Inhibition of Plasmodium falciparum CDPK1 by conditional expression of its J-domain demonstrates a key role in schizont development

2013 ◽  
Vol 452 (3) ◽  
pp. 433-441 ◽  
Author(s):  
Mauro F. Azevedo ◽  
Paul R. Sanders ◽  
Efrosinia Krejany ◽  
Catherine Q. Nie ◽  
Ping Fu ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Fusion Protein ◽  
Fluorescent Protein ◽  
Gene Knockout ◽  
Parasite Development ◽  
Calcium Dependent ◽  
Intracellular Parasites ◽  
Conditional Expression ◽  
Green Fluorescent

PfCDPK1 [Plasmodium falciparum CDPK1 (calcium-dependent protein kinase 1)] is highly expressed in parasite asexual blood and mosquito stages. Its role is still poorly understood, but unsuccessful gene knockout attempts suggest that it is essential for parasite replication and/or RBC (red blood cell) invasion. In the present study, by tagging endogenous CDPK1 with GFP (green fluorescent protein), we demonstrate that CDPK1 localizes to the parasite plasma membrane of replicating and invasive forms as well as very young intracellular parasites and does not appear to be exported into RBCs. Although a knockdown of endogenous CDPK1 was achieved using a destabilization domain, parasites tolerated reduced expression without displaying a phenotype. Because of this, the PfCDPK1 auto-inhibitory J (junction) domain was explored as a means of achieving inducible and specific inhibition. Under in vitro conditions, a fusion protein comprising a J–GFP fusion specifically bound to PfCDPK1 and inhibited its activity. This fusion protein was conditionally expressed in P. falciparum asexual blood stages under the regulation of a DD (destabilization domain) (J–GFP–DD). We demonstrate that J–GFP–DD binds to CDPK1 and that this results in the arrest of parasite development late in the cell cycle during early schizogony. These data point to an early schizont function for PfCDPK1 and demonstrate that conditionally expressing auto-inhibitory regions can be an effective way to address the function of Plasmodium enzymes.

scholarly journals Characterization of the Streptococcal C5a Peptidase Using a C5a-Green Fluorescent Protein Fusion Protein Substrate

2000 ◽  
Vol 182 (11) ◽  
pp. 3254-3258 ◽  
Author(s):  
D. K. Stafslien ◽  
P. P. Cleary
Keyword(s):  
Green Fluorescent Protein ◽  
Fusion Protein ◽  
Fluorescent Protein ◽  
High Sensitivity ◽  
Dependent Manner ◽  
Protein Fusion ◽  
Group B Streptococci ◽  
Mutant Proteins ◽  
Green Fluorescent

ABSTRACT A glutathione-S-transferase (GST)–C5a–green fluorescent protein (GFP) fusion protein was designed for use as a substrate for the streptococcal C5a peptidase (SCPA). The substrate was immobilized on a glutathione-Sepharose affinity matrix and used to measure wild-type SCPA activity in the range of 0.8 to 800 nM. The results of the assay demonstrated that SCPA is highly heat stable and has optimal activity on the synthetic substrate at or above pH 8.0. SCPA activity was unaffected by 0.1 to 10 mM Ca2+, Mg2+, and Mn2+ but was inhibited by the same concentrations of Zn2+. The assay shows high sensitivity to ionic strength; NaCl inhibits SCPA cleavage of GST-C5a-GFP in a dose-dependent manner. Based on previously published computer homology modeling, four substitutions were introduced into the putative active site of SCPA: Asp130-Ala, His193-Ala, Asn295-Ala, and Ser512-Ala. All four mutant proteins had over 1,000-fold less proteolytic activity on C5a in vitro, as determined both by the GFP assay described here and by a polymorphonuclear cell adherence assay. In addition, recombinant SCPA1 and SCPA49, from two distinct lineages of Streptococcus pyogenes (group A streptococci), and recombinant SCPB, fromStreptococcus agalactiae (group B streptococci), were compared in the GFP assay. The three enzymes had similar activities, all cleaving approximately 6 mol of C5a mmol of SCP−1liter−1 min−1.

