A short LysM protein with high molecular diversity from an arbuscular mycorrhizal fungus,Rhizophagus irregularis

Arbuscular mycorrhizal (AM) fungi form an intimate symbiosis with roots of more than 80% of land plants without eliciting a significant defense response, and how they do so is yet to be determined. Typically, plants mount a defense response upon sensing chitin in fungal walls, and to counteract this response, plant-pathogenic fungi secrete small effector proteins with chitin-binding LysM domains. In the AM fungus, Rhizophagus irregularis, a small, putatively-secreted LysM protein, which we refer to as RiSLM, is among the most highly expressed effector-like proteins during symbiosis. Here, we show that RiSLM expression is reduced during non-functional symbiosis with Medicago mutants, mtpt4-2 and vapyrin. We demonstrate that RiSLM can bind to both chitin and chitosan, and we model the protein-ligand interaction to identify possible binding sites. Finally, we have identified RiSLM homologs in five published R. irregularis isolate genomes and demonstrate that the gene is subject to a high rate of evolution and is experiencing positive selection, while still conserving putative function. Our results present important clues for elucidating a role for a LysM effector, RiSLM, in AM symbiosis.     

Spatially Distinct Pools of TORC1 Balance Protein Homeostasis

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Fractionation of plant protoplast types by iso-osmotic density gradient centrifugation

Summary A simple effective technique for the fractionation of protoplast populations is described. Protoplasts are separated by low-speed centrifugation in an iso-osmotic, discontinuous density gradient system on the basis of differences in their buoyant densities. At a constant osmolality of 660±20 mOs/kg H₂O, the gradients provide a density range from 1.017 to 1.069 g/cm³ at 20 °C which corresponds to the buoyant densities of most protoplast types studied. Characteristics of the KMC/S-density gradient system and factors affecting the fractionation were investigated. Protoplasts were isolated from various tissues and cultivars of tobacco, barley, wheat, rye, oat and maize. Their density-dependent distribution profiles in KMC/S-gradients and their average buoyant densities were determined under standardized conditions. Great differences in the buoyant densities were found between protoplasts of different tissues. Mixed populations of two types of protoplasts, differing in buoyant density by about 15–20 mg/cm³, were separated to give highly purified fractions. Factors affecting the buoyant densities of protoplasts have been investigated. Ploidy level and species differences did not significantly affect the fractionation profiles. However, an age-dependent variation in the average buoyant density of tobacco mesophyll protoplasts was observed. Fractionation of tobacco mesophyll protoplasts and their subsequent regeneration to plants demonstrates the practicability and physiological compatibility of the KMC/S-density gradient system under sterile conditions. The morphogenetic potential of protoplasts was not affected by the separation procedure or the gradient components.     

Inositol 1,4,5-trisphosphate receptor-isoform diversity in cell death and survival

Cell-death and -survival decisions are critically controlled by intracellular Ca^2 + homeostasis and dynamics at the level of the endoplasmic reticulum (ER). Inositol 1,4,5-trisphosphate (IP₃) receptors (IP₃Rs) play a pivotal role in these processes by mediating Ca^2 + flux from the ER into the cytosol and mitochondria. Hence, it is clear that many pro-survival and pro-death signaling pathways and proteins affect Ca^2 + signaling by directly targeting IP₃R channels, which can happen in an IP₃R-isoform-dependent manner. In this review, we will focus on how the different IP₃R isoforms (IP₃R1, IP₃R2 and IP₃R3) control cell death and survival. First, we will present an overview of the isoform-specific regulation of IP₃Rs by cellular factors like IP₃, Ca^2 +, Ca^2 +-binding proteins, adenosine triphosphate (ATP), thiol modification, phosphorylation and interacting proteins, and of IP₃R-isoform specific expression patterns. Second, we will discuss the role of the ER as a Ca^2 + store in cell death and survival and how IP₃Rs and pro-survival/pro-death proteins can modulate the basal ER Ca^2 + leak. Third, we will review the regulation of the Ca^2 +-flux properties of the IP₃R isoforms by the ER-resident and by the cytoplasmic proteins involved in cell death and survival as well as by redox regulation. Hence, we aim to highlight the specific roles of the various IP₃R isoforms in cell-death and -survival signaling. This article is part of a Special Issue entitled: Calcium signaling in health and disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.     

Microbial βγ-crystallins

βγ-Crystallins have emerged as a superfamily of structurally homologous proteins with representatives across the domains of life. A major portion of this superfamily is constituted by members from microorganisms. This superfamily has also been recognized as a novel group of Ca²⁺-binding proteins with huge diversity. The βγ domain shows variable properties in Ca²⁺ binding, stability and association with other domains. The various members present a series of evolutionary adaptations culminating in great diversity in properties and functions. Most of the predicted βγ-crystallins are yet to be characterized experimentally. In this review, we outline the distinctive features of microbial βγ-crystallins and their position in the βγ-crystallin superfamily.     

