Alternative Splicing of the Aflatoxin-Associated Baeyer–Villiger Monooxygenase from Aspergillus flavus: Characterisation of MoxY Isoforms

Aflatoxins are carcinogenic mycotoxins that are produced by the filamentous fungus Aspergillus flavus, a contaminant of numerous food crops. Aflatoxins are synthesised via the aflatoxin biosynthesis pathway, with the enzymes involved encoded by the aflatoxin biosynthesis gene cluster. MoxY is a type I Baeyer–Villiger monooxygenase (BVMO), responsible for the conversion of hydroxyversicolorone (HVN) and versicolorone (VN) to versiconal hemiacetal acetate (VHA) and versiconol acetate (VOAc), respectively. Using mRNA data, an intron near the C-terminus was identified that is alternatively spliced, creating two possible MoxY isoforms which exist in vivo, while analysis of the genomic DNA suggests an alternative start codon leading to possible elongation of the N-terminus. These four variants of the moxY gene were recombinantly expressed in Escherichia coli, and their activity evaluated with respect to their natural substrates HVN and VN, as well as surrogate ketone substrates. Activity of the enzyme is absolutely dependent on the additional 22 amino acid residues at the N-terminus. Two MoxY isoforms with alternative C-termini, MoxYAltN and MoxYAltNC, converted HVN and VN, in addition to a range of ketone substrates. Stability and flavin-binding data suggest that MoxYAltN is, most likely, the dominant isoform. MoxYAltNC is generated by intron splicing, in contrast to intron retention, which is the most prevalent type of alternative splicing in ascomycetes. The alternative C-termini did not alter the substrate acceptance profile, or regio- or enantioselectivity of the enzyme, but did significantly affect the solubility and stability.

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A fusion protein with improved thrombolytic effect and low bleeding risk

Thrombosis and Haemostasis ◽

10.1160/th09-04-0235 ◽

2009 ◽

Vol 102 (12) ◽

pp. 1194-1203 ◽

Cited By ~ 3

Author(s):

Lisheng Wang ◽

Qinglin Zhang ◽

Yide Qin ◽

Chutse Wu ◽

Xiudong Wang ◽

...

Keyword(s):

Fusion Protein ◽

Anticoagulant Activity ◽

Bleeding Risk ◽

Fibrin Clot ◽

Clot Lysis ◽

Recognition Sequence ◽

Linker Peptide ◽

C Terminus ◽

N Terminus

SummaryTo resolve the therapeutic dilemma between efficacy of thrombolysis and bleeding risk associated with the use of a combination of thrombolytic and anticoagulant treatments, we created a fusion protein. Staphylokinase was fused to the N-terminus of hirudin using thrombin recognition sequence as linker peptide, resulting in a fusion protein STH.We hypothesised that STH would be cleaved by thrombin at the thrombus site, releasing staphylokinase and hirudin to perform bifunctionally, and attenuating bleeding risk. SDS-PAGE andWestern blot analyses indicated that the linker peptide could be specially recognised and cleaved by thrombin. Amidolytic and thromboelastogram assays showed that the N-terminus of hirudin in STH was blocked by staphylokinase and linker peptide, impeding hirudin’s anticoagulant activity. Once cleaved, STH displayed 35.7% of the anticoagulant activity of equimolar hirudin and exhibited anticoagulant effects in the fibrin clot lysis assay.Thrombin-binding and fibrin clot lysis assays showed that the C-terminus of hirudin retained its high affinity for thrombin. Moreover, STH showed improved thrombolytic effects and a lower bleeding risk in animals. Thus, STH may have the capacity to perform bifunctionally and release anticoagulant activity in a thrombus-targeted manner in vivo, which may reduce the bleeding risk that often accompanies high thrombolytic efficacy in the treatment of thromboembolic diseases.

