scholarly journals C-terminal tripeptide Ser-Asn-Leu (SNL) of human D-aspartate oxidase is a functional peroxisome-targeting signal

1998 ◽  
Vol 336 (2) ◽  
pp. 367-371 ◽  
Author(s):  
Leen AMERY ◽  
Chantal BREES ◽  
Myriam BAES ◽  
Chiaki SETOYAMA ◽  
Retsu MIURA ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Amino Acid ◽  
Fluorescent Protein ◽  
Consensus Sequence ◽  
C Terminus ◽  
Targeting Signal ◽  
Fluorescent Pattern ◽  
Green Fluorescent ◽  
Positively Charged

The functionality of the C-terminus (Ser-Asn-Leu; SNL) of human d-aspartate oxidase, an enzyme proposed to have a role in the inactivation of synaptically released d-aspartate, as a peroxisome-targeting signal (PTS1) was investigated in vivoand in vitro. Bacterially expressed human d-aspartate oxidase was shown to interact with the human PTS1-binding protein, peroxin protein 5 (PEX5p). Binding was gradually abolished by carboxypeptidase treatment of the oxidase and competitively inhibited by a Ser-Lys-Leu (SKL)-containing peptide. After transfection of mouse fibroblasts with a plasmid encoding green fluorescent protein (GFP) extended by PKSNL (the C-terminal pentapeptide of the oxidase), a punctate fluorescent pattern was evident. The modified GFP co-localized with peroxisomal thiolase as shown by indirect immunofluorescence. On transfection in fibroblasts lacking PEX5p receptor, GFP–PKSNL staining was cytosolic. Peroxisomal import of GFP extended by PGSNL (replacement of the positively charged fourth-last amino acid by glycine) seemed to be slower than that of GFP–PKSNL, whereas extension by PKSNG abolished the import of the modified GFP. Taken together, these results indicate that SNL, a tripeptide not fitting the PTS1 consensus currently defined in mammalian systems, acts as a functional PTS1 in mammalian systems, and that the consensus sequence, based on this work and that of other groups, has to be broadened to (S/A/C/K/N)-(K/R/H/Q/N/S)-L.

scholarly journals Functional Regions of the Pseudomonas aeruginosa Cytotoxin ExoU

2005 ◽  
Vol 73 (1) ◽  
pp. 573-582 ◽  
Author(s):  
Shira D. P. Rabin ◽  
Alan R. Hauser
Keyword(s):  
Amino Acids ◽  
Pseudomonas Aeruginosa ◽  
Green Fluorescent Protein ◽  
Amino Acid ◽  
Hela Cells ◽  
Fluorescent Protein ◽  
Host Cells ◽  
Phospholipase Activity ◽  
C Terminus ◽  
Green Fluorescent

ABSTRACT ExoU, a potent patatin-like phospholipase, causes rapid cell death following its injection into host cells by the Pseudomonas aeruginosa type III secretion system. To better define regions of ExoU required for cytotoxicity, transposon-based linker insertion mutagenesis followed by site-directed mutagenesis of individual residues was employed by using a Saccharomyces cerevisiae model system. Random insertion of five amino acids identified multiple regions within ExoU that are required for cell killing. Five regions were chosen for further characterization: three corresponded to the oxyanion hole, hydrolase motif, and catalytic aspartate motif of the patatin-like domain within the N-terminal half of ExoU; one corresponded to an uncharacterized part of the patatin-like domain; and one corresponded to a region near the C terminus. Specific individual amino acid substitutions in each of the four N-terminal regions prevented killing of yeast and significantly reduced phospholipase activity. Whereas five amino acid insertions in the fifth region near the C terminus markedly reduced cytotoxicity and phospholipase activity, substitution of individual amino acids did not abolish either activity. To determine whether each of the five identified regions of ExoU was also essential for cytotoxicity in human cells, representative mutant forms of ExoU fused to green fluorescent protein were expressed in HeLa cells. These variants of ExoU were readily visualized and caused minimal cytotoxicity to HeLa cells, while wild-type ExoU fused to green fluorescent protein induced significant cell lysis and no detectable fluorescence. Thus, a minimum of five regions, including one which is well removed from the patatin-like domain, are required for the cytotoxicity and phospholipase activity of ExoU.

scholarly journals Μελέτη της σχέσης δομή - λειτουργία του κύριου μεταφορέα προλίνης (PrnB) στο μύκητα Aspergillus nidulans

2002 ◽  
Author(s):  
Στέφανος Ταβουλάρης
Keyword(s):  
Green Fluorescent Protein ◽  
Amino Acid ◽  
Aspergillus Nidulans ◽  
Fluorescent Protein ◽  
Transmembrane Segments ◽  
Green Fluorescent

