evolutionary origin. Pederin is linked to beetle-induced dermatitis in humans, and pederin family members possess potent antitumor activity caused by selective inhibition of the eukaryotic ribosome. Their biosynthesis is accomplished by a polyketide/nonribosomal peptide synthetase machinery employing an unusual trans-acyltransferase mechanism. Here, we report a novel pederin type compound, cusperin, from the free-living cyanobacterium Cuspidothrix issatschenkoi (earlier Aphanizomenon). The chemical structure of cusperin is similar to that of nosperin recently isolated from the lichen cya…

Model of a six immunoglobulin-like domain fragment of filamin A (16-21) built using residual dipolar couplings.

Filamins are actin-binding proteins that participate in a wide range of cell functions, including cell morphology, locomotion, membrane protein localization, and intracellular signaling. The three filamin isoforms found in humans, filamins A, B, and C, are highly homologous, and their roles are partly complementary. In addition to actin, filamins interact with dozens of other proteins that have roles as membrane receptors and channels, enzymes, signaling intermediates, and transcription factors. Filamins are composed of an N-terminal actin-binding domain and 24 filamin-type immunoglobulin-like domains (FLN) that form tail-to-tail dimers with their C-terminal FLN domain. Many of the filamin …

Towards Controlled Synthesis of Water-Soluble Gold Nanoclusters : Synthesis and Analysis

Water-soluble gold nanoclusters with well-defined molecular structures and stability possess particular biophysical properties making them excellent candidates for biological applications as well as for fundamental spectroscopic studies. The currently existing synthetic protocols for atomically monodisperse thiolate-protected gold nanoclusters (AuMPCs) have been widely expanded with organothiolates, yet the direct synthesis reports for water-soluble AuMPCs are still deficient. Here, we demonstrate a wet-chemistry pH-controlled synthesis of two large water-soluble nanoclusters utilizing p-mercaptobenzoic acid (pMBA), affording different sizes of plasmonic AuMPCs on the preparative scale (∼7 …

Alternative Biosynthetic Starter Units Enhance the Structural Diversity of Cyanobacterial Lipopeptides

Puwainaphycins (PUWs) and minutissamides (MINs) are structurally analogous cyclic lipopeptides possessing cytotoxic activity. Both types of compound exhibit high structural variability, particularly in the fatty acid (FA) moiety. Although a biosynthetic gene cluster responsible for synthesis of several PUW variants has been proposed in a cyanobacterial strain, the genetic background for MINs remains unexplored. Herein, we report PUW/MIN biosynthetic gene clusters and structural variants from six cyanobacterial strains. Comparison of biosynthetic gene clusters indicates a common origin of the PUW/MIN hybrid nonribosomal peptide synthetase and polyketide synthase. Surprisingly, the biosynthet…

1H, 13C and 15N NMR chemical shift assignments of cAMP-regulated phosphoprotein-19 and -16 (ARPP-19 and ARPP-16)

Protein Phosphatase 2A, PP2A, the principal Serine/threonine phosphatase, has major roles in broad range of signaling pathways that include regulation of cell cycle, cell proliferation and neuronal signaling. The loss of function of PP2A is linked with many human diseases, like cancer and neurodegenerative disorders. Protein phosphatase 2A (PP2A) functions as tumor suppressor and its tumor suppressor activity is inhibited by the overexpression of PP2A inhibitor proteins in most of the cancers. ARPP-19/ARPP-16 has been identified as one of the potential PP2A inhibitor proteins. Here, we report the resonance assignment of backbone 1H, 13C and 15N atoms of human ARPP-19 and ARPP-16 proteins. T…

Author response: Arabidopsis RCD1 coordinates chloroplast and mitochondrial functions through interaction with ANAC transcription factors

Interaction mechanism of endogenous PP2A inhibitor protein ENSA with PP2A

The vast diversity of protein phosphatase 2A (PP2A) holoenzyme composition ensures its multifaceted role in the regulation of cellular growth and signal transduction. In several pathological conditions, such as cancer, PP2A is inhibited by endogenous inhibitor proteins. Several PP2A inhibitor proteins have been identified, one of which is α-endosulfine (ENSA). ENSA inhibits PP2A activity when it is phosphorylated at Ser67 by Greatwall (Gwl) kinase. The role of ENSA in PP2A inhibition is rather well characterized, but knowledge of the mechanism of inhibition is scarce. In this study, we have performed comprehensive structural characterization of ENSA, and its interaction with PP2A A- and var…

