Putative Processing Enzyme Research Articles

The putative vacuolar processing enzyme gene TaVPE3cB is a candidate gene for wheat stem pith-thickness

Key messageThe vacuolar processing enzyme gene TaVPE3cB is identified as a candidate gene for a QTL of wheat pith-thickness on chromosome 3B by BSR-seq and differential expression analyses.The high pith-thickness (PT) of the wheat stem could greatly enhance stem mechanical strength, especially the basal internodes which support the heavier upper part, such as upper stems, leaves and spikes. A QTL for PT in wheat was previously discovered on 3BL in a double haploid population of ‘Westonia’ × ‘Kauz’. Here, a bulked segregant RNA-seq analysis was applied to identify candidate genes and develop associated SNP markers for PT. In this study, we aimed at screening differentially expressed genes (DEGs) and SNPs in the 3BL QTL interval. Sixteen DEGs were obtained based on BSR-seq and differential expression analyses. Twenty-four high-probability SNPs in eight genes were identified by comparing the allelic polymorphism in mRNA sequences between the high PT and low PT samples. Among them, six genes were confirmed to be associated with PT by qRT-PCR and sequencing. A putative vacuolar processing enzyme gene TaVPE3cB was screened out as a potential PT candidate gene in Australian wheat ‘Westonia’. A robust SNP marker associated with TaVPE3cB was developed, which can assist in the introgression of TaVPE3cB.b in wheat breeding programs. In addition, we also discussed the function of other DEGs which may be related to pith development and programmed cell death (PCD). A five-level hierarchical regulation mechanism of stem pith PCD in wheat was proposed.

Small RNAs Asserting Big Roles in Mycobacteria.

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb), with 10.4 million new cases per year reported in the human population. Recent studies on the Mtb transcriptome have revealed the abundance of noncoding RNAs expressed at various phases of mycobacteria growth, in culture, in infected mammalian cells, and in patients. Among these noncoding RNAs are both small RNAs (sRNAs) between 50 and 350 nts in length and smaller RNAs (sncRNA) < 50 nts. In this review, we provide an up-to-date synopsis of the identification, designation, and function of these Mtb-encoded sRNAs and sncRNAs. The methodological advances including RNA sequencing strategies, small RNA antagonists, and locked nucleic acid sequence-specific RNA probes advancing the studies on these small RNA are described. Initial insights into the regulation of the small RNA expression and putative processing enzymes required for their synthesis and function are discussed. There are many open questions remaining about the biological and pathogenic roles of these small non-coding RNAs, and potential research directions needed to define the role of these mycobacterial noncoding RNAs are summarized.

Tropical vibes from Sri Lanka - cyclotides from Viola betonicifolia by transcriptome and mass spectrometry analysis

Cyclotides are an extremely stable class of peptides, ubiquitously distributed in Violaceae. The aim of the present study was to investigate the presence of cyclotides in Sri Lankan Violaceae plants, using combined tools of transcriptomics and mass spectrometry. New cyclotides were discovered for the first time in the wild flora of Sri Lanka, within Viola betonicifolia, a plant used in traditional medicine as an antimicrobial. Plant extracts prepared in small scale from Viola betonicifolia were first subjected to LC-MS analysis. Subsequent transcriptome de novo sequencing of Viola betonicifolia uncovered 25 new (vibe 1–25) and three known (varv A/kalata S, viba 17, viba 11) peptide sequences from Möbius and bracelet cyclotide subfamilies as well as hybrid cyclotides. Among the transcripts, putative linear acyclotide sequences (vibe 4, vibe 10, vibe 11 and vibe 22) that lack a conserved asparagine or aspartic acid vital for cyclisation were also present. Four asparagine endopeptidases (AEPs), VbAEP1-4 were found within the Viola betonicifolia transcriptome, including a peptide asparaginyl ligase (PAL), potentially involved in cyclotide backbone cyclisation, showing >93% sequence homology to Viola yedoensis peptide asparaginyl ligases, VyPALs. In addition, we identified two protein disulfide isomerases (PDIs), VbPDI1-2, likely involved in cyclotide oxidative folding, having high sequence homology (>74%) with previously reported Rubiaceae and Violaceae PDIs. The current study highlights the ubiquity of cyclotides in Violaceae as well as the utility of transcriptomic analysis for cyclotides and their putative processing enzyme discovery. The high variability of cyclotide sequences in terms of loop sizes and residues in V. betonicifolia showcase the cyclotide structure as an adaptable scaffold as well as their importance as a combinatorial library, implicated in plant defense.

