scholarly journals Artificial 64-Residue HIV-1 Enhancer-Binding Peptide Is a Potent Inhibitor of Viral Replication in HIV-1-Infected Cells

2011 ◽  
Vol 2011 ◽  
pp. 1-13 ◽  
Author(s):  
Mouhssin Oufir ◽  
Leslie R. Bisset ◽  
Stefan R. K. Hoffmann ◽  
Gongda Xue ◽  
Stephan Klauser ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
T Antigen ◽  
Inhibitory Peptide ◽  
Sv40 Large T Antigen ◽  
Binding Peptide ◽  
C Terminus ◽  
Arginine Residues ◽  
Green Fluorescent ◽  
Hiv 1

An artificial HIV-1 enhancer-binding peptide was extended by nine consecutive arginine residues at the C-terminus and by the nuclear localization signal of SV40 large T antigen at the N-terminus. The resulting synthetic 64-residue peptide was found to bind to the two enhancers of the HIV-1 long terminal repeat, cross the plasma membrane and the nuclear envelope of human cells, and suppress the HIV-1 enhancer-controlled expression of a green fluorescent protein reporter gene. Moreover, HIV-1 replication is inhibited by this peptide in HIV-1-infected CEM-GFP cells as revealed by HIV-1 p24 ELISA and real-time RT-PCR of HIV-1 RNA. Rapid uptake of this intracellular stable and inhibitory peptide into the cells implies that this peptide may have the potential to attenuate HIV-1 replicationin vivo.

scholarly journals The C-terminal Domain Is the Primary Determinant of Histone H1 Binding to Chromatinin Vivo

2004 ◽  
Vol 279 (19) ◽  
pp. 20028-20034 ◽  
Author(s):  
Michael J. Hendzel ◽  
Melody A. Lever ◽  
Ellen Crawford ◽  
John P. H. Th'ng
Keyword(s):  
Amino Acid ◽  
Fluorescent Protein ◽  
Point Mutations ◽  
Histone H1 ◽  
Single Amino Acid ◽  
Kinetic Measurements ◽  
Terminal Domain ◽  
C Terminus ◽  
Green Fluorescent

We have used a combination of kinetic measurements and targeted mutations to show that the C-terminal domain is required for high-affinity binding of histone H1 to chromatin, and phosphorylations can disrupt binding by affecting the secondary structure of the C terminus. By measuring the fluorescence recovery after photo-bleaching profiles of green fluorescent protein-histone H1 proteins in living cells, we find that the deletion of the N terminus only modestly reduces binding affinity. Deletion of the C terminus, however, almost completely eliminates histone H1.1 binding. Specific mutations of the C-terminal domain identified Thr-152 and Ser-183 as novel regulatory switches that control the binding of histone H1.1in vivo. It is remarkable that the single amino acid substitution of Thr-152 with glutamic acid was almost as effective as the truncation of the C terminus to amino acid 151 in destabilizing histone H1.1 bindingin vivo. We found that modifications to the C terminus can affect histone H1 binding dramatically but have little or no influence on the charge distribution or the overall net charge of this domain. A comparison of individual point mutations and deletion mutants, when reviewed collectively, cannot be reconciled with simple charge-dependent mechanisms of C-terminal domain function of linker histones.

scholarly journals Utilization of green fluorescent protein as a marker for studying the expression and turnover of the monocarboxylate permease Jen1p of Saccharomyces cerevisiae

2002 ◽  
Vol 363 (3) ◽  
pp. 737-744 ◽  
Author(s):  
Sandra PAIVA ◽  
Arthur L. KRUCKEBERG ◽  
Margarida CASAL
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Reporter Protein ◽  
C Terminus ◽  
Lactate Transporter ◽  
Green Fluorescent ◽  
Endocytic Pathways

