Crystal Structures of Glutaminyl Cyclases (QCs) from Drosophila melanogaster Reveal Active Site Conservation between Insect and Mammalian QCs

Glutaminyl cyclases (QCs), which catalyze the formation of pyroglutamic acid (pGlu) at the N-terminus of a variety of peptides and proteins, have attracted particular attention for their potential role in Alzheimer's disease. In a transgenic Drosophila melanogaster (Dm) fruit fly model, oral application of the potent competitive QC inhibitor PBD150 was shown to reduce the burden of pGlu-modified Aβ. In contrast to mammals such as humans and rodents, there are at least three DmQC species, one of which (isoDromeQC) is localized to mitochondria, whereas DromeQC and an isoDromeQC splice variant possess signal peptides for secretion. Here we present the recombinant expression, characterization, and crystal structure determination of mature DromeQC and isoDromeQC, revealing an overall fold similar to that of mammalian QCs. In the case of isoDromeQC, the putative extended substrate binding site might be affected by the proximity of the N-terminal residues. PBD150 inhibition of DromeQC is roughly 1 order of magnitude weaker than that of the human and murine QCs. The inhibitor binds to isoDromeQC in a fashion similar to that observed for human QCs, whereas it adopts alternative binding modes in a DromeQC variant lacking the conserved cysteines near the active center and shows a disordered dimethoxyphenyl moiety in wild-type DromeQC, providing an explanation for the lower affinity. Our biophysical and structural data suggest that isoDromeQC and human QC are similar with regard to functional aspects. The two Dm enzymes represent a suitable model for further in-depth analysis of the catalytic mechanism of animal QCs, and isoDromeQC might serve as a model system for the structure-based design of potential AD therapeutics.     

Isolation and characterization of the glutaminyl cyclases from Solanum tuberosum and Arabidopsis thaliana: implications for physiological functions

Glutaminyl cyclases (QCs) catalyze the formation of pyroglutamic acid at the N-terminus of several peptides and proteins. On the basis of the amino acid sequence of Carica papaya QC, we identified cDNAs of the putative counterparts from Solanum tuberosum and Arabidopsis thaliana. Upon expression of the corresponding cDNAs from both plants via the secretory pathway of Pichia pastoris, two active QC proteins were isolated. The specificity of the purified proteins was assessed using various substrates with different amino acid composition and length. Highest specificities were observed with substrates possessing large hydrophobic residues adjacent to the N-terminal glutamine and for fluorogenic dipeptide surrogates. However, compared to Carica papaya QC, the specificity constants were approximately one order of magnitude lower for most of the QC substrates analyzed. The QCs also catalyzed the conversion of N-terminal glutamic acid to pyroglutamic acid, but with approximately 10(5)- to 10(6)-fold lower specificity. The ubiquitous distribution of plant QCs prompted a search for potential substrates in plants. Based on database entries, numerous proteins, e.g., pathogenesis-related proteins, were found that carry a pyroglutamate residue at the N-terminus, suggesting QC involvement. The putative relevance of QCs and pyroglutamic acid for plant defense reactions is discussed.     

Glutaminyl cyclase knock-out mice exhibit slight hypothyroidism but no hypogonadism: implications for enzyme function and drug development

Glutaminyl cyclases (QCs) catalyze the formation of pyroglutamate (pGlu) residues at the N terminus of peptides and proteins. Hypothalamic pGlu hormones, such as thyrotropin-releasing hormone and gonadotropin-releasing hormone are essential for regulation of metabolism and fertility in the hypothalamic pituitary thyroid and gonadal axes, respectively. Here, we analyzed the consequences of constitutive genetic QC ablation on endocrine functions and on the behavior of adult mice. Adult homozygous QC knock-out mice are fertile and behave indistinguishably from wild type mice in tests of motor function, cognition, general activity, and ingestion behavior. The QC knock-out results in a dramatic drop of enzyme activity in the brain, especially in hypothalamus and in plasma. Other peripheral organs like liver and spleen still contain QC activity, which is most likely caused by its homolog isoQC. The serum gonadotropin-releasing hormone, TSH, and testosterone concentrations were not changed by QC depletion. The serum thyroxine was decreased by 24% in homozygous QC knock-out animals, suggesting a mild hypothyroidism. QC knock-out mice were indistinguishable from wild type with regard to blood glucose and glucose tolerance, thus differing from reports of thyrotropin-releasing hormone knock-out mice significantly. The results suggest a significant formation of the hypothalamic pGlu hormones by alternative mechanisms, like spontaneous cyclization or conversion by isoQC. The different effects of QC depletion on the hypothalamic pituitary thyroid and gonadal axes might indicate slightly different modes of substrate conversion of both enzymes. The absence of significant abnormalities in QC knock-out mice suggests the presence of a therapeutic window for suppression of QC activity in current drug development.     

