Acireductone Dioxygenase 1 (ARD1) Is an Effector of the Heterotrimeric G Protein Subunit in Arabidopsis

Heterotrimeric G protein complexes are conserved from plants to mammals, but the complexity of each system varies. Arabidopsis thaliana contains one Gα, one Gβ (AGB1), and at least three Gγ subunits, allowing it to form three versions of the heterotrimer. This plant model is ideal for genetic studies because mammalian systems contain hundreds of unique heterotrimers. The activation of these complexes promotes interactions between both the Gα subunit and the Gβγ dimer with enzymes and scaffolds to propagate signaling to the cytoplasm. However, although effectors of Gα and Gβ are known in mammals, no Gβ effectors were previously known in plants. Toward identifying AGB1 effectors, we genetically screened for dominant mutations that suppress Gβ-null mutant (agb1-2) phenotypes. We found that overexpression of acireductone dioxygenase 1 (ARD1) suppresses the 2-day-old etiolated phenotype of agb1-2. ARD1 is homologous to prokaryotic and eukaryotic ARD proteins; one function of ARDs is to operate in the methionine salvage pathway. We show here that ARD1 is an active metalloenzyme, and AGB1 and ARD1 both control embryonic hypocotyl length by modulating cell division; they also may contribute to the production of ethylene, a product of the methionine salvage pathway. ARD1 physically interacts with AGB1, and ARD enzymatic activity is stimulated by AGB1 in vitro. The binding interface on AGB1 was deduced using a comparative evolutionary approach and tested using recombinant AGB1 mutants. A possible mechanism for AGB1 activation of ARD1 activity was tested using directed mutations in a loop near the substrate-binding site

Insect Cell Glycosylation and Its Impact on the Functionality of a Recombinant Intracrystalline Nacre Protein, AP24

The impacts of glycosylation on biomineralization protein function are largely unknown. This is certainly true for the mollusk shell, where glycosylated intracrystalline proteins such as AP24 (Haliotis rufescens) exist but their functions and the role of glycosylation remain elusive. To assess the effect of glycosylation on protein function, we expressed two recombinant variants of AP24: an unglycosylated bacteria-expressed version (rAP24N) and a glycosylated insect cell-expressed version (rAP24G). Our findings indicate that rAP24G is expressed as a single polypeptide containing variations in glycosylation that create microheterogeneity in rAP24G molecular masses. These post-translational modifications incorporate O- and N-glycans and anionic monosialylated and bisialylated, and monosulfated and bisulfated monosaccharides on the protein molecules. AFM and DLS experiments confirm that both rAP24N and rAP24G aggregate to form protein phases, with rAP24N exhibiting a higher degree of aggregation, compared to rAP24G. With regard to functionality, we observe that both recombinant proteins exhibit similar behavior within <i>in vitro</i> calcium carbonate mineralization assays and potentiometric titrations. However, rAP24G modifies crystal growth directions and is a stronger nucleation inhibitor, whereas rAP24N exhibits higher mineral phase stabilization and nanoparticle containment. We believe that the post-translational addition of anionic groups (via sialylation and sulfation), along with modifications to the protein surface topology, may explain the changes in glycosylated rAP24G aggregation and mineralization behavior, relative to rAP24N

The Dynamic Structure of α‑Synuclein Multimers

α-Synuclein, a protein that forms ordered aggregates in the brains of patients with Parkinson’s disease, is intrinsically disordered in the monomeric state. Several studies, however, suggest that it can form soluble multimers <i>in vivo</i> that have significant secondary structure content. A number of studies demonstrate that α-synuclein can form β-strand-rich oligomers that are neurotoxic, and recent observations argue for the existence of soluble helical tetrameric species <i>in cellulo</i> that do not form toxic aggregates. To gain further insight into the different types of multimeric states that this protein can adopt, we generated an ensemble for an α-synuclein construct that contains a 10-residue N-terminal extension, which forms multimers when isolated from <i>Escherichia coli</i>. Data from NMR chemical shifts and residual dipolar couplings were used to guide the construction of the ensemble. Our data suggest that the dominant state of this ensemble is a disordered monomer, complemented by a small fraction of helical trimers and tetramers. Interestingly, the ensemble also contains trimeric and tetrameric oligomers that are rich in β-strand content. These data help to reconcile seemingly contradictory observations that indicate the presence of a helical tetramer <i>in cellulo</i> on the one hand, and a disordered monomer on the other. Furthermore, our findings are consistent with the notion that the helical tetrameric state provides a mechanism for storing α-synuclein when the protein concentration is high, thereby preventing non-membrane-bound monomers from aggregating