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Different expression levels of two KgmB-His fusion proteins

Journal of the Serbian Chemical Society ◽

10.2298/jsc0512401m ◽

2005 ◽

Vol 70 (12) ◽

pp. 1401-1407 ◽

Cited By ~ 3

Author(s):

Sandra Markovic ◽

Sandra Vojnovic ◽

Milija Jovanovic ◽

Branka Vasiljevic

Keyword(s):

Recombinant Proteins ◽

Fusion Proteins ◽

Kanamycin Resistance ◽

Expression Vectors ◽

E Coli ◽

C Terminus ◽

N Terminus ◽

Different Levels ◽

Streptomyces Tenebrarius

The KgmB methylase from Streptomyces tenebrarius was expressed and purified using the QIAexpress System. Two expression vectors were made: pQEK-N, which places a (His)6 tag at the N-terminus, and pQEK-C, which places a (His)6 tag at the C-terminus of the recombinant KgmB protein. Kanamycin resistance of the E. coli cells containing either the pQEK-N or the pQEK-C recombinant plasmids confirmed the functionality of both KgmB-His fusion proteins in vivo. Interestingly, different levels of expression were observed between these two recombinant proteins. Namely, KgmB methylase with the (His)6 tag at the N-terminus showed a higher level of expression. Purification of the (His)6-tagged proteins using Ni-NTA affinity chromatography was performed under native conditions and the KgmB methylase with (His)6 tag at the N-terminus was purified to homogeneity >95 %. The recombinant KgmB protein was detected on a Western blot using anti-Sgm antibodies.

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Cdc18p can block mitosis by two independent mechanisms

Journal of Cell Science ◽

10.1242/jcs.111.20.3101 ◽

1998 ◽

Vol 111 (20) ◽

pp. 3101-3108 ◽

Cited By ~ 2

Author(s):

E. Greenwood ◽

H. Nishitani ◽

P. Nurse

Keyword(s):

Dna Replication ◽

S Phase ◽

Checkpoint Control ◽

Replication Checkpoint ◽

Mitotic Kinase ◽

C Terminus ◽

N Terminus ◽

Dna Replication Checkpoint ◽

Checkpoint Genes

The DNA replication checkpoint is required to maintain the integrity of the genome, inhibiting mitosis until S phase has been successfully completed. The checkpoint preventing premature mitosis in Schizosaccharomyces pombe relies on phosphorylation of the tyrosine-15 residue on cdc2p to prevent its activation and hence mitosis. The cdc18 gene is essential for both generating the DNA replication checkpoint and the initiation of S phase, thus providing a key role for the overall control and coordination of the cell cycle. We show that the C terminus of the protein is capable of both initiating DNA replication and the checkpoint function of cdc18p. The C terminus of cdc18p acts upstream of the DNA replication checkpoint genes rad1, rad3, rad9, rad17, hus1 and cut5 and requires the wee1p/mik1p tyrosine kinases to block mitosis. The N terminus of cdc18p can also block mitosis but does so in the absence of the DNA replication checkpoint genes and the wee1p/mik1p kinases therefore acting downstream of these genes. Because the N terminus of cdc18p associates with cdc2p in vivo, we suggest that by binding the cdc2p/cdc13p mitotic kinase directly, it exerts an effect independently of the normal checkpoint control, probably in an unphysiological manner.

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Interaction of the Endocytic Scaffold Protein Pan1 with the Type I Myosins Contributes to the Late Stages of Endocytosis

Molecular Biology of the Cell ◽

10.1091/mbc.e07-05-0436 ◽

2007 ◽

Vol 18 (8) ◽

pp. 2893-2903 ◽

Cited By ~ 29

Author(s):

Sarah L. Barker ◽

Linda Lee ◽

B. Daniel Pierce ◽

Lymarie Maldonado-Báez ◽

David G. Drubin ◽

...