Proteases Shape the Chlamydomonas Secretome: Comparison to Classical Neuropeptide Processing Machinery.

The recent identification of catalytically active peptidylglycine α-amidating monooxygenase (PAM) in Chlamydomonas reinhardtii, a unicellular green alga, suggested the presence of a PAM-like gene and peptidergic signaling in the last eukaryotic common ancestor (LECA). We identified prototypical neuropeptide precursors and essential peptide processing enzymes (subtilisin-like prohormone convertases and carboxypeptidase B-like enzymes) in the C. reinhardtii genome. Reasoning that sexual reproduction by C. reinhardtii requires extensive communication between cells, we used mass spectrometry to identify proteins recovered from the soluble secretome of mating gametes, and searched for evidence that the putative peptidergic processing enzymes were functional. After fractionation by SDS-PAGE, signal peptide-containing proteins that remained intact, and those that had been subjected to cleavage, were identified. The C. reinhardtii mating secretome contained multiple matrix metalloproteinases, cysteine endopeptidases, and serine carboxypeptidases, along with one subtilisin-like proteinase. Published transcriptomic studies support a role for these proteases in sexual reproduction. Multiple extracellular matrix proteins (ECM) were identified in the secretome. Several pherophorins, ECM glycoproteins homologous to the Volvox sex-inducing pheromone, were present; most contained typical peptide processing sites, and many had been cleaved, generating stable N- or C-terminal fragments. Our data suggest that subtilisin endoproteases and matrix metalloproteinases similar to those important in vertebrate peptidergic and growth factor signaling play an important role in stage transitions during the life cycle of C. reinhardtii.

Characterization of the Ashbya gossypii secreted N-glycome and genomic insights into its N-glycosylation pathway

The riboflavin producer Ashbya gossypii is a filamentous hemiascomycete, closely related to the yeast Saccharomyces cerevisiae, that has been used as a model organism to study fungal developmental biology. It has also been explored as a host for the expression of recombinant proteins. However, although N-glycosylation plays important roles in protein secretion, morphogenesis, and the development of multicellular organisms, the N-glycan structures synthesised by A. gossypii had not been elucidated. In this study, we report the first characterization of A. gossypii N-glycans and provide valuable insights into their biosynthetic pathway. By combined matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry profiling and nuclear magnetic resonance (NMR) spectroscopy we determined that the A. gossypii secreted N-glycome is characterized by high-mannose type structures in the range Man4–18GlcNAc2, mostly containing neutral core-type N-glycans with 8–10 mannoses. Cultivation in defined minimal media induced the production of acidic mannosylphosphorylated N-glycans, generally more elongated than the neutral N-glycans. Truncated neutral N-glycan structures similar to those found in other filamentous fungi (Man4–7GlcNAc2) were detected, suggesting the possible existence of trimming activity in A. gossypii. Homologs for all of the S. cerevisiae genes known to be involved in the endoplasmatic reticulum and Golgi N-glycan processing were found in the A. gossypii genome. However, processing of N-glycans by A. gossypii differs considerably from that by S. cerevisiae, allowing much shorter N-glycans. Genes for two putative N-glycan processing enzymes were identified, that did not have homologs in S. cerevisiae.

Functional Expression and Characterization of Dipeptidyl Peptidase IV from the Black-Bellied HornetVespa basalisin Sf21 Insect Cells

The maturation of mastoparan B, the major toxin peptide in the venom of Vespa basalis, requires enzymatic cleavage of its prosequence presumably via sequential liberation of dipeptides. The putative processing enzyme, dipeptidyl peptidase IV, was expressed as a glycosylated His-tag fusion protein (rDPP-IV) via the baculovirus expression system. rDPP-IV purified by one-step nickel-affinity chromatography was verified by Western blot and LC-MS/MS analysis. The k(cat)/K(m) of rDPP-IV was determined to be in the range of 10-500 mM(-1)·S(-1) for five synthetic substrates. The optimal temperature and pH for rDPP-IV were determined to be 50 °C and pH 9. Enzymatic activity of rDPP-IV was significantly reduced by 80 and 60% in the presence of sitagliptin and phenylmethylsulfonyl fluoride respectively.