CXCR2 Inverse Agonism Detected by Arrestin Redistribution

2009 ◽  
Vol 14 (9) ◽  
pp. 1076-1091 ◽  
Author(s):  
Simone Kredel ◽  
Michael Wolff ◽  
Jörg Wiedenmann ◽  
Barbara Moepps ◽  
G. Ulrich Nienhaus ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fusion Protein ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Large Degree ◽  
Distribution Patterns ◽  
Coated Pits ◽  
Inverse Agonism ◽  
Green Fluorescent

To study CXCR2 modulated arrestin redistribution, the authors employed arrestin as a fusion protein containing either the Aequorea victoria—derived enhanced green fluorescent protein (EGFP) or a recently developed mutant of eqFP611, a red fluorescent protein derived from Entacmaea quadricolor. This mutant, referred to as RFP611, had earlier been found to assume a dimeric quarternary structure. It was therefore employed in this work as a “tandem” (td) construct for pseudo monomeric fusion protein labeling. Both arrestin fusion proteins, containing either td RFP611 (Arr td RFP611) or enhanced green fluorescent protein (EGFP; Arr EGFP), were found to colocalize with internalized fluorescently labeled Gro α a few minutes after Gro α addition. Intriguingly, however, Arr td RFP611 and Arr EGFP displayed distinct cellular distribution patterns in the absence of any CXCR2 activating ligand. Under these conditions, Arr td RFP611 showed a largely homoge neous cytosolic distribution, whereas Arr EGFP segregated, to a large degree, into granular spots. These observations indi cate a higher sensitivity of Arr EGFP to the constitutive activity of CXCR2 and, accordingly, an increased arrestin redistribution to coated pits and endocytic vesicles. In support of this interpretation, the authors found the known CXCR2 antagonist Sch527123 to act as an inverse agonist with respect to Arr EGFP redistribution. The inverse agonistic properties of Sch527123 were confirmed in vitro in a guanine nucleotide binding assay, revealing an IC50 value similar to that observed for Arr EGFP redistribution. Thus, the redistribution assay, when based on Arr EGFP, enables the profiling of antagonistic test compounds with respect to inverse agonism. When based on Arr td RFP611, the assay may be employed to study CXCR2 agonism or neutral antagonism. ( Journal of Biomolecular Screening 2009:1076 1091)

scholarly journals Identification of the actin-binding domain of Ins(1,4,5)P3 3-kinase isoform B (IP3K-B)

2004 ◽  
Vol 382 (1) ◽  
pp. 353-362 ◽  
Author(s):  
Maria A. BREHM ◽  
Isabell SCHREIBER ◽  
Uwe BERTSCH ◽  
Albrecht WEGNER ◽  
Georg W. MAYR
Keyword(s):  
Endoplasmic Reticulum ◽  
Fusion Protein ◽  
Fluorescent Protein ◽  
Direct Interaction ◽  
Actin Binding ◽  
Binding Domains ◽  
Isolated Segment ◽  
Green Fluorescent ◽  
Isoform B

Dewaste et al. [Dewaste, Moreau, De Smedt, Bex, De Smedt, Wuytaack, Missiaen and Erneux (2003) Biochem. J. 374, 41–49] showed that over-expressed EGFP (enhanced green fluorescent protein) fused to Ins(1,4,5)P3 3-kinase B (IP3K-B) co-localizes with the cytoskeleton, as well as with the endoplasmic reticulum and the plasma membrane. The domains responsible for these subcellular localizations are not yet identified. For the endogenous enzyme, we confirmed both actin and endoplasmic reticulum localization by employing a high affinity antibody against IP3K-B. F-actin targeting is exclusively dependent on the non-catalytic N-terminal region of IP3K-B. By expressing fragments of this N-terminal domain as EGFP-fusion proteins and inspecting transfected cells by confocal microscopy, we characterized a distinct 63-amino-acid domain comprising amino acids 108–170 of the enzyme which is responsible for F-actin targeting. A truncation of this fragment from both sides revealed that the full size of this segment is essential for this function. Deletion of this segment in a full-length over-expressed IP3K-B–EGFP-fusion protein completely abolished F-actin interaction. Direct interaction of this actin-binding segment with only F-actin, but not with G-actin, was observed in vitro using a bacterially expressed, affinity-purified GST (glutathione S-transferase)–Rattus norvegicus IP3K (aa 108–170) fusion protein. Helix-breaking mutations within this isolated segment abolished the F-actin binding properties both in vitro and when over-expressed in cells, indicating that an intact secondary structure is essential for actin targeting. The segment shows sequence similarities to the actin-binding region in IP3K-A, but no similarity to other actin-binding domains.