Το γονίδιο prnB του μύκητα Aspergillus nidulans κωδικοποιεί ένα ειδικό για την προλίνη μεταφορέα. Ο μεταφορέας αυτός είναι μια διαμεμβρανική πρωτεΐνη με κυτταροπλασματικό αμινοτελικό και καρβοξυτελικό άκρο, που αποτελείται από 12 πιθανά διαμεμβρανικά τμήματα α-έλικας (TMS: Transmembrane segments), τα οποία συνδέονται μεταξύ τους με μικρές υδρόφιλες θηλειές (L: loops). Η πρωτεΐνη PrnB ανήκει στην οικογένεια μεταφορέων APC (Amino Acid Polyamine Organocation) μέλη της οποίας εντοπίζονται σε προκαρυωτικούς και ευκαρυωτικούς οργανισμούς. Στην παρούσα διδακτορική διατριβή μελετήθηκαν οι σχέσεις δομής-λειτουργίας του μεταφορέα PrnB μέσω της γενετικής, μοριακής και βιοχημικής μελέτης στελεχών του Α. nidulans που εκφράζουν μεταλλαγμένες πρωτεΐνες PrnB. Οι εν λόγω μεταλλαγές είτε απομονώθηκαν γενετικά είτε κατασκευάσθηκαν με in vitro μεταλλαξιγένεση. Τα αποτελέσματα έδειξαν ότι οι παρερμηνεύσιμες μεταλλαγές που επηρεάζουν άμεσα ή έμμεσα τη λειτουργία της πρωτεΐνης PrnB εντοπίζονται στα L2-TMS3 (prnB6, prnB144), TMS6 (prnBKL, prnBKE, prnB119), L6-TMS7 (prnB81), L8-TMS9-L9 (prnB117, prnB206, prnB508) και L10-TMS11 (prnB411) τμήματά της. Από τις παραπάνω μεταλλαγές ιδιαίτερο ρόλο στη λειτουργία της πρωτεΐνης PrnB φαίνεται να διαδραματίζουν τα αμινοξέα Κ245 και F248 του TMS6. Η Κ245 είναι ένα από τα δύο θετικά φορτισμένα αμινοξέα που εντοπίζονται μέσα σε διαμεμβρανικά τμήματα της PrnB ενώ η F248 είναι αυστηρά συντηρημένη σε όλα τα μέλη της οικογένειας APC που είναι καταχωρημένα στην τράπεζα δεδομένων SWISS-PROT. Αλλαγή των αμινοξέων αυτών προκαλεί σημαντική μείωση της χημικής συγγένειας της PrnB για την προλίνη. Επιπλέον, σημαντικό ρόλο στη λειτουργία της PrnB φαίνεται να έχει η περιοχή L8-TMS9, η οποία αντιστοιχεί στη σημαντική για τη λειτουργία μεταφορέων αμινοξέων συναινετική αμφιπαθική περιοχή CAR (Consensus Amphipathic Region). Διπλασιασμός 2 και 3 αμινοξέων στην εν λόγω περιοχή προκαλεί πλήρη απώλεια λειτουργίας και σημαντική μεταβολή των τιμών Km και Vmax της PrnB, αντίστοιχα. Ενδιαφέρον αποτελεί το γεγονός ότι αντίστοιχα με τα παραπάνω τμήματα άλλων μελών της οικογένειας μεταφορέων APC έχει δειχθεί ότι είναι σημαντικά για τη λειτουργία των μεταφορέων αυτών. Εν κατακλείδι, τα παραπάνω αποτελέσματα υποδεικνύουν ότι οι περιοχές TMS6 και L8-TMS9 του μεταφορέα PrnB αποτελούν μέρος της οδού πρόσδεσης ή/και μεταφοράς της προλίνης διαμέσω της κυτταρικής μεμβράνης. Στα πλαίσια εμβάθυνσης των σχέσεων δομής - λειτουργίας του μεταφορέα PrnB καθώς και για τη μελέτη της in vivo τοπογένεσής του χρησιμοποιήθηκε η πρωτεΐνη GFP (Green Fluorescent Protein). Ένας αριθμός χιμαιρικών γονιδίων prnB - gfp κατασκευάσθηκαν, ενσωματώθηκαν στο γενετικό τόπο του γονιδίου prnB, εκφράστηκαν από το φυσιολογικό παρακινητή του, και τα αντίστοιχα χιμαιρικά μόρια PrnB - GFP εντοπίστηκαν in vivo. Τα χιμαιρικά αυτά μόρια φέρουν 2, 4 ή 8 συνδετικά αμινοξέα μεταξύ των πρωτεϊνών PrnB και GFP. Τα εν λόγω αμινοξέα δεν τροποποιούν τη δευτεροταγή δομή των χιμαιρικών μορίων, καθώς επιλέχθηκαν να μην είναι συμβατά με το σχηματισμό στοιχείων δευτεροταγούς δομής. Βρέθηκε ότι μόνο τα χιμαιρικά μόρια με 4 συνδετικά αμινοξέα είναι πλήρως λειτουργικά στους 25°C (φυσιολογική θερμοκρασία ανάπτυξης του μύκητα). Τα αποτελέσματα αυτά δείχνουν ότι ο αριθμός και το είδος των συνδετικών αμινοξέων μεταξύ των δύο μελών των χιμαιρικών μορίων είναι σημαντικός για τη "φυσιολογική" τοπογένεσή τους στην κυτταρική μεμβράνη. Επιπλέον, η χρήση της πρωτεΐνης GFP για τον in vivo εντοπισμό του διαμεμβρανικού μεταφορέα PrnB είναι δυνατή χωρίς να απαιτείται η υπερέκφραση του τελευταίου. Τέλος, η μέθοδος που αναπτύχθηκε για την κατασκευή χιμαιρικών PrnB-GFP μορίων δίνει τη δυνατότητα ενσωμάτωσης και έκφρασης οποιουδήποτε prnB- αλληλομόρφου. Αυτό έχει ως συνέπεια το διαχωρισμό των μεταλλαγών που επηρεάζουν άμεσα τη λειτουργία της πρωτεΐνης PrnB από αυτές που επηρεάζουν την τοπογένεσή της.