Keyword(s):

Late Stage ◽

Actin Polymerization ◽

Fluorescent Protein ◽

Temperature Sensitive ◽

Type I ◽

Reading Frame ◽

Rich Domain ◽

C Terminus

The yeast endocytic scaffold Pan1 contains an uncharacterized proline-rich domain (PRD) at its carboxy (C)-terminus. We report that the pan1-20 temperature-sensitive allele has a disrupted PRD due to a frame-shift mutation in the open reading frame of the domain. To reveal redundantly masked functions of the PRD, synthetic genetic array screens with a pan1ΔPRD strain found genetic interactions with alleles of ACT1, LAS17 and a deletion of SLA1. Through a yeast two-hybrid screen, the Src homology 3 domains of the type I myosins, Myo3 and Myo5, were identified as binding partners for the C-terminus of Pan1. In vitro and in vivo assays validated this interaction. The relative timing of recruitment of Pan1-green fluorescent protein (GFP) and Myo3/5-red fluorescent protein (RFP) at nascent endocytic sites was revealed by two-color real-time fluorescence microscopy; the type I myosins join Pan1 at cortical patches at a late stage of internalization, preceding the inward movement of Pan1 and its disassembly. In cells lacking the Pan1 PRD, we observed an increased lifetime of Myo5-GFP at the cortex. Finally, Pan1 PRD enhanced the actin polymerization activity of Myo5–Vrp1 complexes in vitro. We propose that Pan1 and the type I myosins interactions promote an actin activity important at a late stage in endocytic internalization.

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Surface Signaling in Ferric Citrate Transport Gene Induction: Interaction of the FecA, FecR, and FecI Regulatory Proteins

Journal of Bacteriology ◽

10.1128/jb.182.3.637-646.2000 ◽

2000 ◽

Vol 182 (3) ◽

pp. 637-646 ◽

Cited By ~ 88

Author(s):

Sabine Enz ◽

Susanne Mahren ◽

Uwe H. Stroeher ◽

Volkmar Braun

Keyword(s):

Nitrilotriacetic Acid ◽

Ferric Citrate ◽

Binding Assays ◽

Surface Binding ◽

Citrate Transport ◽

C Terminus ◽

N Terminus ◽

Surface Signaling

ABSTRACT In Escherichia coli, transcription of the ferric citrate transport genes fecABCDE is controlled by a novel signal transduction mechanism that starts at the cell surface. Binding of ferric citrate to the outer membrane protein FecA initiates a signal that is transmitted by FecR across the cytoplasmic membrane into the cytoplasm where FecI, the sigma factor, is activated. Interaction between the signaling proteins was demonstrated by utilizing two methods. In in vitro binding assays, FecR that was His tagged at the N terminus [(His)10-FecR] and bound to a Ni-nitrilotriacetic acid agarose column was able to retain FecA, and FecR that was His tagged at the C terminus [FecR-(His)6] retained FecI on the column. An N-terminally truncated, induction-negative but transport-active FecA protein did not bind to (His)10-FecR. The in vivo assay involved the determination of the FecA, FecR, and FecI interacting domains with the bacterial two-hybrid Lex-based system. FecA1–79 interacts with FecR101–317 and FecR1–85 interacts with FecI1–173. These data clearly support a model that proposes interaction of the periplasmic N terminus of FecA with the periplasmic C-terminal portion of FecR and interaction of the cytoplasmic N terminus of FecR with FecI, which results in FecI activation.