A Unique Carboxylic-Acid Hydrogen-Bond Network (CAHBN) Confers Glutaminyl Cyclase Activity on M28 Family Enzymes

Proteins with sequence or structure similar to those of di-Zn exopeptidases are usually classified as the M28-family enzymes, including the mammalian-type glutaminyl cyclases (QCs). QC catalyzes protein N-terminal pyroglutamate formation, a posttranslational modification important under many physiological and pathological conditions, and is a drug target for treating neurodegenerative diseases, cancers and inflammatory disorders. Without functional characterization, mammalian QCs and their orthologs remain indistinguishable at the sequence and structure levels from other M28-family proteins, leading to few reported QCs. Here, we show that a low-barrier carboxylic-acid hydrogen-bond network (CAHBN) is required for QC activity and discriminates QCs from M28-family peptidases. We demonstrate that the CAHBN-containing M28 peptidases deposited in the PDB are indeed QCs. Our analyses identify several thousands of QCs from the three domains of life, and we enzymatically and structurally characterize several. For the first time, the interplay between a CAHBN and the binuclear metal-binding center of mammalian QCs is made clear. We found that the presence or absence of CAHBN is a key discriminator for the formation of either the mono-Zn QCs or the di-Zn exopeptidases. Our study helps explain the possible roles of QCs in life.     

Comparative large scale characterization of plant versus mammal proteins reveals similar and idiosyncratic N-α-acetylation features

N-terminal modifications play a major role in the fate of proteins in terms of activity, stability, or subcellular compartmentalization. Such modifications remain poorly described and badly characterized in proteomic studies, and only a few comparison studies among organisms have been made available so far. Recent advances in the field now allow the enrichment and selection of N-terminal peptides in the course of proteome-wide mass spectrometry analyses. These targeted approaches unravel as a result the extent and nature of the protein N-terminal modifications. Here, we aimed at studying such modifications in the model plant Arabidopsis thaliana to compare these results with those obtained from a human sample analyzed in parallel. We applied large scale analysis to compile robust conclusions on both data sets. Our data show strong convergence of the characterized modifications especially for protein N-terminal methionine excision, co-translational N-α-acetylation, or N-myristoylation between animal and plant kingdoms. Because of the convergence of both the substrates and the N-α-acetylation machinery, it was possible to identify the N-acetyltransferases involved in such modifications for a small number of model plants. Finally, a high proportion of nuclear-encoded chloroplast proteins feature post-translational N-α-acetylation of the mature protein after removal of the transit peptide. Unlike animals, plants feature in a dedicated pathway for post-translational acetylation of organelle-targeted proteins. The corresponding machinery is yet to be discovered.     

Inhibition of glutaminyl cyclase attenuates cell migration modulated by monocyte chemoattractant proteins

QC (glutaminyl cyclase) catalyses the formation of N-terminal pGlu (pyroglutamate) in peptides and proteins. pGlu formation in chemoattractants may participate in the regulation of macrophage activation and migration. However, a clear molecular mechanism for the regulation is lacking. The present study examines the role of QC-mediated pGlu formation on MCPs (monocyte chemoattractant proteins) in inflammation. We demonstrated in vitro the pGlu formation on MCPs by QC using MS. A potent QC inhibitor, PBD150, significantly reduced the N-terminal uncyclized-MCP-stimulated monocyte migration, whereas pGlu-containing MCP-induced cell migration was unaffected. QC small interfering RNA revealed a similar inhibitory effect. Lastly, we demonstrated that inhibiting QC can attenuate cell migration by lipopolysaccharide. These results strongly suggest that QC-catalysed N-terminal pGlu formation of MCPs is required for monocyte migration and provide new insights into the role of QC in the inflammation process. Our results also suggest that QC could be a drug target for some inflammatory disorders.     

