YY1 Binding to Regulatory Elements That Lack Enhancer Activity Promotes Locus Folding and Gene Activation.

Enhancers activate their cognate promoters over huge distances but how enhancer/promoter interactions become established is not completely understood. There is strong evidence that cohesin-mediated loop extrusion is involved but this does not appear to be a universal mechanism. Here, we identify an element within the mouse immunoglobulin lambda (Igλ) light chain locus, HSCλ1, that has characteristics of active regulatory elements but lacks intrinsic enhancer or promoter activity. Remarkably, knock-out of the YY1 binding site from HSCλ1 reduces Igλ transcription significantly and disrupts enhancer/promoter interactions, even though these elements are >10 kb from HSCλ1. Genome-wide analyses of mouse embryonic stem cells identified 2671 similar YY1-bound, putative genome organizing elements that lie within CTCF/cohesin loop boundaries but that lack intrinsic enhancer activity. We suggest that such elements play a fundamental role in locus folding and in facilitating enhancer/promoter interactions.

Circular RNAs Repertoire and Expression Profile during Brassica rapa Pollen Development.

Circular RNAs (circRNAs) are covalently closed RNA molecules generated by the back-splicing of exons from linear precursor mRNAs. Though various linear RNAs have been shown to play important regulatory roles in many biological and developmental processes, little is known about the role of their circular counterparts. In this study, we performed high-throughput RNA sequencing to delineate the expression profile and potential function of circRNAs during the five stages of pollen development in Brassica rapa. A total of 1180 circRNAs were detected in pollen development, of which 367 showed stage-specific expression patterns. Functional enrichment and metabolic pathway analysis showed that the parent genes of circRNAs were mainly involved in pollen-related molecular and biological processes such as mitotic and meiotic cell division, DNA processes, protein synthesis, protein modification, and polysaccharide biosynthesis. Moreover, by predicting the circRNA-miRNA network from our differentially expressed circRNAs, we found 88 circRNAs with potential miRNA binding sites, suggesting their role in post-transcriptional regulation of the genes. Finally, we confirmed the back-splicing sites of nine selected circRNAs using divergent primers and Sanger sequencing. Our study presents the systematic analysis of circular RNAs during pollen development and forms the basis of future studies for unlocking complex gene regulatory networks underpinning reproduction in flowering plants.

SARS-CoV-2 attachment to host cells is possibly mediated via RGD-integrin interaction in a calcium-dependent manner and suggests pulmonary EDTA chelation therapy as a novel treatment for COVID 19.

SARS-CoV-2 is a highly contagious virus that has caused serious health crisis world-wide resulting into a pandemic situation. As per the literature, the SARS-CoV-2 is known to exploit humanACE2 receptors (similar toprevious SARS-CoV-1) for gaining entry into the host cell for invasion, infection, multiplication and pathogenesis. However, considering the higher infectivity of SARS-CoV-2 along with the complex etiology and pathophysiological outcomes seen in COVID-19 patients, it seems that there may be an alternate receptor for SARS-CoV-2. I performed comparative protein sequence analysis, database based gene expression profiling, bioinformatics based molecular docking using authentic tools and techniques for unveiling the molecular basis of high infectivity of SARS-CoV-2 as compared to previous known coronaviruses. My study revealed that SARS-CoV-2 (previously known as 2019-nCoV) harbors a RGD motif in its receptor binding domain (RBD) and the motif is absent in all other previously known SARS-CoVs. The RGD motif is well known for its role in cell-attachment and cell-adhesion. My hypothesis is that the SARS-CoV-2 may be (via RGD) exploiting integrins, that have high expression in lungs and all other vital organs, for invading host cells. However, an experimental verification is required. The expression of ACE2, which is a known receptor for SARS-CoV-2, was found to be negligible in lungs. I assume that higher infectivity of SARS-CoV-2 could be due to this RGD-integrin mediated acquired cell-adhesive property. Gene expression profiling revealed that expression of integrins is significantly high in lung cells, in particular αvß6, α5ß1, αvß8 and an ECM protein, ICAM1. The molecular docking experiment showed the RBD of spike protein binds with integrins precisely at RGD motif in a similar manner as a synthetic RGD peptide binds to integrins as found by other researchers. SARS-CoV-2 spike protein has a number of phosphorylation sites that can induce cAMP, PKC, Tyr signaling pathways. These pathways either activate calcium ion channels or get activated by calcium. In fact, integrins have calcium & metal binding sites that were predicted around and in vicinity of RGD-integrin docking site in our analysis which suggests that RGD-integrins interaction possibly occurs in calcium-dependent manner. The higher expression of integrins in lungs along with their previously known high binding affinity (~KD = 4.0 nM) for virus RGD motif could serve as a possible explanation for high infectivity of SARS-CoV-2. On the contrary, human ACE2 has lower expression in lungs and its high binding affinity (~KD = 15 nM) for spike RBD alone could not manifest significant virus-host attachment. This suggests that besides human ACE2, an additional or alternate receptor for SARS-CoV-2 is likely to exist. A highly relevant evidence never reported earlier which corroborate in favor of RGD-integrins mediated virus-host attachment is an unleashed cytokine storm which causes due to activation of TNF-α and IL-6 activation; and integrins role in their activation is also well established. Altogether, the current study has highlighted possible role of calcium and other divalent ions in RGD-integrins interaction for virus invasion into host cells and suggested that lowering divalent ion in lungs could avert virus-host cells attachment.