HACANCOi : a new Hα-detected experiment for backbone resonance assignment of intrinsically disordered proteins

AbstractUnidirectional coherence transfer is highly efficient in intrinsically disordered proteins (IDPs). Their elevated ps-ns timescale dynamics ensures long transverse (T2) relaxation times allowing sophisticated coherence transfer pathway selection in comparison to folded proteins. 1Hα-detection ensures non-susceptibility to chemical exchange with the solvent and enables chemical shift assignment of consecutive proline residues, typically abundant in IDPs. However, many IDPs undergo a disorder-to-order transition upon interaction with their target protein, which leads to the loss of the favorable relaxation properties. Long coherence transfer routes now result in prohibitively large dec…

Dynamic Stabilization of the Ligand-Metal Interface in Atomically Precise Gold Nanoclusters Au68 and Au144 Protected by meta-Mercaptobenzoic Acid

Ligand-stabilized, atomically precise gold nanoclusters with a metal core of a uniform size of just 1–3 nm constitute an interesting class of nanomaterials with versatile possibilities for applications due to their size-dependent properties and modifiable ligand layers. The key to extending the usability of the clusters in applications is to understand the chemical bonding in the ligand layer as a function of cluster size and ligand structure. Previously, it has been shown that monodispersed gold nanoclusters, stabilized by meta-mercaptobenzoic acid (m-MBA or 3-MBA) ligands and with sizes of 68–144 gold atoms, show ambient stability. Here we show that a combination of nuclear magnetic reson…

Role of two sequence motifs of mesencephalic astrocyte-derived neurotrophic factor in its survival-promoting activity

AbstractMesencephalic astrocyte-derived neurotrophic factor (MANF) is a prosurvival protein that protects the cells when applied intracellularly in vitro or extracellularly in vivo. Its protective mechanisms are poorly known. Here we studied the role of two short sequence motifs within the carboxy-(C) terminal domain of MANF in its neuroprotective activity: the CKGC sequence (a CXXC motif) that could be involved in redox reactions, and the C-terminal RTDL sequence, an endoplasmic reticulum (ER) retention signal. We mutated these motifs and analyzed the antiapoptotic effect and intracellular localization of these mutants of MANF when overexpressed in cultured sympathetic or sensory neurons. …

Fine-tuning the extent and dynamics of binding cleft opening as a potential general regulatory mechanism in parvulin-type peptidyl prolyl isomerases

AbstractParvulins or rotamases form a distinct group within peptidyl prolyl cis-trans isomerases. Their exact mode of action as well as the role of conserved residues in the family are still not unambiguously resolved. Using backbone S2 order parameters and NOEs as restraints, we have generated dynamic structural ensembles of three distinct parvulins, SaPrsA, TbPin1 and CsPinA. The resulting ensembles are in good agreement with the experimental data but reveal important differences between the three enzymes. The largest difference can be attributed to the extent of the opening of the substrate binding cleft, along which motional mode the three molecules occupy distinct regions. Comparison w…

Production of High Amounts of Hepatotoxin Nodularin and New Protease Inhibitors Pseudospumigins by the Brazilian Benthic Nostoc sp. CENA543

Nostoc is a cyanobacterial genus, common in soils and a prolific producer of natural products. This research project aimed to explore and characterize Brazilian cyanobacteria for new bioactive compounds. Here we report the production of hepatotoxins and new protease inhibitors from benthic Nostoc sp. CENA543 isolated from a small, shallow, saline-alkaline lake in the Nhecolandia, Pantanal wetland area in Brazil. Nostoc sp. CENA543 produces exceptionally high amounts of nodularin-R. This is the first free-living Nostoc that produces nodularin at comparable levels as the toxic, bloom-forming, Nodularia spumigena. We also characterized pseudospumigins A-F, which are a novel family of linear te…

Evolutionary plasticity of SH3 domain binding by Nef proteins of the HIV-1/SIVcpz lentiviral lineage