Green fluorescent protein (GFP) from Aequorea victoria was used as an in vivo reporter protein when fused to the C-terminus of the Jen1 lactate permease of Saccharomyces cerevisiae. The Jen1 protein tagged with GFP is a functional lactate transporter with a cellular abundance of 1670 molecules/cell, and a catalytic-centre activity of 123s−1. It is expressed and tagged to the plasma membrane under induction conditions. The factors involved in proper localization and turnover of Jen1p were revealed by expression of the Jen1p—GFP fusion protein in a set of strains bearing mutations in specific steps of the secretory and endocytic pathways. The chimaeric protein Jen1p—GFP is targeted to the plasma membrane via a Sec6-dependent process; upon treatment with glucose, it is endocytosed via END3 and targeted for degradation in the vacuole. Experiments performed in a Δdoa4 mutant strain showed that ubiquitination is associated with the turnover of the permease.

scholarly journals HIV-1 Vpr Oligomerization but Not That of Gag Directs the Interaction between Vpr and Gag

2009 ◽  
Vol 84 (3) ◽  
pp. 1585-1596 ◽  
Author(s):  
Joëlle V. Fritz ◽  
Denis Dujardin ◽  
Julien Godet ◽  
Pascal Didier ◽  
Jan De Mey ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Cell Physiology ◽  
Fluorescence Lifetime Imaging ◽  
M Cell ◽  
C Terminus ◽  
Green Fluorescent ◽  
Hiv 1

ABSTRACT During HIV-1 assembly, the viral protein R (Vpr) is incorporated into newly made viral particles via an interaction with the C-terminal domain of the Gag polyprotein precursor Pr55Gag. Vpr has been implicated in the nuclear import of newly made viral DNA and subsequently in its transcription. In addition, Vpr can affect the cell physiology by causing G2/M cell cycle arrest and apoptosis. Vpr can form oligomers, but their roles have not yet been investigated. We have developed fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer-based assays to monitor the interaction between Pr55Gag and Vpr in HeLa cells. To that end, we used enhanced green fluorescent protein-Vpr that can be incorporated into the virus and tetracysteine (TC)-tagged Pr55Gag-TC. This TC motif is tethered to the C terminus of Pr55Gag and does not interfere with Pr55Gag trafficking and the assembly of virus-like particles (VLPs). Results show that the Pr55Gag-Vpr complexes accumulated mainly at the plasma membrane. In addition, results with Pr55Gag-TC mutants confirm that the 41LXXLF domain of Gag-p6 is essential for Pr55Gag-Vpr interaction. We also report that Vpr oligomerization is crucial for Pr55Gag recognition and its accumulation at the plasma membrane. On the other hand, Pr55Gag-Vpr complexes are still formed when Pr55Gag carries mutations impairing its multimerization. These findings suggest that Pr55Gag-Vpr recognition and complex formation occur early during Pr55Gag assembly.

scholarly journals Crp79p, Like Mex67p, Is an Auxiliary mRNA Export Factor inSchizosaccharomyces pombe

2002 ◽  
Vol 13 (8) ◽  
pp. 2571-2584 ◽  
Author(s):  
Anjan G. Thakurta ◽  
William A. Whalen ◽  
Jin Ho Yoon ◽  
Anekella Bharathi ◽  
Libor Kozak ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Fluorescent Protein ◽  
Mrna Export ◽  
Nuclear Pore ◽  
Rna Recognition Motif ◽  
Synthetic Lethal ◽  
Export Activity ◽  
C Terminus ◽  
Green Fluorescent

The export of mRNA from the nucleus to the cytoplasm involves interactions of proteins with mRNA and the nuclear pore complex. We isolated Crp79p, a novel mRNA export factor from the same synthetic lethal screen that led to the identification of spMex67p inSchizosaccharomyces pombe. Crp79p is a 710-amino-acid-long protein that contains three RNA recognition motif domains in tandem and a distinct C-terminus. Fused to green fluorescent protein (GFP), Crp79p localizes to the cytoplasm. Like Mex67p, Crp79-GFP binds poly(A)+ RNA in vivo, shuttles between the nucleus and the cytoplasm, and contains a nuclear export activity at the C-terminus that is Crm1p-independent. All of these properties are essential for Crp79p to promote mRNA export. Crp79p import into the nucleus depends on the Ran system. A domain of spMex67p previously identified as having a nuclear export activity can functionally substitute for the nuclear export activity at the C-terminus of Crp79p. Although both Crp79p and spMex67p function to export mRNA, Crp79p does not substitute for all of spMex67p functions and probably is not a functional homologue of spMex67p. We propose that Crp79p is a nonessential mRNA export carrier in S. pombe.