Linked Production of Pyroglutamate-Modified Proteins via Self-Cleavage of Fusion Tags with TEV Protease and Autonomous N-Terminal Cyclization with Glutaminyl Cyclase In Vivo

Overproduction of N-terminal pyroglutamate (pGlu)-modified proteins utilizing Escherichia coli or eukaryotic cells is a challenging work owing to the fact that the recombinant proteins need to be recovered by proteolytic removal of fusion tags to expose the N-terminal glutaminyl or glutamyl residue, which is then converted into pGlu catalyzed by the enzyme glutaminyl cyclase. Herein we describe a new method for production of N-terminal pGlu-containing proteins in vivo via intracellular self-cleavage of fusion tags by tobacco etch virus (TEV) protease and then immediate N-terminal cyclization of passenger target proteins by a bacterial glutaminyl cyclase. To combine with the sticky-end PCR cloning strategy, this design allows the gene of target proteins to be efficiently inserted into the expression vector using two unique cloning sites (i.e., SnaB I and Xho I), and the soluble and N-terminal pGlu-containing proteins are then produced in vivo. Our method has been successfully applied to the production of pGlu-modified enhanced green fluorescence protein and monocyte chemoattractant proteins. This design will facilitate the production of protein drugs and drug target proteins that possess an N-terminal pGlu residue required for their physiological activities.     

Structures of glycosylated mammalian glutaminyl cyclases reveal conformational variability near the active center

Formation of N-terminal pyroglutamate (pGlu or pE) from glutaminyl or glutamyl precursors is catalyzed by glutaminyl cyclases (QC). As the formation of pGlu-amyloid has been linked with Alzheimer's disease, inhibitors of QCs are currently the subject of intense development. Here, we report three crystal structures of N-glycosylated mammalian QC from humans (hQC) and mice (mQC). Whereas the overall structures of the enzymes are similar to those reported previously, two surface loops in the neighborhood of the active center exhibit conformational variability. Furthermore, two conserved cysteine residues form a disulfide bond at the base of the active center that was not present in previous reports of hQC structure. Site-directed mutagenesis suggests a structure-stabilizing role of the disulfide bond. At the entrance to the active center, the conserved tryptophan residue, W(207), which displayed multiple orientations in previous structure, shows a single conformation in both glycosylated human and murine QCs. Although mutagenesis of W(207) into leucine or glutamine altered substrate conversion significantly, the binding constants of inhibitors such as the highly potent PQ50 (PBD150) were minimally affected. The crystal structure of PQ50 bound to the active center of murine QC reveals principal binding determinants provided by the catalytic zinc ion and a hydrophobic funnel. This study presents a first comparison of two mammalian QCs containing typical, conserved post-translational modifications.     

Emerging molecular mechanisms that power and regulate the anastral mitotic spindle of flowering plants

Flowering plants, lacking centrosomes as well as dynein, assemble their mitotic spindle via a pathway that is distinct visually and molecularly from that of animals and yeast. The molecular components underlying mitotic spindle assembly and function in plants are beginning to be discovered. Here, we review recent evidence suggesting the preprophase band in plants functions analogously to the centrosome in animals in establishing spindle bipolarity, and we review recent progress characterizing the roles of specific motor proteins in plant mitosis. Loss of function of certain minus-end-directed KIN-14 motor proteins causes a broadening of the spindle pole; whereas, loss of function of a KIN-5 causes the formation of monopolar spindles, resembling those formed when the homologous motor protein (e.g., Eg5) is knocked out in animal cells. We present a phylogeny of the kinesin-5 motor domain, which shows deep divergence among plant sequences, highlighting possibilities for specialization. Finally, we review information concerning the roles of selected structural proteins at mitosis as well as recent findings concerning regulation of M-phase in plants. Insight into the mitotic spindle will be obtained through continued comparison of mitotic mechanisms in a diversity of cells.     

N-Terminal pyroglutamate formation of Aβ38 and Aβ40 enforces oligomer formation and potency to disrupt hippocampal long-term potentiation

Pyroglutamate (pGlu)-modified amyloid peptides have been identified in sporadic and familial forms of Alzheimer's disease (AD) and the inherited disorders familial British and Danish Dementia (FBD and FDD). In this study, we characterized the aggregation of amyloid-β protein Aβ37, Aβ38, Aβ40, Aβ42 and ADan species in vitro, which were modified by N-terminal pGlu (pGlu-Aβ3-x, pGlu-ADan) or possess the intact N-terminus (Aβ1-x, ADan). The pGlu-modification confers rapid formation of oligomers and short fibrillar aggregates. In accordance with these observations, the pGlu-modified Aβ38, Αβ40 and Αβ42 species inhibit hippocampal long term potentiation of synaptic response, but pGlu-Aβ3-42 showing the highest effect. Among the unmodified Aβ peptides, only Aβ1-42 exhibites such propensity, which was similar to pGlu-Aβ3-38 and pGlu-Aβ3-40. Likewise, the amyloidogenic peptide pGlu-ADan impaired synaptic potentiation more pronounced than N-terminal unmodified ADan. The results were validated using conditioned media from cultivated HEK293 cells, which express APP variants favoring the formation of Aβ1-x, Aβ3-x or N-truncated pGlu-Aβ3-x species. Hence, we show that the ability of different amyloid peptides to impair synaptic function apparently correlates to their potential to form oligomers as a common mechanism. The pGlu-modification is apparently mediating a higher surface hydrophobicity, as shown by 1-anilinonaphtalene-8-sulfonate fluorescence, which enforces potential to interfere with neuronal physiology.     