scholarly journals Characterization of PRMT1 from Plasmodium falciparum

2009 ◽  
Vol 421 (1) ◽  
pp. 107-118 ◽  
Author(s):  
Qi Fan ◽  
Jun Miao ◽  
Long Cui ◽  
Liwang Cui
Keyword(s):  
Plasmodium Falciparum ◽  
Fluorescent Protein ◽  
Arginine Methylation ◽  
In Vitro Assays ◽  
Parasite Development ◽  
Type I ◽  
Histone H4 ◽  
Turnover Rates ◽  
Post Translational Modification

Arginine methylation is a post-translational modification that affects many cellular processes in eukaryotes. The malaria parasite Plasmodium falciparum encodes three conserved PRMTs (protein arginine N-methyltransferases). We have determined that PfPRMT1 (P. falciparum PRMT1) has authentic type I PRMT activity to form monomethylarginines and asymmetric dimethylarginines. Compared with mammalian PRMT1s, PfPRMT1 possesses a distinctive N-terminal sequence that is ∼50 amino acids longer and is essential for enzyme activity. Recombinant PfPRMT1 methylated histones H4 and H2A and several conserved substrates involved in RNA metabolism, including fibrillarin, poly(A)-binding protein II, ribosomal protein S2 and a putative splicing factor. Using synthetic peptides and MS, we determined target arginines in several substrates and studied the enzyme kinetics. Whereas the kinetic parameters of recombinant PfPRMT1 on an H4 peptide and S-adenosylmethionine were similar to those of mammalian PRMT1s, PfPRMT1 had much higher substrate-turnover rates. In the histone H4 N-terminus, PfPRMT1 could methylate only Arg3, a mark for transcription activation. Western blotting detected dynamic dimethylation of H4-Arg3 during parasite development, suggesting that histone-arginine methylation may play a conserved role in chromatin-mediated gene regulation. Consistent with the presence of potential substrates in both the cytoplasm and nucleus, green fluorescent protein-tagged PfPRMT1 and untagged PfPRMT1 were localized in both cellular compartments, with the majority in the cytoplasm. in vitro assays showed that PfPRMT1 could be inhibited by several small-molecule inhibitors, with IC50-values in the sub-micromolar range. Most of these compounds also effectively inhibited parasite growth, suggesting that parasite PRMTs are promising targets for developing antiparasitic drugs.

scholarly journals Three Distinct Mechanisms for Translocation and Activation of the δ Subspecies of Protein Kinase C

1998 ◽  
Vol 18 (9) ◽  
pp. 5263-5271 ◽  
Author(s):  
Shiho Ohmori ◽  
Yasuhito Shirai ◽  
Norio Sakai ◽  
Motoko Fujii ◽  
Hiroaki Konishi ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Fusion Protein ◽  
Kinase Activity ◽  
Fluorescent Protein ◽  
Simultaneous Treatment ◽  
Kinase C ◽  
Green Fluorescent ◽  
Number Of Cells