ABSTRACTThe accessory protein Nef of human and simian immunodeficiency viruses (HIV and SIV) is an important pathogenicity factor known to interact with cellular protein kinases and other signaling proteins. A canonical SH3 domain binding motif in Nef is required for most of these interactions. For example, HIV-1 Nef activates the tyrosine kinase Hck by tightly binding to its SH3 domain. An archetypal contact between a negatively charged SH3 residue and a highly conserved arginine in Nef (Arg77) plays a key role here. Combining structural analyses with functional assays, we here show that Nef proteins have also developed a distinct structural strategy - termed the “R-clamp” - that favors th…

Evolutionary plasticity of SH3 domain binding by Nef proteins of the HIV-1/SIVcpz lentiviral lineage

The accessory protein Nef of human and simian immunodeficiency viruses (HIV and SIV) is an important pathogenicity factor known to interact with cellular protein kinases and other signaling proteins. A canonical SH3 domain binding motif in Nef is required for most of these interactions. For example, HIV-1 Nef activates the tyrosine kinase Hck by tightly binding to its SH3 domain. An archetypal contact between a negatively charged SH3 residue and a highly conserved arginine in Nef (Arg77) plays a key role here. Combining structural analyses with functional assays, we here show that Nef proteins have also developed a distinct structural strategy—termed the "R-clamp”—that favors the formation …

RCD1 Coordinates Chloroplastic and Mitochondrial Electron Transfer through Interaction with ANAC Transcription Factors in Arabidopsis

AbstractSignaling from chloroplasts and mitochondria, both dependent on reactive oxygen species (ROS), merge at the nuclear protein RADICAL-INDUCED CELL DEATH1 (RCD1). ROS produced in the chloroplasts affect the abundance, thiol redox state and oligomerization of RCD1. RCD1 directly interactsin vivowith ANAC013 and ANAC017 transcription factors, which are the mediators of the ROS-related mitochondrial complex III retrograde signa and suppresses activity of ANAC013 and ANAC017. Inactivation ofRCD1leads to increased expression of ANAC013 and ANAC017-regulated genes belonging to the mitochondrial dysfunction stimulon (MDS), including genes for mitochondrial alternative oxidases(AOXs).Accumulat…

Atomic Structures of Two Novel Immunoglobulin-like Domain Pairs in the Actin Cross-linking Protein Filamin

Filamins are actin filament cross-linking proteins composed of an N-terminal actin-binding domain and 24 immunoglobulin-like domains (IgFLNs). Filamins interact with numerous proteins, including the cytoplasmic domains of plasma membrane signaling and cell adhesion receptors. Thereby filamins mechanically and functionally link the cell membrane to the cytoskeleton. Most of the interactions have been mapped to the C-terminal IgFLNs 16–24. Similarly, as with the previously known compact domain pair of IgFLNa20–21, the two-domain fragments IgFLNa16–17 and IgFLNa18–19 were more compact in small angle x-ray scattering analysis than would be expected for two independent domains. Solution state NM…

Dynamic Stabilization of the Ligand-Metal Interface in Atomically Precise Gold Nanoclusters Au68 and Au144 Protected by meta-Mercaptobenzoic Acid

Ligand-stabilized, atomically precise gold nanoclusters with a metal core of a uniform size of just 1-3 nm constitute an interesting class of nanomaterials with versatile possibilities for applications due to their size-dependent properties and modifiable ligand layers. The key to extending the usability of the clusters in applications is to understand the chemical bonding in the ligand layer as a function of cluster size and ligand structure. Previously, it has been shown that monodispersed gold nanoclusters, stabilized by meta-mercaptobenzoic acid (m-MBA or 3-MBA) ligands and with sizes of 68-144 gold atoms, show ambient stability. Here we show that a combination of nuclear magnetic reson…

Identification and structural characterization of LytU, a unique peptidoglycan endopeptidase from the lysostaphin family

AbstractWe introduce LytU, a short member of the lysostaphin family of zinc-dependent pentaglycine endopeptidases. It is a potential antimicrobial agent for S. aureus infections and its gene transcription is highly upregulated upon antibiotic treatments along with other genes involved in cell wall synthesis. We found this enzyme to be responsible for the opening of the cell wall peptidoglycan layer during cell divisions in S. aureus. LytU is anchored in the plasma membrane with the active part residing in the periplasmic space. It has a unique Ile/Lys insertion at position 151 that resides in the catalytic site-neighbouring loop and is vital for the enzymatic activity but not affecting the …

Additional file 4: of Characterization of sulfhydryl oxidase from Aspergillus tubingensis

Absorbance spectra (ca. 10 min) of 5 mM reduced GSH (a.) and 5 mM reduced GSH with AtSOX (b.). Arrow indicates the direction of increased UV adsorption due to enzymatic oxidation of the substrate. (PPTX 395 kb)

A novel structural unit in the N-terminal region of filamins.