Docking study of the precursor peptide of matoparan onto its putative processing enzyme, dipeptidyl peptidase IV: a revisit to molecular ticketing

Stepwise-cleavage process of promastoparans to reach maturity was investigated theoretically by combining ab initio folding and unbounded docking. The comparison between the structures of the promastoparans both before and after docking were examined along with the hydrogen bonding interaction pattern between the dipetidyl peptidase IV (DPPIV) and promastoparans to reveal how the endpoint of this stepwise cleavage is recognized among these promastoparans with highly resemble amino acid sequences. The current approach of folding and docking study provides structural insight on the stepwise cleavage process.

Molecular characterisation and functional interrogation of a local natriuretic peptide system in rodent pituitaries, αT3-1 and LβT2 gonadotroph cells

In the pituitary, C-type natriuretic peptide (CNP) has been implicated as a gonadotroph-specific factor, yet expression of the CNP gene (Nppc) and CNP activity in gonadotrophs is poorly defined. Here, we examine the molecular expression and putative function of a local gonadotroph natriuretic peptide system. Nppc, along with all three natriuretic peptide receptors (Npr1, Npr2 and Npr3), was expressed in both alphaT3-1 and LbetaT2 cells and primary mouse pituitary tissue, yet the genes for atrial-(ANP) and B-type natriuretic peptides (Nppa and Nppb) were much less abundant. Putative processing enzymes of CNP were also expressed in alphaT3-1 cells and primary mouse pituitaries. Transcriptional analyses revealed that the proximal 50 bp of the murine Nppc promoter were sufficient for GNRH responsiveness, in an apparent protein kinase C and calcium-dependent manner. Electrophoretic mobility shift assays showed Sp1/Sp3 proteins form major complexes within this region of the Nppc promoter. CNP protein was detectable in rat anterior pituitaries, and electron microscopy detected CNP immunoreactivity in secretory granules of gonadotroph cells. Pharmacological analyses of natriuretic peptide receptor activity clearly showed ANP and CNP are potent activators of cGMP production. However, functional studies failed to reveal a role for CNP in regulating cell proliferation or LH secretion. Surprisingly, CNP potently stimulated the human glycoprotein hormone alpha-subunit promoter in LbetaT2 cells but not in alphaT3-1 cells. Collectively, these findings support a role for CNP as the major natriuretic peptide of the anterior pituitary, and for gonadotroph cells as the major source of CNP expression and site of action.

Molecular cloning of the precursor polypeptide of mastoparan B and its putative processing enzyme, dipeptidyl peptidase IV, from the black‐bellied hornet, Vespa basalis

Mastoparan B, a cationic toxin, is the major peptide component in the venom of Vespa basalis. Molecular cloning of its cDNA fragment revealed that this toxin was initially synthesized as a precursor polypeptide, containing an N-terminal signal sequence, a prosequence, the mature toxin, and an appendix glycine at C-terminus. Sequence alignment between precursors of mastoparan B and melittin from honeybee venom showed a significant conservation in prosequence. Alternate positions existing in both prosequences were either proline or alanine known as the potential cleaving sites for dipeptidyl peptidase IV. Subsequently, a putative dipeptidyl peptidase IV cDNA fragment was cloned from Vespa basalis venom gland. The prosequence may possibly be removed via sequential liberation of dipeptides during the processing of mastoparan B.

Phylogenetic relationships and gene expression pattern of three different cathepsin L (Ctsl) isoforms in zebrafish: Ctsla is the putative yolk processing enzyme