scholarly journals Development and Characterization of a cDNA-Launch Recombinant Simian Hemorrhagic Fever Virus Expressing Enhanced Green Fluorescent Protein: ORF 2b’ Is Not Required for In Vitro Virus Replication

Viruses ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 632
Author(s):  
Yingyun Cai ◽  
Shuiqing Yu ◽  
Ying Fang ◽  
Laura Bollinger ◽  
Yanhua Li ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Cell Lines ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Infectious Clone ◽  
Hemorrhagic Fever ◽  
Simian Hemorrhagic Fever Virus ◽  
Hemorrhagic Fever Virus ◽  
Green Fluorescent

Simian hemorrhagic fever virus (SHFV) causes acute, lethal disease in macaques. We developed a single-plasmid cDNA-launch infectious clone of SHFV (rSHFV) and modified the clone to rescue an enhanced green fluorescent protein-expressing rSHFV-eGFP that can be used for rapid and quantitative detection of infection. SHFV has a narrow cell tropism in vitro, with only the grivet MA-104 cell line and a few other grivet cell lines being susceptible to virion entry and permissive to infection. Using rSHFV-eGFP, we demonstrate that one cricetid rodent cell line and three ape cell lines also fully support SHFV replication, whereas 55 human cell lines, 11 bat cell lines, and three rodent cells do not. Interestingly, some human and other mammalian cell lines apparently resistant to SHFV infection are permissive after transfection with the rSHFV-eGFP cDNA-launch plasmid. To further demonstrate the investigative potential of the infectious clone system, we introduced stop codons into eight viral open reading frames (ORFs). This approach suggested that at least one ORF, ORF 2b’, is dispensable for SHFV in vitro replication. Our proof-of-principle experiments indicated that rSHFV-eGFP is a useful tool for illuminating the understudied molecular biology of SHFV.

scholarly journals Human Keratinocytes Adopt Neuronal Fates After In Utero Transplantation in the Developing Rat Brain

2021 ◽  
Vol 30 ◽  
pp. 096368972097821
Author(s):  
Andrea Tenorio-Mina ◽  
Daniel Cortés ◽  
Joel Esquivel-Estudillo ◽  
Adolfo López-Ornelas ◽  
Alejandro Cabrera-Wrooman ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Ectopic Expression ◽  
Neural Induction ◽  
Human Keratinocytes ◽  
Differentiation Potential ◽  
Neuronal Proteins ◽  
Green Fluorescent