Immunoglobulin-like (Ig) domains are a widely expanded superfamily that act as interaction motifs or as structural spacers in multidomain proteins. Vertebrate filamins (FLNs), which are multifunctional actin-binding proteins, consist of 24 Ig domains. We have recently discovered that in the C-terminal rod 2 region of FLN, Ig domains interact with each other forming functional domain pairs, where the interaction with signaling and transmembrane proteins is mechanically regulated by weak actomyosin contraction forces. Here, we investigated if there are similar inter-domain interactions around domain 4 in the N-terminal rod 1 region of FLN. Protein crystal structures revealed a new type of dom…

Critical Structural Defects Explain Filamin A Mutations Causing Mitral Valve Dysplasia

Mitral valve diseases affect approximately 3% of the population and are the most common reasons for valvular surgery because no drug-based treatments exist. Inheritable genetic mutations have now been established as the cause of mitral valve insufficiency, and four different missense mutations in the filamin A gene (FLNA) have been found in patients suffering from non-syndromic mitral valve dysplasia (MVD). The FLNA protein is expressed, in particular, in endocardial endothelia during fetal valve morphogenesis and is key in cardiac development. The FLNA-MVD causing mutations are clustered in the N-terminal region of FLNA. How the mutations in FLNA modify its structure and function, have mos…

Structural Basis of the High Affinity Interaction between the Alphavirus Nonstructural Protein-3 (nsP3) and the SH3 Domain of Amphiphysin-2

We show that a peptide from Chikungunya virus nsP3 protein spanning residues 1728–1744 binds the amphiphysin-2 (BIN1) Src homology-3 (SH3) domain with an unusually high affinity (Kd 24 nM). Our NMR solution complex structure together with isothermal titration calorimetry data on several related viral and cellular peptide ligands reveal that this exceptional affinity originates from interactions between multiple basic residues in the target peptide and the extensive negatively charged binding surface of amphiphysin-2 SH3. Remarkably, these arginines show no fixed conformation in the complex structure, indicating that a transient or fluctuating polyelectrostatic interaction accounts for this …

Structural Tuning and Conformational Stability of Aromatic Oligoamide Foldamers

A series of aromatic oligoamide foldamers with two or three pyridine-2,6-dicarboxamide units as their main folding motifs and varying aromatic building blocks as linkers have been synthetized to study the effects of the structural variation on the folding properties and conformational stability. Crystallographic studies showed that in the solid state the central linker unit either elongates the helices and more open S-shaped conformations, compresses the helices to more compact conformations or acts as a rigid spacer separating the pyridine-2,6-dicarboxamide units, which for their part add the predictability of the conformational properties. Multidimensional NMR studies showed that, even in…

Integrin cytoplasmic domain and pITAM compete for spleen tyrosine kinase binding

ABSTRACTIn hematopoietic tissues cell-cell communication involves immunoreceptors and specialized cell adhesion receptors that both mediate intracellular signals. Spleen tyrosine kinase (Syk) is a non-receptor tyrosine kinase involved in the downstream signaling of both immunoreceptors tyrosine activation motif (ITAM) receptors and integrin family cell adhesion receptors. Both phosphorylated ITAM (pITAM) and integrins bind to the regulatory domain of Syk composed of two Src homology 2 (SH2) domains. The interaction with pITAM is mediated by binding of a specific phosphotyrosine to each of the SH2 domains, leading to conformational changes and Syk kinase activation. Integrins bind to the int…

Additional file 1: of Characterization of sulfhydryl oxidase from Aspergillus tubingensis