Certain cysteine proteases, such as cathepsin L (Ctsl), have been involved in yolk processing mechanisms in oocytes and embryos of lower vertebrates. In zebrafish ( Danio rerio), three different ctsl genes, ctsla, ctslb and ctslc, have been found in the genome, but their pattern of expression, as well as information on which the encoded enzymes are potentially involved in yolk absorption during embryogenesis, is unknown. Here, phylogenetic and gene structure analysis revealed that zebrafish ctsla and ctslb genes are similar, showing a highly conserved structure in comparison with human ctsl, while ctslc presents different exon organization together with an earlier evolution. Thus, ctslc appears to be evolved from a common ancestral ctsl-like gene, possibly through an early duplication event, whereas ctsla and ctslb may be originated from a second duplication mechanism. Zebrafish ctsla, ctslb and ctslc also showed different patterns of mRNA expression during embryogenesis and in adult tissues. While Ctsla transcripts were accumulated in embryos throughout development and in the adult ovary, those encoding Ctslb were detected only in embryos around the time of hatching as previously reported, and those for Ctslc appeared only in larvae and in some adult tissues, but not in the ovary. In zebrafish and killifish ( Fundulus heteroclitus) embryos, Ctsla mRNA was first detected in blastomers, and later in development it was localized in cells of the yolk syncytial layer, an embryonic structure involved in yolk absorption. These data therefore suggested that Ctsla is most likely the putative protease involved in yolk processing in fish embryos, while Ctslc seems not to be required during early embryogenesis in zebrafish.

Genomic and experimental evidence for a potential sexual cycle in the pathogenic thermal dimorphic fungus Penicillium marneffei

All meiotic genes (except HOP1) and genes encoding putative pheromone processing enzymes, pheromone receptors and pheromone response pathways proteins in Aspergillus fumigatus and Aspergillus nidulans and a putative MAT-1 α box mating-type gene were present in the Penicillium marneffei genome. A putative MAT-2 high-mobility group mating-type gene was amplified from a MAT-1 α box mating-type gene-negative P. marneffei strain. Among 37 P. marneffei patient strains, MAT-1 α box and MAT-2 high-mobility group mating-type genes were present in 23 and 14 isolates, respectively. We speculate that P. marneffei can potentially be a heterothallic fungus that does not switch mating type.

Notch Activation during Endothelial Cell Network Formation in Vitro Targets the Basic HLH Transcription Factor HESR-1 and Downregulates VEGFR-2/KDR Expression

Angiogenesis is essential for normal homeostasis, wound healing, and tumor growth and involves a switch in endothelial cell (EC) phenotype from quiescence to migration, proliferation and network formation, and back to quiescence. The notch signaling pathway is critically involved in cell fate decisions during development, and mice deficient in several notch/notch ligand genes have vascular phenotypes. Here we show that notch signaling is activated during EC capillary-like network formation in vitro and that EC express transcripts for notch 1, notch 4, the notch ligand delta 4, and the putative notch processing enzymes ADAM-10 and presenilin. Expression of dominant negative notch blocks network formation; however, constitutively active notch (NICD) does not induce morphologic changes. Furthermore, both EC network formation and expression of activated notch 1 or notch 4 induce expression of the bHLH transcription factor HESR-1 and downregulate the known HESR-1 target VEGFR-2 (KDR). Notch-mediated reduction in VEGFR-2 expression results in decreased EC proliferation in response to VEGF but not bFGF. These data suggest that HESR-1 may be involved in the phenotypic changes that characterize the progression from EC proliferation and migration to network formation and quiescence.

Forespore signaling is necessary for pro-sigmaK processing during Bacillus subtilis sporulation despite the loss of SpoIVFA upon translational arrest.

The sigmaK checkpoint coordinates gene expression in the mother cell with signaling from the forespore during Bacillus subtilis sporulation. The signaling pathway involves SpoIVB, a serine peptidase produced in the forespore, which is believed to cross the innermost membrane surrounding the forespore and activate a complex of proteins, including BofA, SpoIVFA, and SpoIVFB, located in the outermost membrane surrounding the forespore. Activation of the complex allows proteolytic processing of pro-sigmaK, and the resulting sigmaK RNA polymerase transcribes genes in the mother cell. To investigate activation of the pro-sigmaK processing complex, the level of SpoIVFA in extracts of sporulating cells was examined by Western blot analysis. The SpoIVFA level decreased when pro-sigmaK processing began during sporulation. In extracts of a spoIVB mutant defective in forespore signaling, the SpoIVFA level failed to decrease normally and no processing of pro-sigmaK was observed. Although these results are consistent with a model in which SpoIVFA inhibits processing until the SpoIVB-mediated signal is received from the forespore, we discovered that loss of SpoIVFA was insufficient to allow processing under certain conditions, including static incubation of the culture and continued shaking after the addition of inhibitors of oxidative phosphorylation or translation. Under these conditions, loss of SpoIVFA was independent of spoIVB. The inability to process pro-sigmaK under these conditions was not due to loss of SpoIVFB, the putative processing enzyme, or to a requirement for ongoing synthesis of pro-sigmaK. Rather, it was found that the requirements for shaking of the culture, for oxidative phosphorylation, and for translation could be bypassed by mutations that uncouple processing from dependence on forespore signaling. This suggests that ongoing translation is normally required for efficient pro-sigmaK processing because synthesis of the SpoIVB signal protein is needed to activate the processing complex. When translation is blocked, synthesis of SpoIVB ceases, and the processing complex remains inactive despite the loss of SpoIVFA. Taken together, the results suggest that SpoIVB signaling activates the processing complex by performing another function in addition to causing loss of SpoIVFA or by causing loss of SpoIVFA in a different way than when translation is blocked. The results also demonstrate that the processing machinery can function in the absence of translation or an electrochemical gradient across membranes.