Pharmacological characterisation of novel adenosine A3 receptor antagonists.

The adenosine A3 receptor (A3R) belongs to a family of four adenosine receptor (AR) subtypes which all play distinct roles throughout the body. A3R antagonists have been described as potential treatments for numerous diseases including asthma. Given the similarity between (adenosine receptors) orthosteric binding sites, obtaining highly selective antagonists is a challenging but critical task. Here we screen 39 potential A3R, antagonists using agonist-induced inhibition of cAMP. Positive hits were assessed for AR subtype selectivity through cAMP accumulation assays. The antagonist affinity was determined using Schild analysis (pA2 values) and fluorescent ligand binding. Structure-activity relationship investigations revealed that loss of the 3-(dichlorophenyl)-isoxazolyl moiety or the aromatic nitrogen heterocycle with nitrogen at α-position to the carbon of carboximidamide group significantly attenuated K18 antagonistic potency. Mutagenic studies supported by molecular dynamic simulations combined with Molecular Mechanics-Poisson Boltzmann Surface Area calculations identified the residues important for binding in the A3R orthosteric site. We demonstrate that K18, which contains a 3-(dichlorophenyl)-isoxazole group connected through carbonyloxycarboximidamide fragment with a 1,3-thiazole ring, is a specific A3R (< 1 µM) competitive antagonist. Finally, we introduce a model that enables estimates of the equilibrium binding affinity for rapidly disassociating compounds from real-time fluorescent ligand-binding studies. These results demonstrate the pharmacological characterisation of a selective competitive A3R antagonist and the description of its orthosteric binding mode. Our findings may provide new insights for drug discovery.

A compendium of genome-wide sequence reads from NBS (nucleotide binding site) domains of resistance genes in the common potato.

SolariX is a compendium of DNA sequence tags from the nucleotide binding site (NBS) domain of disease resistance genes of the common potato, Solanum tuberosum Group Tuberosum. The sequences, which we call NBS tags, for nearly all NBS domains from 91 genomes-representing a wide range of historical and contemporary potato cultivars, 24 breeding programs and 200 years-were generated using just 16 amplification primers and high-throughput sequencing. The NBS tags were mapped to 587 NBS domains on the draft potato genome DM, where we detected an average, over all the samples, of 26 nucleotide polymorphisms on each locus. The total number of NBS domains observed, differed between potato cultivars. However, both modern and old cultivars possessed comparable levels of variability, and neither the individual breeder or country nor the generation or time appeared to correlate with the NBS domain frequencies. Our attempts to detect haplotypes (i.e., sets of linked nucleotide polymorphisms) frequently yielded more than the possible 4 alleles per domain indicating potential locus intermixing during the mapping of NBS tags to the DM reference genome. Mapping inaccuracies were likely a consequence of the differences of each cultivar to the reference genome used, coupled with high levels of NBS domain sequence similarity. We illustrate that the SolariX database is useful to search for polymorphism linked with NBS-LRR R gene alleles conferring specific disease resistance and to develop molecular markers for selection.