Purification of AtSOX as analysed by SDS-PAGE. Molecular weight (MW) standards are shown in lanes 1 and 9. The sample from initial crude cell-free medium is shown in lane 2. As a first purification step was used anion exchange chromatography with a Q Sepharose column. In lane 3 are the unbound proteins, and in the lanes 4â 6 bound and then eluted proteins, from Q Sepharose column. Lane 6: AtSOX containing fractions selected for further purifications steps. In the lanes marked 7 and 8 are shown fractions obtained from the last purification step using anion exchange chromatography with Resource Q column (analysed in a separate SDS-PAGE gel with MW standards in lane 9). (PPTX 137Â kb)

Skeletal Dysplasia Mutations Effect on Human Filamins’ Structure and Mechanosensing

AbstractCells’ ability to sense mechanical cues in their environment is crucial for fundamental cellular processes, leading defects in mechanosensing to be linked to many diseases. The actin cross-linking protein Filamin has an important role in the conversion of mechanical forces into biochemical signals. Here, we reveal how mutations in Filamin genes known to cause Larsen syndrome and Frontometaphyseal dysplasia can affect the structure and therefore function of Filamin domains 16 and 17. Employing X-ray crystallography, the structure of these domains was first solved for the human Filamin B. The interaction seen between domains 16 and 17 is broken by shear force as revealed by steered mo…

Molecular Basis of Filamin A-FilGAP Interaction and Its Impairment in Congenital Disorders Associated with Filamin A Mutations

Background: Mutations in filamin A (FLNa), an essential cytoskeletal protein with multiple binding partners, cause developmental anomalies in humans. Methodology/Principal Findings: We determined the structure of the 23rd Ig repeat of FLNa (IgFLNa23) that interacts with FilGAP, a Rac-specific GTPase-activating protein and regulator of cell polarity and movement, and the effect of the three disease-related mutations on this interaction. A combination of NMR structural analysis and in silico modeling revealed the structural interface details between the C and D b-strands of the IgFLNa23 and the C-terminal 32 residues of FilGAP. Mutagenesis of the predicted key interface residues confirmed the…

CCDC 1555247: Experimental Crystal Structure Determination

Related Article: Riia Annala, Aku Suhonen, Heikki Laakkonen, Perttu Permi, Maija Nissinen|2017|Chem.-Eur.J.|23|16671|doi:10.1002/chem.201703985

filamin a ig-like domains 18-19

CCDC 1555255: Experimental Crystal Structure Determination

Related Article: Riia Annala, Aku Suhonen, Heikki Laakkonen, Perttu Permi, Maija Nissinen|2017|Chem.-Eur.J.|23|16671|doi:10.1002/chem.201703985

CCDC 1555258: Experimental Crystal Structure Determination

Related Article: Riia Annala, Aku Suhonen, Heikki Laakkonen, Perttu Permi, Maija Nissinen|2017|Chem.-Eur.J.|23|16671|doi:10.1002/chem.201703985

Decreased temperature increases the expression of a disordered bacterial late embryogenesis abundant (LEA) protein that enhances natural transformation

Late embryogenesis abundant (LEA) proteins are important players in the management of responses to stressful conditions, such as drought, high salinity, and changes in temperature. Many LEA proteins do not have defined three-dimensional structures, so they are intrinsically disordered proteins (IDPs) and are often highly hydrophilic. Although LEA-like sequences have been identified in bacterial genomes, the functions of bacterial LEA proteins have been studied only recently. Sequence analysis of outer membrane interleukin receptor I (BilRI) from the oral pathogen Aggregatibacter actinomycetemcomitans indicated that it shared sequence similarity with group 3/3b/4 LEA proteins. Comprehensive …

CCDC 1555253: Experimental Crystal Structure Determination

Related Article: Riia Annala, Aku Suhonen, Heikki Laakkonen, Perttu Permi, Maija Nissinen|2017|Chem.-Eur.J.|23|16671|doi:10.1002/chem.201703985

CCDC 1555260: Experimental Crystal Structure Determination

Related Article: Riia Annala, Aku Suhonen, Heikki Laakkonen, Perttu Permi, Maija Nissinen|2017|Chem.-Eur.J.|23|16671|doi:10.1002/chem.201703985

CCDC 1555245: Experimental Crystal Structure Determination

Related Article: Riia Annala, Aku Suhonen, Heikki Laakkonen, Perttu Permi, Maija Nissinen|2017|Chem.-Eur.J.|23|16671|doi:10.1002/chem.201703985

