Cellular Chaperone Function of Intrinsically Disordered Dehydrin ERD14

Disordered plant chaperones play key roles in helping plants survive in harsh conditions, and they are indispensable for seeds to remain viable. Aside from well-known and thoroughly characterized globular chaperone proteins, there are a number of intrinsically disordered proteins (IDPs) that can also serve as highly effective protecting agents in the cells. One of the largest groups of disordered chaperones is the group of dehydrins, proteins that are expressed at high levels under different abiotic stress conditions, such as drought, high temperature, or osmotic stress. Dehydrins are characterized by the presence of different conserved sequence motifs that also serve as the basis for their categorization. Despite their accepted importance, the exact role and relevance of the conserved regions have not yet been formally addressed. Here, we explored the involvement of each conserved segment in the protective function of the intrinsically disordered stress protein (IDSP) A. thaliana’s Early Response to Dehydration (ERD14). We show that segments that are directly involved in partner binding, and others that are not, are equally necessary for proper function and that cellular protection emerges from the balanced interplay of different regions of ERD14.     

Genome-Wide Identification,Characterization and Expression Analysis of Soybean CHYR Gene Family

The CHYR (CHY ZINC-FINGER AND RING FINGER PROTEIN) proteins have been functionally characterized in iron regulation and stress response in Arabidopsis, rice and Populus. However, their roles in soybean have not yet been systematically investigated. Here, in this study, 16 GmCHYR genes with conserved Zinc_ribbon, CHY zinc finger and Ring finger domains were obtained and divided into three groups. Moreover, additional 2–3 hemerythrin domains could be found in the N terminus of Group III. Phylogenetic and homology analysis of CHYRs in green plants indicated that three groups might originate from different ancestors. Expectedly, GmCHYR genes shared similar conserved domains/motifs distribution within the same group. Gene expression analysis uncovered their special expression patterns in different soybean tissues/organs and under various abiotic stresses. Group I and II members were mainly involved in salt and alkaline stresses. The expression of Group III members was induced/repressed by dehydration, salt and alkaline stresses, indicating their diverse roles in response to abiotic stress. In conclusion, our work will benefit for further revealing the biological roles of GmCHYRs.     

Proteins Binding to the Carbohydrate HNK-1: Common Origins?

The human natural killer (HNK-1) carbohydrate plays important roles during nervous system development, regeneration after trauma and synaptic plasticity. Four proteins have been identified as receptors for HNK-1: the laminin adhesion molecule, high-mobility group box 1 and 2 (also called amphoterin) and cadherin 2 (also called N-cadherin). Because of HNK-1′s importance, we asked whether additional receptors for HNK-1 exist and whether the four identified proteins share any similarity in their primary structures. A set of 40,000 sequences homologous to the known HNK-1 receptors was selected and used for large-scale sequence alignments and motif searches. Although there are conserved regions and highly conserved sites within each of these protein families, there was no sequence similarity or conserved sequence motifs found to be shared by all families. Since HNK-1 receptors have not been compared regarding binding constants and since it is not known whether the sulfated or non-sulfated part of HKN-1 represents the structurally crucial ligand, the receptors are more heterogeneous in primary structure than anticipated, possibly involving different receptor or ligand regions. We thus conclude that the primary protein structure may not be the sole determinant for a bona fide HNK-1 receptor, rendering receptor structure more complex than originally assumed.     