Human skin contains keratinocytes in the epidermis. Such cells share their ectodermal origin with the central nervous system (CNS). Recent studies have demonstrated that terminally differentiated somatic cells can adopt a pluripotent state, or can directly convert its phenotype to neurons, after ectopic expression of transcription factors. In this article we tested the hypothesis that human keratinocytes can adopt neural fates after culturing them in suspension with a neural medium. Initially, keratinocytes expressed Keratins and Vimentin. After neural induction, transcriptional upregulation of NESTIN, SOX2, VIMENTIN, SOX1, and MUSASHI1 was observed, concomitant with significant increases in NESTIN detected by immunostaining. However, in vitro differentiation did not yield the expression of neuronal or astrocytic markers. We tested the differentiation potential of control and neural-induced keratinocytes by grafting them in the developing CNS of rats, through ultrasound-guided injection. For this purpose, keratinocytes were transduced with lentivirus that contained the coding sequence of green fluorescent protein. Cell sorting was employed to select cells with high fluorescence. Unexpectedly, 4 days after grafting these cells in the ventricles, both control and neural-induced cells expressed green fluorescent protein together with the neuronal proteins βIII-Tubulin and Microtubule-Associated Protein 2. These results support the notion that in vivo environment provides appropriate signals to evaluate the neuronal differentiation potential of keratinocytes or other non-neural cell populations.

scholarly journals Cellular Targets of Functional and Dysfunctional Mutants of Tobacco Mosaic Virus Movement Protein Fused to Green Fluorescent Protein

2000 ◽  
Vol 74 (23) ◽  
pp. 11339-11346 ◽  
Author(s):  
Vitaly Boyko ◽  
Jessica van der Laak ◽  
Jacqueline Ferralli ◽  
Elena Suslova ◽  
Myoung-Ok Kwon ◽  
...  
Keyword(s):  
Amino Acids ◽  
Green Fluorescent Protein ◽  
Tobacco Mosaic Virus ◽  
Inclusion Bodies ◽  
Fluorescent Protein ◽  
Movement Protein ◽  
Leading Edge ◽  
Intercellular Transport ◽  
C Terminus ◽  
Green Fluorescent

ABSTRACT Intercellular transport of tobacco mosaic virus (TMV) RNA involves the accumulation of virus-encoded movement protein (MP) in plasmodesmata (Pd), in endoplasmic reticulum (ER)-derived inclusion bodies, and on microtubules. The functional significance of these interactions in viral RNA (vRNA) movement was tested in planta and in protoplasts with TMV derivatives expressing N- and C-terminal deletion mutants of MP fused to the green fluorescent protein. Deletion of 55 amino acids from the C terminus of MP did not interfere with the vRNA transport function of MP:GFP but abolished its accumulation in inclusion bodies, indicating that accumulation of MP at these ER-derived sites is not a requirement for function in vRNA intercellular movement. Deletion of 66 amino acids from the C terminus of MP inactivated the protein, and viral infection occurred only upon complementation in plants transgenic for MP. The functional deficiency of the mutant protein correlated with its inability to associate with microtubules and, independently, with its absence from Pd at the leading edge of infection. Inactivation of MP by N-terminal deletions was correlated with the inability of the protein to target Pd throughout the infection site, whereas its associations with microtubules and inclusion bodies were unaffected. The observations support a role of MP-interacting microtubules in TMV RNA movement and indicate that MP targets microtubules and Pd by independent mechanisms. Moreover, accumulation of MP in Pd late in infection is insufficient to support viral movement, confirming that intercellular transport of vRNA relies on the presence of MP in Pd at the leading edge of infection.

In vitro and in vivo evaluation of a green fluorescent protein-HSV1-TK dual-function pet reporter gene

2001 ◽  
Vol 44 (S1) ◽  
pp. S339-S341
Author(s):  
K. E. Luker ◽  
G. D. Luker ◽  
C. M. Pica ◽  
J. L. Dahlheimer ◽  
T. J. Fahrner ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Reporter Gene ◽  
Fluorescent Protein ◽  
Dual Function ◽  
In Vivo Evaluation ◽  
Green Fluorescent

Peroxisome targeting signal of rat liver acyl-coenzyme A oxidase resides at the carboxy terminus

1989 ◽  
Vol 9 (1) ◽  
pp. 83-91
Author(s):  
S Miyazawa ◽  
T Osumi ◽  
T Hashimoto ◽  
K Ohno ◽  
S Miura ◽  
...  
Keyword(s):  
Amino Acid ◽  
Rat Liver ◽  
Coenzyme A ◽  
Terminal Region ◽  
Proteinase K ◽  
Amino Acid Residues ◽  
C Terminus ◽  
Targeting Signal ◽  
Acyl Coenzyme A