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Functional Characterization of the Interaction of Ste50p with Ste11p MAPKKK inSaccharomyces cerevisiae

Molecular Biology of the Cell ◽

10.1091/mbc.10.7.2425 ◽

1999 ◽

Vol 10 (7) ◽

pp. 2425-2440 ◽

Cited By ~ 58

Author(s):

Cunle Wu ◽

Ekkehard Leberer ◽

David Y. Thomas ◽

Malcolm Whiteway

Keyword(s):

Protein Kinase ◽

Functional Characterization ◽

Hog Pathway ◽

C Terminus ◽

Extracellular Signal ◽

Osmotic Stress Response ◽

N Terminus

The Saccharomyces cerevisiae Ste11p protein kinase is a homologue of mammalian MAPK/extracellular signal-regulated protein kinase kinase kinases (MAPKKKs or MEKKs) as well as theSchizosaccharomyces pombe Byr2p kinase. Ste11p functions in several signaling pathways, including those for mating pheromone response and osmotic stress response. The Ste11p kinase has an N-terminal domain that interacts with other signaling molecules to regulate Ste11p function and direct its activity in these pathways. One of the Ste11p regulators is Ste50p, and Ste11p and Ste50p associate through their respective N-terminal domains. This interaction relieves a negative activity of the Ste11p N terminus, and removal of this negative function is required for Ste11p function in the high-osmolarity glycerol (HOG) pathway. The Ste50p/Ste11p interaction is also important (but not essential) for Ste11p function in the mating pathway; in this pathway binding of the Ste11p N terminus with both Ste50p and Ste5p is required, with the Ste5p association playing the major role in Ste11p function. In vitro, Ste50p disrupts an association between the catalytic C terminus and the regulatory N terminus of Ste11p. In addition, Ste50p appears to modulate Ste11p autophosphorylation and is itself a substrate of the Ste11p kinase. Therefore, both in vivo and in vitro data support a role for Ste50p in the regulation of Ste11p activity.

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Atg15 in Saccharomyces cerevisiae consists of two functionally distinct domains

Molecular Biology of the Cell ◽

10.1091/mbc.e20-07-0500 ◽

2021 ◽

pp. mbc.E20-07-0500

Author(s):

Eri Hirata ◽

Kyo Shirai ◽

Tatsuya Kawaoka ◽

Kosuke Sato ◽

Fumito Kodama ◽

...

Keyword(s):

Transmembrane Domain ◽

Multivesicular Body ◽

Catalytic Triad ◽

Double Membrane ◽

Terminal Domain ◽

C Terminus ◽

N Terminus ◽

Membrane Bound ◽

Important Residue

Autophagy is a cellular degradation system widely conserved among eukaryotes. During autophagy, cytoplasmic materials fated for degradation are compartmentalized in double membrane–bound organelles called autophagosomes. After fusing with the vacuole, their inner membrane–bound structures are released into the vacuolar lumen to become autophagic bodies and eventually degraded by vacuolar hydrolases. Atg15 is a lipase essential for disintegration of autophagic body membranes and has a transmembrane domain at the N-terminus and a lipase domain at the C-terminus. However, the roles of both domains in vivo are not well understood. In this study, we found that the N-terminal domain alone can travel to the vacuole via the multivesicular body pathway, and that targeting of the C-terminal lipase domain to the vacuole is required for degradation of autophagic bodies. Moreover, we found that the C-terminal domain could disintegrate autophagic bodies when it was transported to the vacuole via the Pho8 pathway instead of the multivesicular body pathway. Finally, we identified H435 as one of the residues composing the putative catalytic triad, and W466 as an important residue for degradation of autophagic bodies. This study may provide a clue to understanding how the C-terminal lipase domain recognizes autophagic bodies to degrade them. [Media: see text] [Media: see text]

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Alternative splicing takes charge of interleukin-22 and interferon lambda 4 signaling

10.31237/osf.io/sca8k ◽

2018 ◽

Author(s):

Chrissie Lim

Keyword(s):