Secretory carrier membrane proteins

Secretory carrier membrane proteins (SCAMPs) are a family of integral membrane proteins that play roles in mediating exocytosis in animal cells. However, relatively little is known about the subcellular localization, trafficking, and function of SCAMPs in plants. Several recent studies in plant cells indicate that plant SCAMPs share many similarities with their mammalian homologs although there are differences. In this review, we will first summarize and compare animal and plant SCAMPs in terms of their subcellular localization, trafficking, and possible functions. We will then present a phylogenetic analysis of plant and animal SCAMPs. Finally, we will present expression analysis on selective Arabidopsis SCAMPs in the hope of pointing to directions for functional characterization of plant SCAMPs in the future.     

Isoaspartate-containing amyloid precursor protein-derived peptides alter efficacy and specificity of potential beta-secretases

Neuritic plaques of Alzheimer patients are composed of multiple protein components. Among them, the amyloid beta-peptides (Abeta) 1-40/42 and further N- and C-terminally modified fragments of Abeta are highly abundant. Most prominent are the isoaspartate (isoAsp)-Abeta peptides and pyroglutamyl (pGlu)-Abeta. While pGlu-Abeta can only be formed from an N-terminal glutamate by glutaminyl cyclase, spontaneous isoAsp-isomerization cannot occur at an N-terminal aspartate of peptides. This means that isoAsp-Abeta formation must precede proteolysis of the amyloid precursor protein (APP). Abeta generation from APP by beta- and gamma-secretases initiates the amyloid peptide aggregation and deposition process. Two aspartate proteases have been identified as secretases: BACE-1 (beta-site amyloid precursor protein cleaving enzyme) and the intramembrane gamma-secretase multiprotein complex. However, recent evidence supports more than one beta-secretase initiating this cascade. Formation of Abeta1-40/42 was predominantly studied by expression of mutated human APP sequences in cell culture and transgenic animals, generating Abeta fragments that did not contain such multiple posttranslational modifications as in Alzheimer's disease. This prompted us to investigate the catalytic turnover of Asp- or isoAsp-containing APP-derived peptide sequences by BACE-1 and cathepsin B, another potential beta-secretase. While cathepsin B is more effective than BACE-1 in processing the Asp-containing peptide derivatives, only cathepsin B can cleave the isoAsp-containing peptides, which occurs with high catalytic efficiency.     

Borate Transport and Cell Growth and Proliferation: Not Only in Plants

Boron is an abundant mineral essential for the life cycle of plants and may play a role in animal development and growth. Very little is known about boron homeostasis in plant and animal cells and the physiological roles of boron in animals. The recent identification of boron transporters, BOR1 in plants and NaBC1 in mammals, and that NaBC1 functions as an electrogenic Na+-coupled borate transporter essential for cell growth and proliferation open the way to probe the roles of boron in cellular function and physiology.     

Substrate specificity of glutaminyl cyclases from plants and animals

Glutaminyl cyclases (QC) catalyze the intramolecular cyclization of N-terminal glutamine residues of peptides and proteins. For a comparison of the substrate specificity of human and papaya QC enzymes, a novel continuous assay was established by adapting an existing discontinuous method. Specificity constants (kcat/Km) of dipeptides and dipeptide surrogates were higher for plant QC, whereas the selectivity for oligopeptides was similar for both enzymes. However, only the specificity constants of mammalian QC were dependent on size and composition of the substrates. Specificity constants of both enzymes were equally pH-dependent in the acidic pH-region, revealing a pKa value identical to the pKa of the substrate, suggesting similarities in the substrate conversion mode. Accordingly, both QCs converted the L-beta homoglutaminyl residue in the peptide H-beta homoGln-Phe-Lys-Arg-Leu-Ala-NH2 and the glutaminyl residues of the branched peptide H-Gln-Lys(Gln)-Arg-Leu-Ala-NH2 as well as the partially cyclized peptide H-Gln-cyclo(N epsilon-Lys-Arg-Pro-Ala-Gly-Phe). In contrast, only QC from C. papaya was able to cyclize a methylated glutamine residue, while this compound did not even inhibit human QC-catalysis, suggesting distinct substrate recognition pattern. The conversion of the potential physiological substrates [Gln1]-gastrin, [Gln1]-neurotensin and [Gln1]-fertilization promoting peptide indicates that human QC may play a key role in posttranslational modification of most if not all pGlu-containing hormones.     