To identify the topogenic signal of peroxisomal acyl-coenzyme A oxidase (AOX) of rat liver, we carried out in vitro import experiments with mutant polypeptides of the enzyme. Full-length AOX and polypeptides that were truncated at the N-terminal region were efficiently imported into peroxisomes, as determined by resistance to externally added proteinase K. Polypeptides carrying internal deletions in the C-terminal region exhibited much lower import activities. Polypeptides that were truncated or mutated at the extreme C terminus were totally import negative. When the five amino acid residues at the extreme C terminus were attached to some of the import-negative polypeptides, the import activities were rescued. Moreover, the C-terminal 199 and 70 amino acid residues of AOX directed fusion proteins with two bacterial enzymes to peroxisomes. These results are interpreted to mean that the peroxisome targeting signal of AOX residues at the C terminus and the five or fewer residues at the extreme terminus have an obligatory function in targeting. The C-terminal internal region also has an important role for efficient import, possibly through a conformational effect.

scholarly journals Enhanced Green Fluorescent Protein as Selectable Marker of Retroviral-Mediated Gene Transfer in Immature Hematopoietic Bone Marrow Cells

Blood ◽  
1997 ◽  
Vol 90 (9) ◽  
pp. 3304-3315 ◽  
Author(s):  
Marti F.A. Bierhuizen ◽  
Yvonne Westerman ◽  
Trudi P. Visser ◽  
Wati Dimjati ◽  
Albertus W. Wognum ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Green Fluorescent Protein ◽  
Gene Transfer ◽  
Fluorescent Protein ◽  
Bone Marrow Cells ◽  
Retroviral Vector ◽  
Egfp Expression ◽  
Green Fluorescent ◽  
Marrow Cells

Abstract The further improvement of gene transfer into hematopoietic stem cells and their direct progeny will be greatly facilitated by markers that allow rapid detection and efficient selection of successfully transduced cells. For this purpose, a retroviral vector was designed and tested encoding a recombinant version of the Aequorea victoria green fluorescent protein that is enhanced for high-level expression in mammalian cells (EGFP). Murine cell lines (NIH 3T3, Rat2) and bone marrow cells transduced with this retroviral vector demonstrated a stable green fluorescence signal readily detectable by flow cytometry. Functional analysis of the retrovirally transduced bone marrow cells showed EGFP expression in in vitro clonogenic progenitors (GM-CFU), day 13 colony-forming unit-spleen (CFU-S), and in peripheral blood cells and marrow repopulating cells of transplanted mice. In conjunction with fluorescence-activated cell sorting (FACS) techniques EGFP expression could be used as a marker to select for greater than 95% pure populations of transduced cells and to phenotypically define the transduced cells using antibodies directed against specific cell-surface antigens. Detrimental effects of EGFP expression were not observed: fluorescence intensity appeared to be stable and hematopoietic cell growth was not impaired. The data show the feasibility of using EGFP as a convenient and rapid reporter to monitor retroviral-mediated gene transfer and expression in hematopoietic cells, to select for the genetically modified cells, and to track these cells and their progeny both in vitro and in vivo.

scholarly journals Recombinant Green Fluorescent Protein-Expressing Human Cytomegalovirus as a Tool for Screening Antiviral Agents

2000 ◽  
Vol 44 (6) ◽  
pp. 1588-1597 ◽  
Author(s):  
Manfred Marschall ◽  
Martina Freitag ◽  
Sigrid Weiler ◽  
Gabriele Sorg ◽  
Thomas Stamminger
Keyword(s):  
Green Fluorescent Protein ◽  
Human Cytomegalovirus ◽  
Fluorescent Protein ◽  
Antiviral Agents ◽  
Inhibitory Effects ◽  
Plaque Reduction Assay ◽  
Human Fibroblast Cultures ◽  
Antiviral Activities ◽  
Green Fluorescent

ABSTRACT A recombinant human cytomegalovirus (AD169-GFP) expressing green fluorescent protein was generated by homologous recombination. Infection of human fibroblast cultures with AD169-GFP virus produced stable and readily detectable amounts of GFP signals which were quantitated by automated fluorometry. Hereby, high levels of sensitivity and reproducibility could be achieved, compared to those with the conventional plaque reduction assay. Antiviral activities were determined for four reference compounds as well as a set of putative novel cytomegalovirus inhibitors. The results obtained were exactly in line with the known characteristics of reference compounds and furthermore revealed distinct antiviral activities of novel in vitro inhibitors. The fluorometric data could be confirmed by GFP-based flow cytometry and fluorescence microscopy. In addition, laboratory virus variants derived from the recombinant AD169-GFP virus provided further possibilities for study of the characteristics of drug resistance. The GFP-based antiviral assay appeared to be very reliable for measuring virus-inhibitory effects in concentration- and time-dependent fashions and might also be adaptable for high-throughput screenings of cytomegalovirus-specific antiviral agents.

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