An Oligomeric C‑RING Nacre Protein Influences Prenucleation Events and Organizes Mineral Nanoparticles

The mollusk shell nacre layer integrates mineral phases with macromolecular components such as intracrystalline proteins. However, the roles performed by intracrystalline proteins in calcium carbonate nucleation and subsequent postnucleation events (e.g., organization of mineral deposits) in the nacre layer are not known. We find that AP7, a nacre intracrystalline C-RING protein, self-assembles to form amorphous protein oligomers and films on mica that further assemble into larger aggregates or phases in the presence of Ca²⁺. Using solution nuclear magnetic resonance spectroscopy, we determine that the protein assemblies are stabilized by interdomain interactions involving the aggregation-prone T31–N66 C-terminal C-RING domain but are destabilized by the labile nature of the intrinsically disordered D1–T19 AA N-terminal sequence. Thus, the dynamic, amorphous nature of the AP7 assemblies can be traced to the molecular behavior of the N-terminal sequence. Using potentiometric methods, we observe that AP7 protein phases prolong the time interval for prenucleation cluster formation but neither stabilize nor destabilize ACC clusters. Time-resolved flow cell scanning transmission electron microscopy mineralization studies confirm that AP7 protein phases delay the onset of nucleation and assemble and organize mineral nanoparticles into ring-shaped branching clusters in solution. These phenomena are not observed in protein-deficient assays. We conclude that C-RING AP7 protein phases modulate the time period for early events in nucleation and form strategic associations with forming mineral nanoparticles that lead to mineral organization

Interdisciplinary Linguistic and Psychiatric Research on Language Disorders

Interdisciplinary Linguistic and Psychiatric Research on Language Disorders is a collection of scientific papers presented at the International Scientific Workshop on Clinical Linguistics, held on 20 November 2018 at the Education Centre of the University Psychiatric Hospital Vrapče.The Erdeljac &amp; Sekulić Sović research group in clinical linguistics, based at the Department of Linguistics, Faculty of Humanities and Social Sciences, University of Zagreb, in collaboration with psychiatrists from the Department of Biological Psychiatry and Psychogeriatrics and the Department of Diagnostics and Intensive Care, both at the University Psychiatric Hospital Vrapče, present a unique example of an academic publication designed to spotlight ongoing research on semantic processing in individuals diagnosed with psychosis spectrum disorders who are native speakers of Croatian.A further value of this book lies in the co-authors’ contributions, written by specialists in clinical linguistics and psychiatry to expand the focus of research in clinical linguistics to other domains of language disorders while showcasing the research being undertaken at prominent institutions such as University College London, the University of Cologne, Johannes Gutenberg University Mainz, Philipps University Marburg, the University of Belgrade, the University of Novi Sad, and Massachusetts Institute of Technology.Jana Willer GoldUniversity College LondonEditor-in-chief Objavljivanje ove knjige potpomognuto je sredstvima projekta Clinical linguistics: Psycholinguistic parameters in lexical-semantic processingin patients with schizophrenia i sredstvima Klinike za psihijatriju Vrapče.Interdisciplinary Linguistic and Psychiatric Research on Language Disorders is a collection of scientific papers presented at the International Scientific Workshop on Clinical Linguistics, held on 20 November 2018 at the Education Centre of the University Psychiatric Hospital Vrapče.The Erdeljac &amp; Sekulić Sović research group in clinical linguistics, based at the Department of Linguistics, Faculty of Humanities and Social Sciences, University of Zagreb, in collaboration with psychiatrists from the Department of Biological Psychiatry and Psychogeriatrics and the Department of Diagnostics and Intensive Care, both at the University Psychiatric Hospital Vrapče, present a unique example of an academic publication designed to spotlight ongoing research on semantic processing in individuals diagnosed with psychosis spectrum disorders who are native speakers of Croatian.A further value of this book lies in the co-authors’ contributions, written by specialists in clinical linguistics and psychiatry to expand the focus of research in clinical linguistics to other domains of language disorders while showcasing the research being undertaken at prominent institutions such as University College London, the University of Cologne, Johannes Gutenberg University Mainz, Philipps University Marburg, the University of Belgrade, the University of Novi Sad, and Massachusetts Institute of Technology.Jana Willer GoldUniversity College LondonEditor-in-chief Objavljivanje ove knjige potpomognuto je sredstvima projekta Clinical linguistics: Psycholinguistic parameters in lexical-semantic processingin patients with schizophrenia i sredstvima Klinike za psihijatriju Vrapče

