scholarly journals Cell Wall Targeting of Laccase of Cryptococcus neoformans during Infection of Mice

2006 ◽  
Vol 75 (2) ◽  
pp. 714-722 ◽  
Author(s):  
Scott R. Waterman ◽  
Moshe Hacham ◽  
John Panepinto ◽  
Guowu Hu ◽  
Soowan Shin ◽  
...  
Keyword(s):  
Cell Wall ◽  
Fusion Protein ◽  
Cryptococcus Neoformans ◽  
Fluorescent Protein ◽  
Pathogenic Fungus ◽  
Expression Studies ◽  
Green Fluorescent ◽  
The Brain ◽  
Mouse Lungs

ABSTRACT Laccase is a major virulence factor of the pathogenic fungus Cryptococcus neoformans, which afflicts both immunocompetent and immunocompromised individuals. In the present study, laccase was expressed in C. neoformans lac1Δ cells as a fusion protein with an N-terminal green fluorescent protein (GFP) using C. neoformans codon usage. The fusion protein was robustly localized to the cell wall at physiological pH, but it was mislocalized at low pH. Structural analysis of the laccase identified a C-terminal region unique to C. neoformans, and expression studies showed that the region was required for efficient transport to the cell wall both in vitro and during infection of mouse lungs. During infection of mice, adherence to alveolar macrophages was also associated with a partial mislocalization of GFP-laccase within cytosolic vesicles. In addition, recovery of cryptococcal cells from lungs of two strains of mice (CBA/J and Swiss Albino) later in infection was also associated with cytosolic mislocalization, but cells from the brain showed almost exclusive localization to cell walls, suggesting that there was more efficient cell wall targeting during infection of the brain. These data suggest that host cell antifungal defenses may reduce effective cell wall targeting of laccase during infection of the lung but not during infection of the brain, which may contribute to a more predominant role for the enzyme during infection of the brain.

Cryptococcus neoformans Differential Gene Expression Detected In Vitro and In Vivo with Green Fluorescent Protein

1999 ◽  
Vol 67 (4) ◽  
pp. 1812-1820
Author(s):  
Maurizio del Poeta ◽  
Dena L. Toffaletti ◽  
Thomas H. Rude ◽  
Sara D. Sparks ◽  
Joseph Heitman ◽  
...  
Keyword(s):  
Gene Expression ◽  
Green Fluorescent Protein ◽  
Cryptococcus Neoformans ◽  
Differential Gene Expression ◽  
Fluorescent Protein ◽  
Differential Gene ◽  
Green Fluorescent

Nuclear localization of angiotensinogen in astrocytes

2005 ◽  
Vol 288 (2) ◽  
pp. R539-R546 ◽  
Author(s):  
Mikhiela Sherrod ◽  
Xuebo Liu ◽  
Xiaoji Zhang ◽  
Curt D. Sigmund
Keyword(s):  
Fusion Protein ◽  
Nuclear Localization ◽  
Fluorescent Protein ◽  
Primary Cultures ◽  
Endocrine Function ◽  
Astrocytoma Cell ◽  
Protein Encoding ◽  
Localization Signal ◽  
Green Fluorescent ◽  
The Brain

In the brain, angiotensinogen (AGT) is primarily expressed in astrocytes; brain ANG II derived from locally produced AGT has been shown to influence blood pressure. To better understand the molecular basis of AGT expression in the brain, we identified a human astrocytoma cell line, CCF-STTG1, that expresses endogenous AGT mRNA and produces AGT protein. Studies examining CCF-STTG1 cell AGT after N- and O-glycosidase suggest that AGT may not be posttranslationally modified by glycosylation in these cells as it is in plasma. Small amounts of AGT (5% of HepG2) were detected in the culture medium, suggesting a low rate of AGT secretion. Immunocytochemical examination of AGT in CCF-STTG1 cells revealed mainly nuclear localization. Although this has not been previously reported, it is consistent with nuclear localization of other serpin family members. To examine this further, we generated a fusion protein consisting of green fluorescent protein (GFP) and human AGT and examined subcellular localization by confocal microscopy after confirming expression of the fusion protein by Western blot. In CCF-STTG1 cells, a control GFP construct lacking AGT was mainly localized in the cytoplasm, whereas the GFP-AGT fusion protein was primarily localized in the nucleus. To map the location of a potential nuclear localization signal, overlapping 500-bp fragments of human AGT cDNA were fused in frame downstream of GFP. Although four of the fusion proteins exhibited either perinuclear or cytoplasmic localization, one fusion protein encoding the COOH terminus of AGT was localized in the nucleus. Importantly, nuclear localization of human AGT was confirmed in primary cultures of glial cells isolated from transgenic mice expressing the human AGT under the control of its own endogenous promoter. Our results suggest that AGT may have a novel intracellular role in the brain apart from its predicted endocrine function.