ABSTRACT We expressed δ subspecies of protein kinase C (δ-PKC) fused with green fluorescent protein (GFP) in CHO-K1 cells and observed the movement of this fusion protein in living cells after three different stimulations. The δ-PKC–GFP fusion protein had enzymological characteristics very similar to those of the native δ-PKC and was present throughout the cytoplasm in CHO-K1 cells. ATP at 1 mM caused a transient translocation of δ-PKC–GFP to the plasma membrane approximately 30 s after the stimulation and a sequent retranslocation to the cytoplasm within 3 min. A tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA; 1 μM), induced a slower translocation of δ-PKC–GFP, and the translocation was unidirectional. Concomitantly, the kinase activity of δ-PKC–GFP was increased by these two stimulations, when the kinase activity of the immunoprecipitated δ-PKC–GFP was measured in vitro in the absence of PKC activators such as phosphatidylserine and diacylglycerol. Hydrogen peroxide (H2O2; 5 mM) failed to translocate δ-PKC–GFP but increased its kinase activity more than threefold. δ-PKC–GFP was strongly tyrosine phosphorylated when treated with H2O2 but was tyrosine phosphorylated not at all by ATP stimulation and only slightly by TPA treatment. Both TPA and ATP induced the translocation of δ-PKC–GFP even after treatment with H2O2. Simultaneous treatment with TPA and H2O2 further activated δ-PKC–GFP up to more than fivefold. TPA treatment of cells overexpressing δ-PKC–GFP led to an increase in the number of cells in G2/M phase and of dikaryons, while stimulation with H2O2 increased the number of cells in S phase and induced no significant change in cell morphology. These results indicate that at least three different mechanisms are involved in the translocation and activation of δ-PKC.

scholarly journals PML/RARα fusion protein expression in normal human hematopoietic progenitors dictates myeloid commitment and the promyelocytic phenotype

Blood ◽  
2000 ◽  
Vol 96 (4) ◽  
pp. 1531-1537 ◽  
Author(s):  
Francesco Grignani ◽  
Mauro Valtieri ◽  
Marco Gabbianelli ◽  
Vania Gelmetti ◽  
Rosanna Botta ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Fluorescent Protein ◽  
Single Cells ◽  
Retroviral Vectors ◽  
Leukemic Cells ◽  
Cellular Mechanisms ◽  
Fusion Protein Expression ◽  
Vitro Model ◽  
Green Fluorescent

The role of fusion proteins in acute myeloid leukemia (AML) is well recognized, but the leukemic target cell and the cellular mechanisms generating the AML phenotype are essentially unknown. To address this issue, an in vitro model to study the biologic activity of leukemogenic proteins was established. Highly purified human hematopoietic progenitor cells/stem cells (HPC/HSC) in bulk cells or single cells are transduced with retroviral vectors carrying cDNA of the fusion protein and the green fluorescent protein (GFP), purified to homogeneity and induced into multilineage or unilineage differentiation by specific hematopoietic growth factor (HGF) combinations. Expression of PML/RARα fusion protein in human HPC/HSC dictates the acute promyelocytic leukemia (APL) phenotype, largely through these previously unreported effects: rapid induction of HPC/HSC differentiation to the promyelocytic stage, followed by maturation arrest, which is abolished by retinoic acid; reprogramming of HPC commitment to preferential granulopoietic differentiation, irrespective of the HGF stimulus (transduction of single sibling HPC formally demonstrated this effect); HPC protection from apoptosis induced by HGF deprivation. A PML/RARα mutated in the co-repressor N-CoR/histone deacetylase binding region lost these biologic effects, showing that PML/RARα alters the early hematopoietic program through N-CoR–dependent target gene repression mechanisms. These observations identify the cellular mechanism underlying development of the APL phenotype, showing that the fusion protein directly dictates the specific lineage and differentiation stage of leukemic cells.

scholarly journals PML/RARα fusion protein expression in normal human hematopoietic progenitors dictates myeloid commitment and the promyelocytic phenotype

Blood ◽  
2000 ◽  
Vol 96 (4) ◽  
pp. 1531-1537 ◽  
Author(s):  
Francesco Grignani ◽  
Mauro Valtieri ◽  
Marco Gabbianelli ◽  
Vania Gelmetti ◽  
Rosanna Botta ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Fluorescent Protein ◽  
Single Cells ◽  
Retroviral Vectors ◽  
Leukemic Cells ◽  
Cellular Mechanisms ◽  
Fusion Protein Expression ◽  
Vitro Model ◽  
Green Fluorescent