Real-time tracking reveals catalytic roles for the two DNA binding sites of Rad51.

During homologous recombination, Rad51 forms a nucleoprotein filament on single-stranded DNA to promote DNA strand exchange. This filament binds to double-stranded DNA (dsDNA), searches for homology, and promotes transfer of the complementary strand, producing a new heteroduplex. Strand exchange proceeds via two distinct three-strand intermediates, C1 and C2. C1 contains the intact donor dsDNA whereas C2 contains newly formed heteroduplex DNA. Here, we show that the conserved DNA binding motifs, loop 1 (L1) and loop 2 (L2) in site I of Rad51, play distinct roles in this process. L1 is involved in formation of the C1 complex whereas L2 mediates the C1-C2 transition, producing the heteroduplex. Another DNA binding motif, site II, serves as the DNA entry position for initial Rad51 filament formation, as well as for donor dsDNA incorporation. Our study provides a comprehensive molecular model for the catalytic process of strand exchange mediated by eukaryotic RecA-family recombinases.

Calmodulin is involved in the dual subcellular location of two chloroplast proteins.

Cell compartmentalization is an essential process by which eukaryotic cells separate and control biological processes. Although calmodulins are well-known to regulate catalytic properties of their targets, we show here their involvement in the subcellular location of two plant proteins. Both proteins exhibit a dual location, namely in the cytosol in addition to their association to plastids (where they are known to fulfil their role). One of these proteins, ceQORH, a long-chain fatty acid reductase, was analyzed in more detail, and its calmodulin-binding site was identified by specific mutations. Such a mutated form is predominantly targeted to plastids at the expense of its cytosolic location. The second protein, TIC32, was also shown to be dependent on its calmodulin-binding site for retention in the cytosol. Complementary approaches (bimolecular fluorescence complementation and reverse genetics) demonstrated that the calmodulin isoform CAM5 is specifically involved in the retention of ceQORH in the cytosol. This study identifies a new role for calmodulin and sheds new light on the intriguing CaM-binding properties of hundreds of plastid proteins, despite the fact that no CaM or CaM-like proteins were identified in plastids.

Complementary-sense gene regulation in beet curly top virus-SpCT.

A 278-bp region upstream of the beet curly top virus-SpCT (BCTV-SpCT) C2/C3 genes is necessary for promoter activity and exhibits significant sequence similarity to AL2/3 promoter sequences in tomato golden mosaic virus (TGMV). Maximal expression of the downstream C2/3 genes in BCTV-SpCT requires the presence of the C1 protein, which is supported by observations that mutation of the initiator codon for C1 results in decreased C2/C3 expression. This is similar to TGMV and cabbage leaf curl virus, where AL1 is required for maximal AL2/3 expression. Together, these data suggest a common strategy for complementary-sense gene regulation amongst curtoviruses and begomoviruses.

LINE-1 Evasion of Epigenetic Repression in Humans.

Epigenetic silencing defends against LINE-1 (L1) retrotransposition in mammalian cells. However, the mechanisms that repress young L1 families and how L1 escapes to cause somatic genome mosaicism in the brain remain unclear. Here we report that a conserved Yin Yang 1 (YY1) transcription factor binding site mediates L1 promoter DNA methylation in pluripotent and differentiated cells. By analyzing 24 hippocampal neurons with three distinct single-cell genomic approaches, we characterized and validated a somatic L1 insertion bearing a 3' transduction. The source (donor) L1 for this insertion was slightly 5' truncated, lacked the YY1 binding site, and was highly mobile when tested in vitro. Locus-specific bisulfite sequencing revealed that the donor L1 and other young L1s with mutated YY1 binding sites were hypomethylated in embryonic stem cells, during neurodifferentiation, and in liver and brain tissue. These results explain how L1 can evade repression and retrotranspose in the human body.