Isolation and molecular analysis of six taproot expressed genes from sugar beet

The taproot from sugar beet (Beta vulgaris L.) undergoes a specific developmental process to function as a food storage organ. Suppression Subtractive Hybridization (SSH) was utilized for the isolation of cDNA fragments for taproot expressed genes. Isolation and molecular analysis of six cDNAs encoding the complete gene product revealed that these genes comprise homologues of a drought-inducible linker histone, a homologue of a major latex-like protein, a phosphoenolpyruvate carboxylase kinase, a putative vacuolar processing enzyme, a thaumatin-like protein and an alanine- and glutamic acid-rich protein. All genes are transcribed in taproots while transcription in leaves is low or undetectable.

A sporulation membrane protein tethers the pro-sigmaK processing enzyme to its inhibitor and dictates its subcellular localization.

The developmental transcription factor sigmaK is derived from the inactive precursor protein pro-sigmaK by regulated proteolysis during the process of sporulation in the bacterium Bacillus subtilis. The putative pro-sigmaK processing enzyme SpoIVFB is a member of a family of membrane-embedded metalloproteases and is held inactive by two other integral membrane proteins, SpoIVFA and BofA. Herein we show that the processing enzyme and its two regulators exist in a multimeric complex that localizes to the membrane surrounding the developing spore (the forespore). We further show that one of the regulators, SpoIVFA, plays a central role in both the formation of this complex and its subcellular localization. Evidence is presented in support of a model in which SpoIVFA acts as a platform for bringing BofA and SpoIVFB together, whereby BofA inhibits pro-sigmaK processing until a signal has been received from the forespore.

An investigation into the compartmentalization of the sporulation transcription factor sigmaE in Bacillus subtilis.

Sporulation in Bacillus subtilis involves the formation of a polar septum, which divides the sporangium into a mother cell and a forespore. The sigmaE factor, which is encoded within the spoIIG operon, is a cell-specific regulatory protein that directs gene transcription in the mother cell. SigmaE is synthesized as an inactive proprotein pro-sigmaE, which is converted to the mature factor by the putative processing enzyme SpoIIGA. Processing of pro-sigmaE does not commence until after asymmetric division when sigmaE is largely confined to the mother cell. Processing depends on the signalling protein SpoIIR, which delays proteolysis until after polar septation, but the mechanism by which sigmaE is confined to the mother cell is not understood. Previous work favoured a model in which pro-sigmaE localizes to the mother cell face of the polar septum, such that sigmaE would be selectively released into mother cell cytoplasm. Based on the use of green fluorescent protein (GFP) fusions, we now report that pro-sigmaE is distributed approximately uniformly along all membrane surfaces and is not confined to the mother- cell face of the septum. Rather, our results are consistent with a model in which preferential and persistent transcription of the spoIIG operon in the mother cell and degradation of sigmaE in the forespore contribute to the selective accumulation of sigmaE in the mother cell. Persistent transcription of spoIIG after polar septation also contributes to the proper timing of pro-sigmaE processing.

Molecular characterization of putative yolk processing enzymes and their expression during oogenesis and embryogenesis in rainbow trout (Oncorhynchus mykiss).