scholarly journals In Vivo Inactivation of the Mycobacterial Integral Membrane Stearoyl Coenzyme A Desaturase DesA3 by a C-Terminus-Specific Degradation Process

2008 ◽  
Vol 190 (20) ◽  
pp. 6686-6696 ◽  
Author(s):  
Yong Chang ◽  
Gary E. Wesenberg ◽  
Craig A. Bingman ◽  
Brian G. Fox
Keyword(s):  
Coenzyme A ◽  
Fluorescent Protein ◽  
Degradation Process ◽  
Side Chains ◽  
Terminal Sequence ◽  
C Terminus ◽  
A Cell ◽  
Essential Enzyme ◽  
Green Fluorescent

ABSTRACT DesA3 (Rv3229c) from Mycobacterium tuberculosis is a membrane-bound stearoyl coenzyme A Δ9 desaturase that reacts with the oxidoreductase Rv3230c to produce oleic acid. This work provides evidence for a mechanism used by mycobacteria to regulate this essential enzyme activity. DesA3 expressed as a fusion with either a C-terminal His6 or c-myc tag had consistently higher activity and stability than native DesA3 having the native C-terminal sequence of LAA, which apparently serves as a binding determinant for a mycobacterial protease/degradation system directed at DesA3. Fusion of only the last 12 residues of native DesA3 to the C terminus of green fluorescent protein (GFP) was sufficient to make GFP unstable. Furthermore, the comparable C-terminal sequence from the Mycobacterium smegmatis DesA3 homolog Msmeg_1886 also conferred instability to the GFP fusion. Systematic examination revealed that residues with charged side chains, large nonpolar side chains, or no side chain at the last two positions were most important for stabilizing the construct, while lesser effects were observed at the third-from-last position. Using these rules, a combinational substitution of the last three residues of DesA3 showed that either DKD or LEA gave the best enhancement of stability for the modified GFP in M. smegmatis. Moreover, upon mutagenesis of LAA at the C terminus in native DesA3 to either of these tripeptides, the modified enzyme had enhanced catalytic activity and stability. Since many proteases are conserved within bacterial families, it is reasonable that M. tuberculosis will use a similar C-terminal degradation system to posttranslationally regulate the activity of DesA3 and other proteins. Application of these rules to the M. tuberculosis genome revealed that ∼10% the proteins encoded by essential genes may be susceptible to C-terminal proteolysis. Among these, an annotation is known for less than half, underscoring a general lack of understanding of proteins that have only temporal existence in a cell.

scholarly journals A co-translational model to explain the in vivo import of proteins into HeLa cell mitochondria

2004 ◽  
Vol 382 (1) ◽  
pp. 385-392 ◽  
Author(s):  
Abhijit MUKHOPADHYAY ◽  
Li NI ◽  
Henry WEINER
Keyword(s):  
Signal Peptide ◽  
Protein Import ◽  
Fluorescent Protein ◽  
Mitochondrial Protein ◽  
Major Pathway ◽  
Native Proteins ◽  
C Terminus ◽  
N Terminus ◽  
Green Fluorescent

The dual signal approach, i.e. a mitochondrial signal at the N-terminus and an ER (endoplasmic reticulum) or a peroxisomal signal at the C-terminus of EGFP (enhanced green fluorescent protein), was employed in transfected HeLa cells to test for a co-translational import model. The signal peptide from OTC (ornithine transcarbamylase) or arginase II was fused to the N-terminus of EGFP, and an ER or peroxisomal signal was fused to its C-terminus. The rationale was that if the free preprotein remained in the cytosol, it could be distributed between the two organelles by using a post-translational pathway. The resulting fusion proteins were imported exclusively into mitochondria, suggesting that co-translational import occurred. Native preALDH (precursor of rat liver mitochondrial aldehyde dehydrogenase), preOTC and rhodanese, each with the addition of a C-terminal ER or peroxisomal signal, were also translocated only to the mitochondria, again showing that a co-translational import pathway exists for these native proteins. Import of preALDHsp–DHFR, a fusion protein consisting of the leader sequence (signal peptide) of preALDH fused to DHFR (dihydrofolate reductase), was studied in the presence of methotrexate, a substrate analogue for DHFR. It was found that 70% of the preALDHsp–DHFR was imported into mitochondria in the presence of methotrexate, implying that 70% of the protein utilized the co-translational import pathway and 30% used the post-translational import pathway. Thus it appears that co-translational import is a major pathway for mitochondrial protein import. A model is proposed to explain how competition between binding factors could influence whether or not a cytosolic carrier protein, such as DHFR, uses the co- or post-translational import pathway.