Structure-based discovery of antiviral inhibitors targeting the E dimer interface of Japanese encephalitis virus.

Flaviviruses are emerging arthropod-borne viruses posing a great threat to human beings worldwide. The E dimer configuration of the flavivirus was prominent during viral assembly, maturation and entry. Neutralization antibodies targeting E dimer played the important role in controlling the flavivirus infection. Previously, the ideal drug target of small molecular inhibitors of JEV was viral proteases and polymerases. The crystal structure of JEV E protein showed a conserved pocket in it is important at membrane fusion step. Recently, a set of anti-virus drugs has been found by virtual screening. Here, we show that the fusion-loop pocket of JEV E protein was a conservative region and an ideal drug target. ChemDiv-3 from virtual screening as the lead compound was found to show a relatively modest inhibition effect for JEV in vitro and in vivo test and could interfere with the formation of JEV sE dimer. ChemDiv-3 interacts with the amino acid residues ASN 313, PRO 314, ALA 315, and VAL 323 in E protein via hydrogen bonds for occupation of the fusion-loop pocket. The key binding sites LYS 312, ALA 513 and THR 317 forming the fusion-loop pocket are the same and other auxiliary sites are similar among the flavivirus. Taken together, the fusion-loop pocket of the flavivirus could be one promising target for drug discovery.

Circular IRE-type RNAs of the NR5A1 gene are formed in adrenocortical cells.

The recently discovered circular RNAs (circRNAs) are mostly formed by back-splicing where the downstream 5' splice site splices to the upstream 3' splice site by conventional pre-mRNA splicing. These circRNAs regulate gene expression by acting as sponges for micro-RNAs or RNA-binding proteins. Here we show that the NR5A1 (previously called Ad4BP or SF-1) gene which is exclusively expressed in the adrenal cortex and steroidogenic tissue can form atypical circRNAs by unconventional splicing. Two stem loops with inositol-requiring protein-1α (IRE1α) cleavage sites are connected by an IRE1α cleavage site to form a circRNA (circIRE RNA). From total RNA of normal human adrenal cortex, we detected a circIRE RNA with connected ends by IRE1α cleavage sites in exon 6 and exon 1 (circIRE NR5A1 ex6-1 RNA). circIRE NR5A1 ex6-1 RNA was not detected in the adrenocortical cancer cell line, H295R. When IRE1α was expressed in H295R cells a different circIRE NR5A1 RNA connecting IRE1-cleavage sites in exon 7 and exon 1 was detected (circIRE NR5A1 ex7-1 RNA). The expression of this circIRE RNA was inhibited by the IRE1 inhibitor 1, STF-083010, implicating that it was formed via the ER stress pathway, where IRE1α is a major factor. This is the first report of this type of circular RNA connected by IRE1-cleavage sites found to be expressed in mammalian cells in a tissue-specific manner. To our surprise, the concomitant expression of NR5A1 was increased by IRE1α implicating that NR5A1 was not subjected to IRE1-dependent decay of mRNA (RIDD) but rather activating a transcriptional regulatory network to cope with ER stress in steroidogenic tissue reminiscent to XBP1 in other tissue. We believe this is the first report of such tissue-specific transcriptional cascade responding to ER stress as well as the novel finding of circular RNAs connected by IRE1α cleavage sites expressed in mammalian tissue.

Identification of a promoter element mediating kisspeptin-induced increases in GnRH gene expression in sheep.