High prevalence of cerebral venous sinus thrombosis (CVST) as presentation of cystathionine beta-synthase deficiency in childhood: Molecular and clinical findings of Turkish probands

Classical homocystinuria is the most commonly inherited disorder of sulfur metabolism, caused by the genetic alterations in human cystathionine beta-synthase (CBS) gene. In this study, we present comprehensive clinical findings and the genetic basis of homocystinuria in a cohort of Turkish patients. Excluding some CBS mutations, detailed genotype–phenotype correlation for different CBS mutations has not been established in literature. We aimed to make clinical subgroups according to main clinical symptoms and discussed these data together with mutational analysis results from our patients. Totally, 16 different mutations were identified; twelve of which had already been reported, and four are novel (p.N93Y, p.L251P, p.D281V and c.829−2A>T). The probands were classified into three major groups according to the clinical symptoms caused by these mutations. A psychomotor delay was the most common diagnostic symptom (n = 12, 46.2% neurological presentation), followed by thromboembolic events (n = 6, 23.1% vascular presentation) and lens ectopia, myopia or marfanoid features (n = 5, 19.2% connective tissue presentation). Pyridoxine responsiveness was 7.7%; however, with partial responsive probands, the ratio was 53.9%.     

Bridging of anions by hydrogen bonds in nest motifs and its significance for Schellman loops and other larger motifs within proteins

The nest is a protein motif of three consecutive amino acid residues with dihedral angles 1,2‐α_Rα_L (RL nests) or 1,2‐α_Lα_R (LR nests). Many nests form a depression in which an anion or δ‐negative acceptor atom is bound by hydrogen bonds from the main chain NH groups. We have determined the extent and nature of this bridging in a database of protein structures using a computer program written for the purpose. Acceptor anions are bound by a pair of bridging hydrogen bonds in 40% of RL nests and 20% of LR nests. Two thirds of the bridges are between the NH groups at Positions 1 and 3 of the motif (N1N3‐bridging)—which confers a concavity to the nest; one third are of the N2N3 type—which does not. In bridged LR nests N2N3‐bridging predominates (14% N1N3: 75% N2N3), whereas in bridged RL nests the reverse is true (69% N1N3: 25% N2N3). Most bridged nests occur within larger motifs: 45% in (hexapeptide) Schellman loops with an additional 4 → 0 hydrogen bond (N1N3), 11% in Schellman loops with an additional 5 → 1 hydrogen bond (N2N3), 12% in a composite structure including a type 1β‐bulge loop and an asx‐ or ST‐ motif (N1N3)—remarkably homologous to the N1N3‐bridged Schellman loop—and 3% in a composite structure including a type 2β‐bulge loop and an asx‐motif (N2N3). A third hydrogen bond is a previously unrecognized feature of Schellman loops as those lacking bridged nests have an additional 4 → 0 hydrogen bond. Proteins 2014; 82:3023–3031. © 2014 Wiley Periodicals, Inc.     

Steric and electronic interactions controlling the cis/trans isomer equilibrium at X‐pro tertiary amide motifs in solution

A systematic understanding of the noncovalent interactions that influence the structures of the cis conformers and the equilibrium between the cis and the trans conformers, of the X‐Pro tertiary amide motifs, is presented based on analyses of ¹H‐, ¹³C‐NMR and FTIR absorption spectra of two sets of homologous peptides, X‐Pro‐Aib‐OMe and X‐Pro‐NH‐Me (where X is acetyl, propionyl, isobutyryl and pivaloyl), in solvents of varying polarities. First, this work shows that the cis conformers of any X‐Pro tertiary amide motif, including Piv‐Pro, are accessible in the new motifs X‐Pro‐Aib‐OMe, in solution. These conformers are uniquely observable by FTIR spectroscopy at ambient temperatures and by NMR spectroscopy from temperatures as high as 273 K. This is made possible by the persistent presence of n_i‐1→π_i* interactions at Aib, which also influence the disappearance of steric effects at these cis X‐Pro rotamers. Second, contrary to conventional understanding, the energy contribution of steric effects to the cis/trans equilibrium at the X‐Pro motifs is found to be nonvariant (0.54 ± 0.02 kcal/mol) with increase in steric bulk on the X group. Third, the current studies provide direct evidence for the weak intramolecular interactions namely the n_i‐1→π_i*, the N_Pro???H_i+1 (C₅a), and the C₇ hydrogen bond that operate and influence the structures, stabilities, and dynamics between different conformational states of X‐Pro tertiary amide motifs. NMR and IR spectral data suggest that the cis conformers of X‐Pro motifs are ensembles of short‐lived rotamers about the C′_X–N_Pro bond. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 66–77, 2014.