An attempt to unify the structure of polymerases

With the great availability of sequences from RNA- and DNA-dependentRNA and DNA polymerases, it has become possible to delineatea few highly conserved regions for various polymerase types.In this work a DNA polymerase sequence from bacteriophage SPO2was found to be homologous to the polymerase domain of the Klenowfragment of polymerase I from Escherichia coli, which is knownto be closely related to those from Staphylococcus pneumoniae,Thermits aquaticus and bacteriophages T7 and T5. The alignmentof the SPO2 polymerase with the other five sequences considerablynarrowed the conserved motifs in these proteins. Three of themotifs matched reasonably all the conserved motifs of anotherDNA polymerase type, characterized by human polymerase a. Itis also possible to find these three motifs in monomeric DNA-dependentRNA polymerases and two of them in DNA polymerase ßand DNA terminal transferases. These latter two motifs alsomatched two of the four motifs recently identified in 84 RNA-dependentpolymerases. From the known tertiary architecture of the Klenowfragment of E.coli pol I, a spatial arrangement can be impliedfor these motifs. In addition, numerous biochemical experimentssuggesting a role for the motifs in a common function (dNTPbinding) also support these inferences. This speculative hypothesis,attempting to unify polymerase structure at least locally, ifnot globally, under the pol I fold, should provide a usefulmodel to direct mutagenesis experiments to probe template andsubstrate specificity in polymerases.     

Two Unconventional Metallothioneins in the Apple Snail Pomacea bridgesii Have Lost Their Metal Specificity during Adaptation to Freshwater Habitats

Metallothioneins (MTs) are a diverse group of proteins responsible for the control of metal homeostasis and detoxification. To investigate the impact that environmental conditions might have had on the metal-binding abilities of these proteins, we have characterized the MTs from the apple snail Pomacea bridgesii, a gastropod species belonging to the class of Caenogastropoda with an amphibious lifestyle facing diverse situations of metal bioavailability. P. bridgesii has two structurally divergent MTs, named PbrMT1 and PbrMT2, that are longer than other gastropod MTs due to the presence of extra sequence motifs and metal-binding domains. We have characterized the Zn(II), Cd(II), and Cu(I) binding abilities of these two MTs after their heterologous expression in E. coli. Our results have revealed that despite their structural differences, both MTs share an unspecific metal-binding character, and a great ability to cope with elevated amounts of different metal ions. Our analyses have also revealed slight divergences in their metal-binding features: PbrMT1 shows a more pronounced Zn(II)-thionein character than PbrMT2, while the latter has a stronger Cu(I)-thionein character. The characterization of these two unconventional PbrMTs supports the loss of the metal-binding specificity during the evolution of the MTs of the Ampullariid family, and further suggests an evolutionary link of this loss with the adaptation of these gastropod lineages to metal-poor freshwater habitats.     

Construction of a dictionary of sequence motifs that characterize groups of related proteins

An automatic procedure is proposed to identify, from the proteinsequence database, conserved amino acid patterns (or sequencemotifs) that are exclusive to a group of functionally relatedproteins. This procedure is applied to the PIR database anda dictionary of sequence motifs that relate to specific superfamiliesconstructed. The motifs have a practical relevance in identifyingthe membership of specific superfamilies without the need toperform sequence database searches in 20% of newly determinedsequences. The sequence motifs identified represent functionallyimportant sites on protein molecules. When multiple blocks existin a single motif they are often close together in the 3-D structure.Furthermore, occasionally these motif blocks were found to besplit by introns when the correlation with exon structures wasexamined.     

Characterization of Class V DyP-Type Peroxidase SaDyP1 from Streptomyces avermitilis and Evaluation of SaDyPs Expression in Mycelium

DyP-type peroxidases are a family of heme peroxidases named for their ability to degrade persistent anthraquinone dyes. DyP-type peroxidases are subclassified into three classes: classes P, I and V. Based on its genome sequence, Streptomyces avermitilis, eubacteria, has two genes presumed to encode class V DyP-type peroxidases and two class I genes. We have previously shown that ectopically expressed SaDyP2, a member of class V, indeed has the characteristics of a DyP-type peroxidase. In this study, we analyzed SaDyP1, a member of the same class V as SaDyP2. SaDyP1 showed high amino acid sequence identity to SaDyP2, retaining a conserved GXXDG motif and catalytic aspartate. SaDyP1 degraded anthraquinone dyes, which are specific substrates of DyP-type peroxidases but not azo dyes. In addition to such substrate specificity, SaDyP1 showed other features of DyP-type peroxidases, such as low optimal pH. Furthermore, immunoblotting using an anti-SaDyP2 polyclonal antibody revealed that SaDyP1 and/or SaDyP2 is expressed in mycelia of wild-type S. avermitilis.     