Gonadotropin-releasing hormone (GnRH) plays an important role in regulating the activities of other components downstream of the hypothalamic-pituitary-gonadal (HPG) axis and maintaining the normal reproductive cycle of animals. However, the molecular mechanisms by which GnRH synthesis and secretion are regulated in sheep remains unclear. In this study, a series of eight recombinant vectors with deletion fragments were constructed and cotransfected with pGL3-Basic and pRL-SV40 into sheep hypothalamic neuronal cells. After treatment with 1 nM kisspeptin, the core promoter of the sheep GnRH gene was identified to be in the region of -1912 bp to -1461 bp by dual-luciferase reporter assay. Bioinformatics analysis showed that there was a binding site for the transcription factor Otx-2 in the core promoter region (-1786 to -1770 bp) that was highly conserved among different species. The expression patterns of Kiss-1, Otx-2 and GnRH in the sheep hypothalamus were the same, and the expression of Kiss-1, Otx-2 and GnRH was significantly higher in the breeding season than in nonbreeding season (P < 0.01). In addition, when hypothalamic neurons were cultured in vitro with kisspeptin, kisspeptin induced the expression of GnRH and Otx-2. In conclusion, these results provide evidence that the core promoter region (-1786 to -1770 bp) of the GnRH gene is involved in the regulation of hypothalamic activity by kisspeptin and that binding of the transcription factor Otx-2 mediates this activation.

Inhibition of TNFα-induced interleukin-6 gene expression by barley (Hordeum vulgare) ethanol extract in BV-2 microglia.

BACKGROUND: Inflammation in the central nervous system is closely associated with pathological neurodegenerative diseases as well as psychiatric disorders. Prolonged activation of microglia can produce many inflammatory mediators, which may result in pathological neurotoxic side effects. Interleukin (IL)-6 serves as a hallmark of the injured brain. OBJECTIVE: Whole grains are known to contain many bioactive components. However, little information is available about anti-neuroinflammatory effects of grains in the CNS. This study aims to investigate the effect of Hordeum vulgare ethanol extract (HVE) on the suppression of IL-6 expression in BV2 microglia. METHODS: Inhibitory effects of HVE on IL-6 expression were analyzed by immunoblot anaysis, immunofluoresce microscopic analysis, reverse transcription-polymerase chain reaction, and luciferase promoter reporter assay. RESULTS: HVE inhibited TNFα-induced phosphorylation of IKKα/ß, IκB, and p65/RelA NF-κB. TNFα-induced IL-6 mRNA expression and promoter activity were reduced by HVE. Point mutation of NF-κB-binding site within the IL-6 gene promoter abolished TNFα-induced reporter activity, whereas exogenous expression of p65 NF-κB enhanced IL-6 promoter activity. CONCLUSION: NF-κB-binding site within the IL-6 promoter region is a HVE target element involved in the inhibition of TNFα-induced IL-6 gene transcription. HVE inhibits TNFα-induced IL-6 expression via suppression of NF-κB signaling in BV2 microglial cells.

Flexibility In Vitro of Amino Acid 226 in the Receptor-Binding Site of an H9 Subtype Influenza A Virus and Its Effect In Vivo on Virus Replication, Tropism, and Transmission.

Influenza A viruses (IAVs) remain a significant public health threat, causing more than 300,000 hospitalizations in the United States during the 2015-2016 season alone. While only a few IAVs of avian origin have been associated with human infections, the ability of these viruses to cause zoonotic infections further increases the public health risk of influenza. Of these, H9N2 viruses in Asia are of particular importance as they have contributed internal gene segments to other emerging zoonotic IAVs. Notably, recent H9N2 viruses have acquired molecular markers that allow for a transition from avian-like to human-like terminal sialic acid (SA) receptor recognition via a single amino acid change at position 226 (H3 numbering), from glutamine (Q226) to leucine (L226), within the hemagglutinin (HA) receptor-binding site (RBS). We sought to determine the plasticity of amino acid 226 and the biological effects of alternative amino acids on variant viruses. We created a library of viruses with the potential of having any of the 20 amino acids at position 226 on a prototypic H9 HA subtype IAV. We isolated H9 viruses that carried naturally occurring amino acids, variants found in other subtypes, and variants not found in any subtype at position 226. Fitness studies in quails revealed that some natural amino acids conferred an in vivo replication advantage. This study shows the flexibility of position 226 of the HA of H9 influenza viruses and the resulting effect of single amino acid changes on the phenotype of variants in vivo and in vitroIMPORTANCE A single amino acid change at position 226 in the hemagglutinin (HA) from glutamine (Q) to leucine (L) has been shown to play a key role in receptor specificity switching in various influenza virus HA subtypes, including H9. We tested the flexibility of amino acid usage and determined the effects of such changes. The results reveal that amino acids other than L226 and Q226 are well tolerated and that some amino acids allow for the recognition of both avian and human influenza virus receptors in the absence of other changes. Our results can inform better avian influenza virus surveillance efforts as well as contribute to rational vaccine design and improve structural molecular dynamics algorithms.