Analysis ofChlamydomonasSF-assemblin by GFP tagging and expression of antisense constructs

2002 ◽  
Vol 115 (7) ◽  
pp. 1511-1522 ◽  
Author(s):  
Karl-Ferdinand Lechtreck ◽  
Jutta Rostmann ◽  
Andrea Grunow
Keyword(s):  
Elevated Temperatures ◽  
Fluorescent Protein ◽  
Heat Stability ◽  
Fiber Formation ◽  
Wild Type ◽  
C Terminus ◽  
Antisense Constructs ◽  
Flagellar Assembly ◽  
Green Fluorescent

Striated fiber assemblin (SF-assemblin or SFA) is the major component of the striated microtubule-associated fibers (SMAFs) in the flagellar basal apparatus of green flagellates. We generated nuclear transformants of Chlamydomonas expressing green fluorescent protein (GFP) fused to the C-terminus of SFA. SFA-GFP assembled into striated fibers that exceeded those of wild-type cells in size by several fold. At elevated temperatures(≥32°C) SFA-GFP was mostly soluble and heat shock depolymerized the SMAFs. C-terminal deletions of 18 or only six residues disturbed the ability of SFA-GFP to polymerize, indicating an important role of the C-terminal domain for fiber formation. The exchange of the penultimate Ser275 with alanine made SFA-GFP highly insoluble, causing aberrant fiber formation and conferring heat stability to the fibers. By contrast, a replacement with glutamic acid increased the solubilty of the molecule, indicating that phosphorylation on Ser275 might control solubility of SFA. In vivo observation of GFP fluorescence showed that SFA-GFP fibers were disassembled during mitosis. In cells overexpressing full-length or truncated SFA-GFP, the amount of wild-type protein was reduced. Elevated temperatures dissolved SFA-GFP fibers and induced the synthesis of SFA, suggesting that cells control both the amount of soluble and polymeric SFA. By expressing constructs consisting of cDNA and genomic DNA for parts of SFA in antiparallel configuration, the amount of SFA was severely reduced. In these strains we observed defects in flagellar assembly, indicating an important role for noncontractile striated roots in the flagella apparatus.

scholarly journals Localization and Functional Analysis of PepI, the Immunity Peptide of Pep5-Producing Staphylococcus epidermidis Strain 5

2004 ◽  
Vol 70 (6) ◽  
pp. 3263-3271 ◽  
Author(s):  
Anja Hoffmann ◽  
Tanja Schneider ◽  
Ulrike Pag ◽  
Hans-Georg Sahl
Keyword(s):  
Amino Acid ◽  
Staphylococcus Epidermidis ◽  
Fluorescent Protein ◽  
Terminal Segment ◽  
Terminal Part ◽  
C Terminus ◽  
Amino Acid Stretch ◽  
Green Fluorescent ◽  
Immunity Mechanism

ABSTRACT Pep5 is a cationic pore-forming lantibiotic produced by Staphylococcus epidermidis strain 5. The producer strain protects itself from the lethal action of its own bacteriocin through the 69-amino-acid immunity peptide PepI. The N-terminal segment of PepI contains a 20-amino-acid stretch of apolar residues, whereas the C terminus is very hydrophilic, with a net positive charge. We used green fluorescent protein (GFP)-PepI fusions to obtain information on its localization in vivo. PepI was found to occur outside the cytoplasm and to accumulate at the membrane-cell wall interface. The extracellular localization appeared essential for conferring immunity. We analyzed the functional role of the specific segments by constructing various mutant peptides, which were also fused to GFP. When the hydrophobic N-terminal segment of PepI was disrupted by introducing charged amino acids, the export of PepI was blocked and clones expressing such mutant peptides were Pep5 sensitive. When PepI was successively shortened at the C terminus, in contrast, its export properties remained unchanged whereas its ability to confer immunity was gradually reduced. The results show that the N-terminal part is required for the transport of PepI and that the C-terminal part is important for conferring the immunity phenotype. A concept based on target shielding is proposed for the PepI immunity mechanism.