赤子爱胜蚓蛋白磷酸酶2A类似激活剂样蛋白cDNA和氨基酸序列分析

目的分析赤子爱胜蚓蛋白磷酸酶2A(protein phosphatase 2A,PP2A)类似激活剂样蛋白的cDNA和氨基酸序列。方法从赤子爱胜蚓cDNA文库中随机测序得到目标cDNA序列,应用DNAMAN、NCBI ORF finder、BLAST、Conserved Domains、GOR、SWISS-MODEL、PDB等基因和蛋白质分析软件进行该目标基因测序及氨基酸序列分析。结果赤子爱胜蚓PP2A类似激活剂样蛋白cDNA序列长547 bp,编码87个氨基酸残基;与该蛋白基因序列相似度较高的多是不同物种中PP2A类似激活剂蛋白基因序列,其中与Crassostrea virginica serine/threonine-protein phosphatase 2A activator-like mRNA核苷酸相似度为67%;与该蛋白氨基酸序列相似度较高的多是不同物种中的PP2A类似激活剂蛋白氨基酸序列,其中与serine/threonine-protein phosphatase 2A activator(Crassostrea gigas)氨基酸相似度为70%;目标序列中存在一段PTPA超家族保守结构域,蛋白质二级结构以α螺旋以及无规则卷曲为主,三级预测结果与二级结构一致。结论本实验分析的赤子爱胜蚓PP2A类似激活剂样蛋白与牡蛎PP2A类似激活剂蛋白及存在于所有真核生物中且高度保守的PTPA同源度较高,可能具有特异性激活肿瘤抑制因子PP2A的间接抑制肿瘤作用。     

Structural Identification of the Pacemaker Cells and Expression of Hyperpolarization-Activated Cyclic Nucleotide-Gated (HCN) Channels in the Heart of the Wild Atlantic Cod,Gadus morhua (Linnaeus, 1758)

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are proteins that contain highly conserved functional domains and sequence motifs that are correlated with their unique biophysical activities, to regulate cardiac pacemaker activity and synaptic transmission. These pacemaker proteins have been studied in mammalian species, but little is known now about their heart distribution in lower vertebrates and c-AMP modulation. Here, we characterized the pacemaker system in the heart of the wild Atlantic cod (Gadus morhua), with respect to primary pacemaker molecular markers. Special focus is given to the structural, ultrastructural and molecular characterization of the pacemaker domain, through the expression of HCN channel genes and the immunohistochemistry of HCN isoforms, including the location of intracardiac neurons that are adjacent to the sinoatrial region of the heart. Similarly to zebrafish and mammals, these neurons are immunoreactive to ChAT, VAChT and nNOS. It has been shown that cardiac pacemaking can be modulated by sympathetic and parasympathetic pathways, and the existence of intracardiac neurons projecting back to the central nervous system provide a plausible link between them.     

PDI Family Members as Guides for Client Folding and Assembly

Complicated and sophisticated protein homeostasis (proteostasis) networks in the endoplasmic reticulum (ER), comprising disulfide catalysts, molecular chaperones, and their regulators, help to maintain cell viability. Newly synthesized proteins inserted into the ER need to fold and assemble into unique native structures to fulfill their physiological functions, and this is assisted by protein disulfide isomerase (PDI) family. Herein, we focus on recent advances in understanding the detailed mechanisms of PDI family members as guides for client folding and assembly to ensure the efficient production of secretory proteins.     

Comparison of 10,11‐Dehydrocurvularin Polyketide Synthases from Alternaria cinerariae and Aspergillus terreus Highlights Key Structural Motifs

Iterative type I polyketide synthases (PKSs) from fungi are multifunctional enzymes that use their active sites repeatedly in a highly ordered sequence to assemble complex natural products. A phytotoxic macrolide with anticancer properties, 10,11‐dehydrocurvularin (DHC), is produced by cooperation of a highly reducing (HR) iterative PKS and a non‐reducing (NR) iterative PKS. We have identified the DHC gene cluster in Alternaria cinerariae, heterologously expressed the active HR PKS (Dhc3) and NR PKS (Dhc5) in yeast, and compared them to corresponding proteins that make DHC in Aspergillus terreus. Phylogenetic analysis and homology modeling of these enzymes identified variable surfaces and conserved motifs that are implicated in product formation.     