Alternative Splicing ◽

Intron Retention ◽

Protein Isoforms ◽

Functional Domain ◽

Type I ◽

Protein Coding ◽

Interleukin 22 ◽

Interferon Lambda ◽

Type Iii Ifn ◽

Overexpression System

Immune responses require the tight control of dose, location, strength and duration through genetic, epigenetic or biochemical regulation. Of these, the generation of alternatively-spliced constructs increases transcriptional and proteomic diversity in post-transcriptional modification, localization and functional domain integrity. Specifically, this thesis explores how splice variation engenders profound differences in the biological functions of interleukin-22 (IL-22) binding protein (IL-22BP) and interferon lambda 4 (IFNλ4), which are both central components of distinct cytokine pathways in mucosal immunity and inflammation. IL-22BP is a soluble receptor for IL-22 that is expressed as three isoforms in humans, though the physiological relevance of the three human isoforms has remained a mystery due to the absence of this variation in mice. We present novel findings that IL-22BPi1 is inactive due to intracellular retention by its unique exon, while IL-22BPi3 is also an antagonist but with differential activity from IL-22BPi2. Importantly, while IL-22BPi3 has widespread expression in steady-state homeostatic conditions, IL-22BPi2 is the only isoform induced by inflammatory TLR2/retinoic acid stimulation, highlighting important spatiotemporal control of the two isoforms that exploit their differential activities. IFNλ4 presents a different mystery in which the protein-coding variant is genetically associated with poorer clearance, but the mechanism for this association remains unclear. We investigated several non-canonical functions proposed by the field, including intrinsic differences in activity of the three protein isoforms and their interference with antiviral activites of other type I or III interferons. Establishing an overexpression system and purifying recombinant proteins, we found that only the full-length isoform is active and exhibits similar effects to canonical type III IFN IFNλ3, without any blockade of other IFN signaling. Simultaneously, functional IFNλ4 expression is suppressed in hepatocytes and dendritic cells through preferential splicing to increase intron retention and expression of inactive isoforms. Therefore, alternative splicing in IFNλ4 is an important mechanism to control IFNλ4 bioactivity. The divergent manners in which alternative splice forms impact the activity of both IL-22BP and IFNλ4 highlight the important contributions of this process to cytokine biology and bigger implications that escape detection by genomic analyses.

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Roles of the Carboxy-Terminal Half of Pseudomonas aeruginosa Major Outer Membrane Protein OprF in Cell Shape, Growth in Low-Osmolarity Medium, and Peptidoglycan Association

Journal of Bacteriology ◽

10.1128/jb.180.14.3556-3562.1998 ◽

1998 ◽

Vol 180 (14) ◽

pp. 3556-3562 ◽

Cited By ~ 57

Author(s):

Eileen G. Rawling ◽

Fiona S. L. Brinkman ◽

Robert E. W. Hancock

Keyword(s):

Pseudomonas Aeruginosa ◽

Membrane Protein ◽

Protein Expression ◽

Outer Membrane ◽

Outer Membrane Protein ◽

Cell Shape ◽

Cell Length ◽

C Terminus ◽

N Terminus

ABSTRACT OprF, the major outer membrane protein of Pseudomonas aeruginosa, is multifunctional in that it can act as a nonspecific porin, plays a role in the maintenance of cell shape, and is required for growth in a low-osmolarity environment. The latter two structural roles of OprF, and OprF’s association with the peptidoglycan, have been proposed to be localized in the carboxy terminus of the protein, based on this region’s similarity to members of the OmpA family of proteins. To determine if this is correct, we constructed a series of C-terminally truncated OprF derivatives and examined their effects on P. aeruginosa cell length and growth in low-osmolarity medium. While the C terminus of OprF was required for wild-type cell length and growth in low-osmolarity medium, expression of the N terminus (first 163 amino acids [aa]) also influenced these phenotypes (compared with OprF deficiency). The first 154 to 164 aa of OprF seemed required for stable protein expression, consistent with the existence of a β-barrel domain in the N terminus of OprF. Greater than 215 aa of the protein were required for strong peptidoglycan association, confirming that residues in the C-terminal end of OprF are required for peptidoglycan binding. OprF deficiency did not affect the in vivo growth of an OprF-deficient strain in a mouse chamber model. Collectively, these data suggest that the C terminus of OprF plays a role in cell length, growth of P. aeruginosa in low-osmolarity media (but not in vivo), and peptidoglycan association, while the N terminus has an influence on the first two characteristics and is additionally important for stable protein expression.