Vitellogenin is the major yolk protein precursor in fish, but little is known about its processing pathway in the oocyte, nor about mobilization of yolk proteins during embryogenesis. In this study we cloned three putative yolk processing enzymes; specifically, cathepsin B and L, and lipoprotein lipase (LPL), from the rainbow trout ovary and determined their patterns of gene expression, together with cathepsin D, during oogenesis and embryogenesis using reverse transcription-polymerase chain reaction. The approximate sizes of both cathepsin B and cathepsin L transcripts were estimated as 1.7-1.8 kilobases by Northern blot analysis. Cathepsin D mRNA and cathepsin L mRNA were expressed constitutively throughout vitellogenesis and embryogenesis, showing the highest levels of expression at around fertilization. Cathepsin B and LPL were expressed exclusively during oogenesis. Quantitatively, expression of cathepsin D mRNA was higher than cathepsin B, cathepsin L, and LPL mRNA throughout the period studied. The different patterns of expression for these genes during oogenesis and embryogenesis signify specific temporal roles in yolk protein processing.

Exfoliatin-Producing Strains Define a Fourth agr Specificity Group in Staphylococcus aureus

The staphylococcal virulon is activated by the density-sensing agr system, which is autoinduced by a short peptide (autoinducing peptide [AIP]) processed from a propeptide encoded by agrD. A central segment of the agr locus, consisting of the C-terminal two-thirds of AgrB (the putative processing enzyme), AgrD, and the N-terminal half of AgrC (the receptor), shows striking interstrain variation. This finding has led to the division of Staphylococcus aureus isolates into three different agr specificity groups and to the division of non-aureus staphylococci into a number of others. The AIPs cross-inhibit the agr responses between groups. We have previously shown that most menstrual toxic shock strains belong to agr specificity group III but that no strong clinical identity has been associated with strains of the other two groups. In the present report, we demonstrate a fourth agr specificity group among S. aureus strains and show that most exfoliatin-producing strains belong to this group. A striking common feature of group IV strains is activation of the agr response early in exponential phase, at least 2 h earlier than in strains of the other groups. This finding raises the question of the biological significance of the agr autoinduction threshold.

Negative regulation of the proteolytic activation of a developmental transcription factor in Bacillus subtilis.

The sporulation transcription factor sigmaK of Bacillus subtilis is controlled by a signal transduction pathway that operates at the level of the proteolytic processing of the inactive precursor protein pro-sigmaK. The conversion of pro-sigmaK to sigmaK requires the putative processing enzyme SpoIVFB and is governed by the regulatory proteins SpoIVFA and BofA. We engineered vegetative cells to carry out processing of pro-sigmaK by inducing the synthesis of the proprotein, a modified form of the putative processing enzyme, and its two regulators during growth. The results showed that (i) modified SpoIVFB was the only sporulation protein necessary to achieve processing of pro-sigmaK; (ii) SpoIVFA stimulated processing, apparently by protecting the processing enzyme from degradation; (iii) BofA inhibited processing in a manner that did not involve degradation of SpoIVFB; and (iv) the inhibition of SpoIVFB by BofA was dependent on SpoIVFA. We conclude that BofA and SpoIVFA act synergistically and are the only two sporulation proteins needed to inhibit the function of SpoIVFB. Our results are consistent with the idea that activation of pro-sigmaK occurs by a reversal of the BofA/SpoIVFA-mediated inhibition of the processing enzyme.

Expression and retinoid modulation of N-arginine dibasic convertase and an aminopeptidase-B in human neuroblastoma cell lines.

Under retinoic acid exposure, the three SK-N-BE(2)-derived human neuroblastoma cell lines, BE(2)-NA, BE(2)-SA and BE(2)-M17 undergo mainly differentiation, apoptosis or continue to proliferate, respectively. We have used this model system to study the modulation of the transcriptional expression of putative processing enzymes, two novel metallopeptidases; i.e. N-arginine dibasic convertase (NRD convertase; EC 3.4,24,61) and an aminopeptidase-B after exposure of the cells either to retinoic acid or to synthetic retinoid analogs. The data indicate that the two respective enzymes are differently modulated in the various cell lines. Whereas aminopeptidase-B expression is enhanced in most cases, NRD convertase appears to undergo opposite regulation in proliferating versus differentiating neuroblastoma cells. It is concluded that both genes might contain retinoic acid regulatory elements (RARE) in their promoters.