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.

scholarly journals Isolation, Characterization, and Localization of a Capsule-Associated Gene, CAP10, of Cryptococcus neoformans

1999 ◽  
Vol 181 (18) ◽  
pp. 5636-5643 ◽  
Author(s):  
Y. C. Chang ◽  
K. J. Kwon-Chung
Keyword(s):  
Cryptococcus Neoformans ◽  
Fluorescent Protein ◽  
Pathogenic Fungus ◽  
Wild Strain ◽  
Extracellular Polysaccharide ◽  
Capsule Formation ◽  
Expression Levels ◽  
Model Studies ◽  
Isolation And Characterization ◽  
Green Fluorescent

ABSTRACT Cryptococcus neoformans is a pathogenic fungus which most commonly affects the central nervous system and causes fatal meningoencephalitis primarily in patients with AIDS. This fungus produces a thick extracellular polysaccharide capsule which is well recognized as a virulence factor. Here, we describe the isolation and characterization of a novel gene, CAP10, which is required for capsule formation. Complementation of the acapsularcap10 mutant produced an encapsulated strain and the deletion of CAP10 from a wild strain resulted in an acapsular phenotype. The molecular mass of the hemagglutinin epitope-tagged Cap10p is about 73 kDa, which is similar to the size predicted from sequence analysis. When CAP10 was fused with a hybrid green fluorescent protein construct, the fluorescence signals appeared as patches in the cytoplasm. Using a reporter gene construct, we found that CAP10 was expressed at high levels in late-stationary-phase cells. In addition, we found that the expression levels of CAP10 are modulated by the transcriptional factorSTE12α. Deletion of STE12α downregulated the expression levels of CAP10 while overexpression ofSTE12α upregulated the expression levels ofCAP10. Animal model studies indicate that deletion of theCAP10 gene results in the loss of virulence, and complementation of the acapsular phenotype of cap10restores virulence. Thus, CAP10 is required for capsule formation and virulence.

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 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 Role of the ESCRT Pathway in Laccase Trafficking and Virulence of Cryptococcus neoformans

2020 ◽  
Vol 88 (7) ◽  
Author(s):  
Yoon-Dong Park ◽  
Shu Hui Chen ◽  
Emma Camacho ◽  
Arturo Casadevall ◽  
Peter R. Williamson
Keyword(s):  
Cell Wall ◽  
Cryptococcus Neoformans ◽  
Fluorescent Protein ◽  
Extracellular Vesicle ◽  
Multivesicular Bodies ◽  
Melanin Production ◽  
Content Type ◽  
Endosomal Sorting ◽  
Green Fluorescent

ABSTRACT The endosomal sorting complex required for transport (ESCRT) plays a crucial role in the transportation and degradation of proteins. We determined that Vps27, a key protein of the ESCRT-0 complex, is required for the transport of the virulence factor laccase to the cell wall in Cryptococcus neoformans. Laccase activity was perturbed, as was melanin production, in vps27Δ strains. In the absence of VPS27, there was an accumulation of multivesicular bodies with vacuolar fragmentation and mistargeting of the vacuolar carboxypeptidase CPY/Prc1, resulting in an extracellular localization. In addition, deletion of VPS27 resulted in a defect in laccase targeting of a Lac1-green fluorescent protein (GFP) fusion to the cell wall with trapping within intracellular puncta; this deletion was accompanied by reduced virulence in a mouse model. However, the actin cytoskeleton remained intact, suggesting that the trafficking defect is not due to defects in actin-related localization. Extracellular vesicle maturation was also defective in the vps27Δ mutant, which had a larger vesicle size as measured by dynamic light scattering. Our data identify cryptococcal VPS27 as a required gene for laccase trafficking and attenuates virulence of C. neoformans in a mouse intravenous (i.v.) meningitis model.

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.

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