Interdisciplinary Linguistic and Psychiatric Research on Language Disorders

Interdisciplinary Linguistic and Psychiatric Research on Language Disorders is a collection of scientific papers presented at the International Scientific Workshop on Clinical Linguistics, held on 20 November 2018 at the Education Centre of the University Psychiatric Hospital Vrapče.The Erdeljac &amp; Sekulić Sović research group in clinical linguistics, based at the Department of Linguistics, Faculty of Humanities and Social Sciences, University of Zagreb, in collaboration with psychiatrists from the Department of Biological Psychiatry and Psychogeriatrics and the Department of Diagnostics and Intensive Care, both at the University Psychiatric Hospital Vrapče, present a unique example of an academic publication designed to spotlight ongoing research on semantic processing in individuals diagnosed with psychosis spectrum disorders who are native speakers of Croatian.A further value of this book lies in the co-authors’ contributions, written by specialists in clinical linguistics and psychiatry to expand the focus of research in clinical linguistics to other domains of language disorders while showcasing the research being undertaken at prominent institutions such as University College London, the University of Cologne, Johannes Gutenberg University Mainz, Philipps University Marburg, the University of Belgrade, the University of Novi Sad, and Massachusetts Institute of Technology.Jana Willer GoldUniversity College LondonEditor-in-chief Objavljivanje ove knjige potpomognuto je sredstvima projekta Clinical linguistics: Psycholinguistic parameters in lexical-semantic processingin patients with schizophrenia i sredstvima Klinike za psihijatriju Vrapče.Interdisciplinary Linguistic and Psychiatric Research on Language Disorders is a collection of scientific papers presented at the International Scientific Workshop on Clinical Linguistics, held on 20 November 2018 at the Education Centre of the University Psychiatric Hospital Vrapče.The Erdeljac &amp; Sekulić Sović research group in clinical linguistics, based at the Department of Linguistics, Faculty of Humanities and Social Sciences, University of Zagreb, in collaboration with psychiatrists from the Department of Biological Psychiatry and Psychogeriatrics and the Department of Diagnostics and Intensive Care, both at the University Psychiatric Hospital Vrapče, present a unique example of an academic publication designed to spotlight ongoing research on semantic processing in individuals diagnosed with psychosis spectrum disorders who are native speakers of Croatian.A further value of this book lies in the co-authors’ contributions, written by specialists in clinical linguistics and psychiatry to expand the focus of research in clinical linguistics to other domains of language disorders while showcasing the research being undertaken at prominent institutions such as University College London, the University of Cologne, Johannes Gutenberg University Mainz, Philipps University Marburg, the University of Belgrade, the University of Novi Sad, and Massachusetts Institute of Technology.Jana Willer GoldUniversity College LondonEditor-in-chief Objavljivanje ove knjige potpomognuto je sredstvima projekta Clinical linguistics: Psycholinguistic parameters in lexical-semantic processingin patients with schizophrenia i sredstvima Klinike za psihijatriju Vrapče

A soluble α-synuclein construct forms a dynamic tetramer

A heterologously expressed form of the human Parkinson disease-associated protein α-synuclein with a 10-residue N-terminal extension is shown to form a stable tetramer in the absence of lipid bilayers or micelles. Sequential NMR assignments, intramonomer nuclear Overhauser effects, and circular dichroism spectra are consistent with transient formation of α-helices in the first 100 N-terminal residues of the 140-residue α-synuclein sequence. Total phosphorus analysis indicates that phospholipids are not associated with the tetramer as isolated, and chemical cross-linking experiments confirm that the tetramer is the highest-order oligomer present at NMR sample concentrations. Image reconstruction from electron micrographs indicates that a symmetric oligomer is present, with three- or fourfold symmetry. Thermal unfolding experiments indicate that a hydrophobic core is present in the tetramer. A dynamic model for the tetramer structure is proposed, based on expected close association of the amphipathic central helices observed in the previously described micelle-associated “hairpin” structure of α-synuclein