Engineered variants of a lipase from Yarrowia lipolytica with improved trypsin resistance for enzyme replacement therapy.

To improve the proteolytic stability of the lipase LIP2 from Yarrowia lipolytica, the peptide bonds susceptible to trypsin in LIP2 were analyzed by tandem mass spectrometry and redesigned by site-directed mutagenesis. Different variants of the enzyme were expressed in Pichia pastoris GS115 and their biochemical properties were subsequently investigated. Although most of the variants were still cleaved by trypsin, some of them did show an evident increase of resistance against proteolytic degradation. The most stable mutant was LIP2-C5, in which five trypsin-cleavage sites were replaced by non-preferred amino acids. Upon incubation with human trypsin for 80 min at 37°C, the mutant LIP2-C5 was found to retain >70% of its initial activity, compared to only 10% for the wild-type.

Adaptation of the FK506 binding protein 1B to hibernation in bats.

Hibernation is an adaptive strategy used by some animals to cope with cold and food shortage. The heart rate, overall energy need, body temperature, and many other physiological functions are greatly reduced during torpor but promptly return to normal levels upon arousal. The heartbeat of torpid bats can be hundreds fold lower than that of active bats, indicating that hibernating bats have a remarkable ability to control excitation-contraction coupling in cardiac muscle. FKBP1B (calstabin 2), a peptidyl-prolyl cis-trans isomerase, is critical for the regulation of excitation-contraction coupling. Whether FKBP1B is adapted to hibernation in bats is not known. Evolutionary analyses showed that the ω values of the Fkbp1b genes of 25 mammalian species are all less than 1, and amino acid sequence alignments revealed that FKBP1B proteins are highly conserved in mammals. The expression of the Fkbp1b gene was found to be elevated at both mRNA and protein levels in two distantly related bats (Rhinolophus ferrumequinum in Yinpterochiroptera and Myotis ricketti in Yangochiroptera) during torpor. Transcription factors such as YY1 and SPs were bioinformatically determined to have a higher binding affinity to the potential regulatory regions of Fkbp1b genes in hibernating than in non-hibernating mammals. This study provides new insights into the molecular evolution of Fkbp1b in adaptation to bat hibernation.

Early life experience drives structural variation of neural genomes in mice.

The brain is a genomic mosaic owing to somatic mutations that arise throughout development. Mobile genetic elements, including retrotransposons, are one source of somatic mosaicism in the brain. Retrotransposition may represent a form of plasticity in response to experience. Here, we use droplet digital polymerase chain reaction to show that natural variations in maternal care mediate the mobilization of long interspersed nuclear element-1 (LINE-1 or L1) retrotransposons in the hippocampus of the mouse brain. Increasing the amount of maternal care blocks the accumulation of L1. Maternal care also alters DNA methylation at YY1 binding sites implicated in L1 activation and affects expression of the de novo methyltransferase DNMT3a. Our observations indicate that early life experience drives somatic variation in the genome via L1 retrotransposons.

Effect of glucocorticoid treatment on BAFF and APRIL expression in patients with immune thrombocytopenia (ITP).