Abstract The role of fusion proteins in acute myeloid leukemia (AML) is well recognized, but the leukemic target cell and the cellular mechanisms generating the AML phenotype are essentially unknown. To address this issue, an in vitro model to study the biologic activity of leukemogenic proteins was established. Highly purified human hematopoietic progenitor cells/stem cells (HPC/HSC) in bulk cells or single cells are transduced with retroviral vectors carrying cDNA of the fusion protein and the green fluorescent protein (GFP), purified to homogeneity and induced into multilineage or unilineage differentiation by specific hematopoietic growth factor (HGF) combinations. Expression of PML/RARα fusion protein in human HPC/HSC dictates the acute promyelocytic leukemia (APL) phenotype, largely through these previously unreported effects: rapid induction of HPC/HSC differentiation to the promyelocytic stage, followed by maturation arrest, which is abolished by retinoic acid; reprogramming of HPC commitment to preferential granulopoietic differentiation, irrespective of the HGF stimulus (transduction of single sibling HPC formally demonstrated this effect); HPC protection from apoptosis induced by HGF deprivation. A PML/RARα mutated in the co-repressor N-CoR/histone deacetylase binding region lost these biologic effects, showing that PML/RARα alters the early hematopoietic program through N-CoR–dependent target gene repression mechanisms. These observations identify the cellular mechanism underlying development of the APL phenotype, showing that the fusion protein directly dictates the specific lineage and differentiation stage of leukemic cells.

scholarly journals The Dynamic Mobility of Histone H1 Is Regulated by Cyclin/CDK Phosphorylation

2003 ◽  
Vol 23 (23) ◽  
pp. 8626-8636 ◽  
Author(s):  
Alejandro Contreras ◽  
Tracy K. Hale ◽  
David L. Stenoien ◽  
Jeffrey M. Rosen ◽  
Michael A. Mancini ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Posttranslational Modifications ◽  
Fluorescent Protein ◽  
Histone H1 ◽  
Open Chromatin ◽  
Nuclear Dynamics ◽  
Cdk2 Activity ◽  
Green Fluorescent

ABSTRACT The linker histone H1 is involved in maintaining higher-order chromatin structures and displays dynamic nuclear mobility, which may be regulated by posttranslational modifications. To analyze the effect of H1 tail phosphorylation on the modulation of the histone's nuclear dynamics, we generated a mutant histone H1, referred to as M1-5, in which the five cyclin-dependent kinase phosphorylation consensus sites were mutated from serine or threonine residues into alanines. Cyclin E/CDK2 or cyclin A/CDK2 cannot phosphorylate the mutant in vitro. Using the technique of fluorescence recovery after photobleaching, we observed that the mobility of a green fluorescent protein (GFP)-M1-5 fusion protein is decreased compared to that of a GFP-wild-type H1 fusion protein. In addition, recovery of H1 correlated with CDK2 activity, as GFP-H1 mobility was decreased in cells with low CDK2 activity. Blocking the activity of CDK2 by p21 expression decreased the mobility of GFP-H1 but not that of GFP-M1-5. Finally, the level and rate of recovery of cyan fluorescent protein (CFP)-M1-5 were lower than those of CFP-H1 specifically in heterochromatic regions. These data suggest that CDK2 phosphorylates histone H1 in vivo, resulting in a more open chromatin structure by destabilizing H1-chromatin interactions.

scholarly journals Cloning and identification of regulatory gene UL76 of human cytomegalovirus

2000 ◽  
Vol 81 (10) ◽  
pp. 2407-2416 ◽  
Author(s):  
Shang-Kwei Wang ◽  
Chang-Yih Duh ◽  
Tai-Tsung Chang
Keyword(s):  
Fusion Protein ◽  
Human Cytomegalovirus ◽  
Fluorescent Protein ◽  
Regulatory Gene ◽  
Regulatory Protein ◽  
Immediate Early ◽  
Free Form ◽  
Infected Cells ◽  
Green Fluorescent