scholarly journals Identification of HI-Like Loop in CELO Adenovirus Fiber for Incorporation of Receptor Binding Motifs

2007 ◽  
Vol 81 (18) ◽  
pp. 9641-9652 ◽  
Author(s):  
Denis Y. Logunov ◽  
Olga V. Zubkova ◽  
Anna S. Karyagina-Zhulina ◽  
Eugenia A. Shuvalova ◽  
Andrei P. Karpov ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Fluorescent Protein ◽  
Low Cost ◽  
Cell Types ◽  
Binding Motif ◽  
Recombinant Viruses ◽  
Rgd Motif ◽  
C Terminus ◽  
Green Fluorescent

ABSTRACT Vectors based on the chicken embryo lethal orphan (CELO) avian adenovirus (Ad) have two attractive properties for gene transfer applications: resistance to preformed immune responses to human Ads and the ability to grow in chicken embryos, allowing low-cost production of recombinant viruses. However, a major limitation of this technology is that CELO vectors demonstrate decreased efficiency of gene transfer into cells expressing low levels of the coxsackie-Ad receptor (CAR). In order to improve the efficacy of gene transfer into CAR-deficient cells, we modified viral tropism via genetic alteration of the CELO fiber 1 protein. The αv integrin-binding motif (RGD) was incorporated at two different sites of the fiber 1 knob domain, within an HI-like loop that we identified and at the C terminus. Recombinant fiber-modified CELO viruses were constructed containing secreted alkaline phosphatase (SEAP) and enhanced green fluorescent protein genes as reporter genes. Our data show that insertion of the RGD motif within the HI-like loop of the fiber resulted in significant enhancement of gene transfer into CAR-negative and CAR-deficient cells. In contrast, CELO vectors containing the RGD motif at the fiber 1 C terminus showed reduced transduction of all cell lines. CELO viruses modified with RGD at the HI-like loop transduced the SEAP reporter gene into rabbit mammary gland cells in vivo with an efficiency significantly greater than that of unmodified CELO vector and similar to that of Ad type 5 vector. These results illustrate the potential for efficient CELO-mediated gene transfer into a broad range of cell types through modification of the identified HI-like loop of the fiber 1 protein.

scholarly journals A Conserved Mechanism of Synaptogyrin Localization

2001 ◽  
Vol 12 (8) ◽  
pp. 2275-2289 ◽  
Author(s):  
Hongjuan Zhao ◽  
Michael L. Nonet
Keyword(s):  
Synaptic Vesicles ◽  
Hippocampal Neurons ◽  
Fluorescent Protein ◽  
Transmembrane Domain ◽  
Mutational Analysis ◽  
C Elegans ◽  
C Terminus ◽  
Green Fluorescent ◽  
Cultured Hippocampal Neurons

We have studied the localization of synaptogyrin family members in vivo. Both native and green fluorescent protein (GFP)-taggedCaenorhabditis elegans synaptogyrin (SNG-1) are expressed in neurons and synaptically localized. Deletion and mutational analysis with the use of GFP-tagged SNG-1 has defined a 38 amino acid sequence within the C terminus of SNG-1 and a single arginine in the cytoplasmic loop between transmembrane domain 2 and 3 that are required for SNG-1 localization. These domains may represent components of signals that target synaptogyrin for endocytosis from the plasma membrane and direct synaptogyrin to synaptic vesicles, respectively. In chimeric studies, these regions were sufficient to relocalize cellugyrin, a nonneuronal form of synaptogyrin, from nonsynaptic regions such as the sensory dendrites and the cell body to synaptic vesicles. Furthermore, GFP-tagged rat synaptogyrin is synaptically localized in neurons of C. elegans and in cultured hippocampal neurons. Similarly, the C-terminal domain of rat synaptogyrin is necessary for localization in hippocampal neurons. Our study suggests that the mechanisms for synaptogyrin localization are likely to be conserved from C. elegans to vertebrates.

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