The Effects of Arbuscular Mycorrhizal Fungi on Direct and Indirect Defense Metabolites of Plantago lanceolata L.

Arbuscular mycorrhizal fungi can strongly influence the metabolism of their host plant, but their effect on plant defense mechanisms has not yet been thoroughly investigated. We studied how the principal direct defenses (iridoid glycosides) and indirect defenses (volatile organic compounds) of Plantago lanceolata L. are affected by insect herbivory and mechanical wounding. Volatile compounds were collected and quantified from mycorrhizal and non-mycorrhizal P. lanceolata plants that underwent three different treatments: 1) insect herbivory, 2) mechanical wounding, or 3) no damage. The iridoids aucubin and catalpol were extracted and quantified from the same plants. Emission of terpenoid volatiles was significantly higher after insect herbivory than after the other treatments. However, herbivore-damaged mycorrhizal plants emitted lower amounts of sesquiterpenes, but not monoterpenes, than herbivore-damaged non-mycorrhizal plants. In contrast, mycorrhizal infection increased the emission of the green leaf volatile (Z)-3-hexenyl acetate in untreated control plants, making it comparable to emission from mechanically wounded or herbivore-damaged plants whether or not they had mycorrhizal associates. Neither mycorrhization nor treatment had any influence on the levels of iridoid glycosides. Thus, mycorrhizal infection did not have any effect on the levels of direct defense compounds measured in P. lanceolata. However, the large decline in herbivore-induced sesquiterpene emission may have important implications for the indirect defense potential of this species.     

Periplaneta americana perception of phytochemical naphthoquinones as allelochemicals

Electrochemistry of malePeriplaneta americana perception of naphthoquinones as allelochemicals is presented. Importance of the oxidation-reduction potential and hydrogen-bonding capabilities of the naphtho-quinone messenger to its exchange of energy with the information-encoding, redox protein of the chemosensory neuron is considered. Evidence that the required information is initially encoded by an electroreactive protein in dendritic membrane is analyzed. A sulfhydryl-disulfide protein-based electrochemical mechanism for the demonstrated linear conversion of a molecular messenger energy state into whole insect behavior (avoidance or feeding inhibition) is elucidated. Even though the information-encoding mechanism is genetically conserved (kept electrochemically similar) within the species, behavioral biotypical differences regarding sensitivity to naphthoquinones are allowed through a group of proteins in the outer aqueous medium in the chemosensory sensillum. Such proteins electrochemically interface the dendritic membrane of the chemosensory neuron in the sensillum with the environment external to the sensillum. These interfacing proteins bind (complex) with chemical messengers (e.g., naphthoquinones) and/or variously degrade them, and thus determine the amount (moles) that is required in the environment to result in stimulation of the underlying sensory neurons and in a changed behavior.     

PRINTS-a protein motif fingerprint database

The PRINTS database of protein ‘fingerprints’ isdescribed. Fingerprints comprise sets of motifs excised fromconserved regions of sequence alignments, their diagnostic poweror potency being refined by iterative database scanning (inthis case the OWL composite sequence database). Generally, themotifs do not overlap, but are separated along a sequence, thoughthey may be contiguous in 3-D space. The use of groups of independent,linearly or spatially separate motifs allows particular proteinfolds and functionalities to be characterized more flexiblyand powerfully than conventional single-component patterns orregular expressions. The current version of the database (4.0)contains 150 entries (encoding >700 motifs), covering a widerange of globular and membrane proteins, modular polypeptidesand so on. The growth of the database is influenced by a numberof factors, e.g. the use of multiple motifs, the maximizationof sequence information through iterative database scanningand the fact that the database searched is a large composite.The information contained within PRINTS is distinct from butcomplementary to the single consensus expressions stored inthe widely used PROSITE dictionary of patterns.