The major immediate-early promoter/enhancer (MIEP, −1139 to +52) of human cytomegalovirus (HCMV) is regulated by cell type-specific transcriptional factors, its own MIE proteins (IE2p40, IE1p55, IE1p72 and IE2p86) as well as viral proteins pUL69, pUL82 and pUL84. To investigate the hypothesis that the regulation of HCMV MIEP is modulated by additional viral genes, HCMV (AD169) genomic sublibraries were constructed and in vitro transient co-transfection assays were performed to assess the ability of these sublibraries to modulate MIEP expression. In this study, enhancement of MIEP expression was exhibited by a number of sublibraries, from one of which a genomic clone was selected for augmentation of expression. Subcloning the insert fragment led to the identification of the responsible locus, UL76. To generate a UL76-specific antibody for immunodetection, the UL76 ORF was constructed as a histidine-tagged fusion protein that was produced in prokaryotic cells. A polyclonal antibody raised against the UL76 fusion protein immunoreacts with a protein of 38 kDa (pUL76) in UL76 ORF-transfected cells. Additionally, pUL76 is present in HCMV-infected cells at the immediate-early to late stages of the reproductive cycle. Characterized by its highly basic composition (predicted pI 11·6), a free form of pUL76 tagged with green fluorescent protein was found to localize exclusively to the nucleus. In this report, pUL76 is defined as a novel regulatory protein that modulates both activation and repression of gene expression, depending on the promoter context and the ratio of transfected effector DNA.

scholarly journals Generation of a Soluble Form of Human Endoglin Fused to Green Fluorescent Protein

2021 ◽  
Vol 22 (20) ◽  
pp. 11282
Author(s):  
Lidia Ruiz-Llorente ◽  
M. Cristina Vega ◽  
Francisco J. Fernández ◽  
Carmen Langa ◽  
Nicholas W. Morrell ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fusion Protein ◽  
Fluorescent Protein ◽  
Extracellular Domain ◽  
Soluble Form ◽  
Migration And Invasion ◽  
Type I ◽  
Juxtamembrane Region ◽  
Green Fluorescent

Endoglin (Eng, CD105) is a type I membrane glycoprotein that functions in endothelial cells as an auxiliary receptor for transforming growth factor β (TGF-β)/bone morphogenetic protein (BMP) family members and as an integrin ligand, modulating the vascular pathophysiology. Besides the membrane-bound endoglin, there is a soluble form of endoglin (sEng) that can be generated by the action of the matrix metalloproteinase (MMP)-14 or -12 on the juxtamembrane region of its ectodomain. High levels of sEng have been reported in patients with preeclampsia, hypercholesterolemia, atherosclerosis and cancer. In addition, sEng is a marker of cardiovascular damage in patients with hypertension and diabetes, plays a pathogenic role in preeclampsia, and inhibits angiogenesis and tumor proliferation, migration, and invasion in cancer. However, the mechanisms of action of sEng have not yet been elucidated, and new tools and experimental approaches are necessary to advance in this field. To this end, we aimed to obtain a fluorescent form of sEng as a new tool for biological imaging. Thus, we cloned the extracellular domain of endoglin in the pEGFP-N1 plasmid to generate a fusion protein with green fluorescent protein (GFP), giving rise to pEGFP-N1/Eng.EC. The recombinant fusion protein was characterized by transient and stable transfections in CHO-K1 cells using fluorescence microscopy, SDS-PAGE, immunodetection, and ELISA techniques. Upon transfection with pEGFP-N1/Eng.EC, fluorescence was readily detected in cells, indicating that the GFP contained in the recombinant protein was properly folded into the cytosol. Furthermore, as evidenced by Western blot analysis, the secreted fusion protein yielded the expected molecular mass and displayed a specific fluorescent signal. The fusion protein was also able to bind to BMP9 and BMP10 in vitro. Therefore, the construct described here could be used as a tool for functional in vitro studies of the extracellular domain of endoglin.

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