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In Vivo Analysis of Erythroid Protein 4.1 Pre-mRNA Splicing Mechanisms: Use of Antisense Morpholinos to Assay Function of Deep Intron Regulatory Elements

Blood ◽

10.1182/blood.v116.21.815.815 ◽

2010 ◽

Vol 116 (21) ◽

pp. 815-815

Author(s):

Marilyn Parra ◽

Xiuli An ◽

Narla Mohandas ◽

John G. Conboy

Keyword(s):

Alternative Splicing ◽

Splice Site ◽

Cultured Cells ◽

Conflicts Of Interest ◽

Regulatory Elements ◽

Protein Isoforms ◽

Start Codon ◽

Exon 2 ◽

Mouse Tissues

Abstract Abstract 815 Erythroid stage-specific alternative splicing plays an essential role in the expression of protein 4.1R isoforms that interact with other skeletal proteins to strengthen the membrane. In late erythroblasts, 4.1R mRNA is processed from pre-mRNA that initiates transcription at alternative first exon 1A (E1A) and splices exclusively to the more distal of two alternative 3' splice sites at exon 2 (E2dis), ~100kb downstream. This splicing event is important because it is required to generate the shorter N-terminal domain characteristic of 80kDa isoforms of 4.1R protein in red cells. We have reported that E1A splicing to E2dis requires two nested intrasplicing events mediated by an essential deep intron element originally annotated as exon 1B. However, these studies employed small minigenes transfected into cultured cells, an artificial system that may not correctly reflect in vivo mechanisms. Here we used an antisense RNA strategy to explore splicing of endogenous full length 4.1R pre-mRNA in tissues of live mice and in primary erythroblasts. Chemically modified oligonucleotides known as vivo-morpholinos (vMOs), introduced via tail vein injection and internalized into selected organs, can base pair with complementary cellular RNA sequences and block function of candidate regulatory motifs. Importantly, two independent vMOs directed against the 4.1R intraexon regulatory element both substantially abrogated intrasplicing in several mouse tissues, robustly switching E1A splicing from E2dis to the proximal 3' splice site in E2 (E2prox). This switch results in inclusion of start codon AUG1 in mature 4.1R mRNA and synthesis of larger isoforms of 4.1R protein. These results were highly sequence-specific, since negative control vMOs directed against other genes did not alter E1A splicing to E2dis. Interestingly, we have recently used vMOs to confirm the existence of a similar deep intron element required for analogous E1A-E2dis splicing in the paralogous 4.1B gene. Together these findings strongly support the in vivo physiological function of deep intron elements in the control of intrasplicing in both 4.1R and 4.1B pre-mRNAs. To test whether the 4.1R intrasplicing mechanism is also active in erythroid cells, we incubated mouse splenic erythroblasts isolated from FVA-treated animals with morpholinos directed against the intraexon. Two independent morpholinos against its 5' splice site and branch point both induced a concentration-dependent switch in E1A splicing from E2dis to E2prox. Control morpholinos had no effect on E1A splicing. Because the splicing switch results in inclusion of alternative translation initiation codon AUG1, it was predicted to induce synthesis of larger isoforms of 4.1R including the N-terminal headpiece known to influence 4.1R binding affinities for other skeletal proteins. Western blot analysis of erythroblast proteins confirmed a switch to expression of larger 4.1R protein isoforms that are not present in normal late stage erythroblasts. Intrasplicing is mediated by deep intron elements, and is essential for accurate physiological splicing of natural 4.1R pre-mRNA in erythroid and other cells. Antisense morpholinos represent a new tool for alternative splicing studies in vivo or in cultured erythroblasts. Disclosures: No relevant conflicts of interest to declare.

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