CCDC 1555251: Experimental Crystal Structure Determination

Related Article: Riia Annala, Aku Suhonen, Heikki Laakkonen, Perttu Permi, Maija Nissinen|2017|Chem.-Eur.J.|23|16671|doi:10.1002/chem.201703985

CCDC 1555256: Experimental Crystal Structure Determination

Related Article: Riia Annala, Aku Suhonen, Heikki Laakkonen, Perttu Permi, Maija Nissinen|2017|Chem.-Eur.J.|23|16671|doi:10.1002/chem.201703985

CCDC 1555244: Experimental Crystal Structure Determination

Related Article: Riia Annala, Aku Suhonen, Heikki Laakkonen, Perttu Permi, Maija Nissinen|2017|Chem.-Eur.J.|23|16671|doi:10.1002/chem.201703985

Additional file 6: of Characterization of sulfhydryl oxidase from Aspergillus tubingensis

For each chromosomal cluster the table shows accession numbers for genes (Accession), scaffold identifier (Scaffold), start and end on the scaffold, direction of the gene (Direction) and Interpro protein domain identifiers found in the genes (Interpro), as details for Additional file 5. (XLSX 20Â kb)

CCDC 1555248: Experimental Crystal Structure Determination

Related Article: Riia Annala, Aku Suhonen, Heikki Laakkonen, Perttu Permi, Maija Nissinen|2017|Chem.-Eur.J.|23|16671|doi:10.1002/chem.201703985

CCDC 1555250: Experimental Crystal Structure Determination

Related Article: Riia Annala, Aku Suhonen, Heikki Laakkonen, Perttu Permi, Maija Nissinen|2017|Chem.-Eur.J.|23|16671|doi:10.1002/chem.201703985

CCDC 1555259: Experimental Crystal Structure Determination

Related Article: Riia Annala, Aku Suhonen, Heikki Laakkonen, Perttu Permi, Maija Nissinen|2017|Chem.-Eur.J.|23|16671|doi:10.1002/chem.201703985

CCDC 1555249: Experimental Crystal Structure Determination

Related Article: Riia Annala, Aku Suhonen, Heikki Laakkonen, Perttu Permi, Maija Nissinen|2017|Chem.-Eur.J.|23|16671|doi:10.1002/chem.201703985

Additional file 5: of Characterization of sulfhydryl oxidase from Aspergillus tubingensis

Details to Fig. 6 Candidate secondary metabolism clusters with SOX enzymes on fungal chromosomes. On the left an approximate phylogenetic tree of the species compiled from literature [54, 55]. On the right a stretch of a scaffold from each species containing the cluster and neighbouring genes. Genes are shown as boxes on the scaffold stretch. NRPS, PKS, P450 and Zn2 are indicated when present. Grey lines connect genes with identical protein domains on adjacent scaffolds (excluding NRPS, PKS, P450, Zn2 and SOX genes) in order to reveal syntenies. Codes above the gene boxes are their identifiers and below them the Interpro protein domain identifiers found in the genes. Panel a. shows the glio…

CCDC 1555254: Experimental Crystal Structure Determination

Related Article: Riia Annala, Aku Suhonen, Heikki Laakkonen, Perttu Permi, Maija Nissinen|2017|Chem.-Eur.J.|23|16671|doi:10.1002/chem.201703985

CCDC 1555252: Experimental Crystal Structure Determination

Related Article: Riia Annala, Aku Suhonen, Heikki Laakkonen, Perttu Permi, Maija Nissinen|2017|Chem.-Eur.J.|23|16671|doi:10.1002/chem.201703985

CCDC 1555257: Experimental Crystal Structure Determination

Related Article: Riia Annala, Aku Suhonen, Heikki Laakkonen, Perttu Permi, Maija Nissinen|2017|Chem.-Eur.J.|23|16671|doi:10.1002/chem.201703985

CCDC 1555246: Experimental Crystal Structure Determination

Related Article: Riia Annala, Aku Suhonen, Heikki Laakkonen, Perttu Permi, Maija Nissinen|2017|Chem.-Eur.J.|23|16671|doi:10.1002/chem.201703985