Hormone modulation of legume‐rhizobial symbiosis

Leguminous plants can establish symbiotic associations with diazotropic rhizobia to form nitrogenfixating nodules, which are classified as determinate or indeterminate based on the persistence of nodule meristem. The formation of nitrogen-fixing nodules requires coordinating rhizobial infection and root nodule organogenesis. The formation of an infection thread and the extent of nodule formation are largely under plant control, but vary with environmental conditions and the physiological state of the host plants. Many achievements in these two areas have been made in recent decades.Phytohormone signaling pathways have gradually emerged as important regulators of root nodule symbiosis. Cytokinin, strigolactones(SLs) and local accumulation of auxin can promote nodule development. Ethylene,jasmonic acid(JA), abscisic acid(ABA) and gibberellic acid(GA) all negatively regulate infection thread formation and nodule development. However, salicylic acid(SA) and brassinosteroids(BRs) have different effects on the formation of these two nodule types. Some peptide hormones are also involved in nodulation. This review summarizes recent findings on the roles of these plant hormones in legume-rhizobial symbiosis, and we propose that DELLA proteins may function as a node to integrate plant hormones to regulate nodulation.     

水稻与拟南芥PHD-finger蛋白的系统分析

PHD-finger(plant homeodomain finger)是具Cys4-His-Cys3特征结构的锌指蛋白结构域,它广泛存在于植物或动物转录调节蛋白中。此类蛋白在植物或动物蛋白质组中具有多个功能各异的家族成员,并在发育中扮演重要的角色。鉴定了水稻44个推定的非冗余PHD-finger蛋白,并与拟南芥蛋白质组中45个该家族成员进行了比较;所构建的2棵进化树涵盖了这89个蛋白质,暗示水稻、拟南芥与人的PHD-finger蛋白中存在共同祖先。     

Cloning, expression, characterization, and crystallization of a glutaminyl cyclase from human bone marrow: a single zinc metalloenzyme

Glutaminyl cyclase (QC) catalyzes the N-terminal pyroglutamate formation of numerous hormones and peptides from their glutaminyl precursor. Pyroglutamate is a posttranslational or cotranslational modification important in many physiological and pathological processes. Here, we present the cloning of a QC cDNA from human bone marrow cDNA library. The protein was expressed in Escherichia coli system with the yields higher than approximately 10 mg/L bacterial culture, using a thioredoxin-tagged expression vector with several modifications. Based on high histidine content ( approximately 5%) of the protein, a convenient purification step by Ni-affinity chromatography was designed, leading to near homogeneity of the purified human QC. The identity of the recombinant human QC was confirmed by mass spectrometry and circular dichroism spectroscopy. The enzyme was active on both synthetic and physiological substrates, and the activity could be inhibited by several imidazole, triazole, and tetrazole derivatives. An atomic absorption analysis demonstrated that human QC contains one zinc ion per protein molecule. We also obtained the human QC crystals, which belong to cubic, tetragonal, and rhombohedral forms. Our works are useful to acquire new insights into human and animal QCs, particularly for future structural analysis and inhibitor designs.     

Signal transduction by plant heterotrimeric G‐protein

Heterotrimeric G‐proteins are complexes that regulate important signalling pathways essential for growth and development in both plants and animals. Although plant cells are composed of the core components (Gα, Gβ and Gγ subunits) found in animal G‐proteins, the complexities of the architecture, function and signalling mechanisms of those in animals are dissimilar to those identified in some plants. Current studies on plant G‐proteins have improved knowledge of the essential physiological and agronomic properties, which when harnessed, could potentially impact global food security. Extensive studies on the molecular mechanisms underlying these properties in diverse plant species will be imperative in improving our current understanding of G‐protein signalling pathways involved in plant growth and development. The advancement of G‐protein signalling networks in distinct plant species could significantly aid in better crop development. This review summarizes current progress, novel discoveries and future prospects for this area in potential crop improvement.