Immune thrombocytopenic purpura (ITP) is an idiopathic bleeding disorder. B cell activating factor (BAFF) and 'A proliferation-inducing ligand' (APRIL) have regulatory effects on B and T cells and may represent relevant factors in the pathogenesis of ITP. Serum levels and gene expression were investigated in ITP patients. Both BAFF and APRIL serum levels were significantly elevated in active ITP. However, gene expression analysis revealed both factors to have a tendency toward downregulation. Glucocorticoid treatment significantly reduced BAFF but not APRIL serum levels, which may be mediated by differences in transcription factor binding sites. The glucocorticoid receptor binding site is present in the BAFF promotor region, but not in the APRIL promotor region. Prednisolone in combination with vitamin D3 may be effective in reducing APRIL serum levels. In conclusion, glucocorticoid treatment exerts different regulatory effects on both BAFF and APRIL, whereas antioxidant supplementation may also be beneficial in reducing serum levels.

A tandem CBM25 domain of α-amylase from Microbacterium aurum as potential tool for targeting proteins to starch granules during starch biosynthesis.

BACKGROUND: Starch-binding domains from carbohydrate binding module family 20 have been used as a tool for starch engineering. Previous studies showed that expression of starch binding domain fusion proteins in planta resulted in modified starch granule structures and physicochemical properties. However, although 13 carbohydrate binding module families have been reported to contain starch-binding domains, only starch-binding domains from carbohydrate binding module family 20 have been well studied and introduced into plants successfully. In this study, two fragments, the tandem CBM25 domain and the tandem CBM25 with multiple fibronectin type III (FN3) domains of the α-amylase enzyme from Microbacterium aurum, were expressed in the tubers of a wild type potato cultivar (cv. Kardal) and an amylose-free (amf) potato mutant. RESULTS: The (CBM25)2 and FN3 protein were successfully accumulated in the starch granules of both Kardal and amf transformants. The accumulation of (CBM25)2 protein did not result in starch morphological alterations in Kardal but gave rise to rough starch granules in amf, while the FN3 resulted in morphological changes of starch granules (helical starch granules in Kardal and rough surface granules in amf) but only at a very low frequency. The starches of the different transformants did not show significant differences in starch size distribution, apparent amylose content, and physico-chemical properties in comparison to that of untransformed controls. CONCLUSION: These results suggest that the starch-binding domains from carbohydrate binding module family 25 can be used as a novel tool for targeting proteins to starch granules during starch biosynthesis without side-effects on starch morphology, composition and properties.

Zn2+-dependent autocatalytic activity of the Bordetella pertussis CyaA-hemolysin.

Proteolytic degradation of the ∼100-kDa isolated RTX (Repeat-in-ToXin) subdomain (CyaA-RTX) of the Bordetella pertussis CyaA-hemolysin (CyaA-Hly) was evidently detected upon solely-prolonged incubation. Here, a truncated CyaA-Hly fragment (CyaA-HP/BI) containing hydrophobic and acylation regions connected with the first RTX block (BI1015-1088) was constructed as a putative precursor for investigating its potential autocatalysis. The 70-kDa His-tagged CyaA-HP/BI fragment which was over-expressed in Escherichia coli as insoluble aggregate was entirely solubilized with 4 M urea. After re-naturation in a Ni2+-NTA affinity column, the purified-refolded CyaA-HP/BI fragment in HEPES buffer (pH 7.4) supplemented with 2 mM CaCl2 was completely degraded upon incubation at 37 °C for 3 h. Addition of 1,10-phenanthroline‒an inhibitor of Zn2+-dependent metalloproteases markedly reduced the extent of degradation for CyaA-HP/BI and CyaA-RTX, but the degradative effect was clearly enhanced by addition of 100 mM ZnCl2. Structural analysis of a plausible CyaA-HP/BI model revealed a potential Zn2+-binding His-Asp cluster located between the acylation region and RTX-BI1015-1088. Moreover, Arg997‒one of the identified cleavage sites of the CyaA-RTX fragment was located in close proximity to the Zn2+-binding catalytic site. Overall results demonstrated for the first time that the observed proteolysis of CyaA-HP/BI and CyaA-RTX fragments is conceivably due to their Zn2+-dependent autocatalytic activity.