scholarly journals Small expression tags enhance bacterial expression of the first three transmembrane segments of the apelin receptor

2014 ◽  
Vol 92 (4) ◽  
pp. 269-278 ◽  
Author(s):  
Aditya Pandey ◽  
Muzaddid Sarker ◽  
Xiang-Qin Liu ◽  
Jan K. Rainey
Keyword(s):  
High Performance ◽  
E Coli ◽  
Transmembrane Segments ◽  
Physiological Processes ◽  
Membrane Mimetic ◽  
Tumour Formation ◽  
Immunodeficiency Virus ◽  
Apelin Receptor ◽  
G Protein Coupled

G-protein coupled receptors (GPCRs) are inherently dynamic membrane protein modulators of various important cellular signaling cascades. The apelin receptor (AR or APJ) is a class A GPCR involved in numerous physiological processes, implicated in angiogenesis during tumour formation and as a CD4 co-receptor for entry of human immunodeficiency virus type 1 (HIV-1) to cells. Due to the lack of efficient methods to produce full-length GPCRs enriched with nuclear magnetic resonance (NMR) active 15N, 13C, and (or) 2H isotopes, small GPCR fragments typically comprising 1–2 transmembrane segments are frequently studied using NMR spectroscopy. Here, we report successful overexpression of transmembrane segments 1–3 of AR (AR_TM1-3) in the C41(DE3) strain of Escherichia coli using an AT-rich gene tag previously reported to enhance cell-free expression yields. The resulting protein, with 6 additional N-terminal residues due to the expression tag, was purified using high-performance liquid chromatography (HPLC). Far UV circular dichroism spectropolarimetry demonstrates that AR_TM1-3 has the predicted ∼40% α-helical character in membrane-mimetic environments. 1H-15N HSQC NMR experiments imply amenability to high-resolution NMR structural characterization and stability in solution for weeks. Notably, this small expression tag approach may also be generally applicable to other membrane proteins that are difficult to express in E. coli.

A Synthetic Peptide Derived From Human Immunodeficiency Virus Type 1 gp120 Downregulates the Expression and Function of Chemokine Receptors CCR5 and CXCR4 in Monocytes by Activating the 7-Transmembrane G-Protein–Coupled Receptor FPRL1/LXA4R

Blood ◽  
1999 ◽  
Vol 94 (4) ◽  
pp. 1165-1173
Author(s):  
Xiyun Deng ◽  
Hirotsugu Ueda ◽  
Shao Bo Su ◽  
Wanghua Gong ◽  
Nancy M. Dunlop ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
G Protein ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Synthetic Peptide ◽  
Immunodeficiency Virus ◽  
And Function ◽  
G Protein Coupled ◽  
Hiv 1

Because envelope gp120 of various strains of human immunodeficiency virus type 1 (HIV-1) downregulates the expression and function of a variety of chemoattractant receptors through a process of heterologous desensitization, we investigated whether epitopes derived from gp120 could mimic the effect. A synthetic peptide domain, designated F peptide, corresponding to amino acid residues 414-434 in the V4-C4 region of gp120 of the HIV-1 Bru strain, potently reduced monocyte binding and chemotaxis response to macrophage inflammatory protein 1β (MIP-1β) and stromal cell-derived factor 1 (SDF-1), chemokines that use the receptors CCR5 and CXCR4, respectively. Further study showed that F peptide by itself is an inducer of chemotaxis and calcium mobilization in human monocytes and neutrophils. In cross-desensitization experiments, among the numerous chemoattractants tested, only the bacterial chemotactic peptide fMLF, when used at high concentrations, partially attenuated calcium mobilization induced by F peptide in phagocytes, suggesting that this peptide domain might share a 7-transmembrane, G-protein–coupled receptor with fMLF. By using cells transfected with cDNAs encoding receptors that interact with fMLF, we found that F peptide uses an fMLF receptor variant, FPRL1, as a functional receptor. The activation of monocytes by F peptide resulted in downregulation of the cell surface expression of CCR5 and CXCR4 in a protein kinase C-dependent manner. These results demonstrate that activation of FPRL1 on human moncytes by a peptide domain derived from HIV-1 gp120 could lead to desensitization of cell response to other chemoattractants. This may explain, at least in part, the initial activation of innate immune responses in HIV-1–infected patients followed by immune suppression.

scholarly journals Microglia Express CCR5, CXCR4, and CCR3, but of These, CCR5 Is the Principal Coreceptor for Human Immunodeficiency Virus Type 1 Dementia Isolates

1999 ◽  
Vol 73 (1) ◽  
pp. 205-213 ◽  
Author(s):  
Andrew V. Albright ◽  
Joseph T. C. Shieh ◽  
Takayuki Itoh ◽  
Benhur Lee ◽  
David Pleasure ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Chemokine Receptors ◽  
Calcium Flux ◽  
Human Adult ◽  
Immunodeficiency Virus ◽  
The Central Nervous System ◽  
Hiv Entry ◽  
G Protein Coupled ◽  
Hiv 1

ABSTRACT Microglia are the main human immunodeficiency virus (HIV) reservoir in the central nervous system and most likely play a major role in the development of HIV dementia (HIVD). To characterize human adult microglial chemokine receptors, we analyzed the expression and calcium signaling of CCR5, CCR3, and CXCR4 and their roles in HIV entry. Microglia expressed higher levels of CCR5 than of either CCR3 or CXCR4. Of these three chemokine receptors, only CCR5 and CXCR4 were able to transduce a signal in microglia in response to their respective ligands, MIP-1β and SDF-1α, as recorded by single-cell calcium flux experiments. We also found that CCR5 is the predominant coreceptor used for infection of human adult microglia by the HIV type 1 dementia isolates HIV-1DS-br, HIV-1RC-br, and HIV-1YU-2, since the anti-CCR5 antibody 2D7 was able to dramatically inhibit microglial infection by both wild-type and single-round luciferase pseudotype reporter viruses. Anti-CCR3 (7B11) and anti-CXCR4 (12G5) antibodies had little or no effect on infection. Last, we found that virus pseudotyped with the DS-br and RC-br envelopes can infect cells transfected with CD4 in conjunction with the G-protein-coupled receptors APJ, CCR8, and GPR15, which have been previously implicated in HIV entry.

scholarly journals Penetration of dapsone into pulmonary lining fluid of human immunodeficiency virus type 1-infected patients.

1997 ◽  
Vol 41 (5) ◽  
pp. 1077-1081 ◽  
Author(s):  
M Cruciani ◽  
G Gatti ◽  
C Mengoli ◽  
A Cazzadori ◽  
L Lazzarini ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
High Performance ◽  
Pneumocystis Carinii ◽  
Primary Prophylaxis ◽  
Apparent Volume ◽  
Prophylactic Regimen ◽  
Immunodeficiency Virus

We studied the penetration of dapsone into the epithelial lining fluid (ELF) of sixteen human immunodeficiency virus type 1-infected patients who had received the drug at a dose of 100 mg twice weekly as primary prophylaxis for Pneumocystis carinii pneumonia. Bronchoscopy, bronchoalveolar lavage (BAL), and venipuncture were performed for each patient at a specific time after administration of the last dose of dapsone. Dapsone concentrations in plasma and BAL were determined by high-performance liquid chromatography. The apparent volume of ELF recovered by BAL was determined by using urea as an endogenous marker. The mean concentrations of dapsone in ELF at 2 h (five patients), 4 h (three patients), 12 h (two patients), 24 h (three patients), and 48 h (three patients) were 0.95, 0.70, 1.55, 0.23, and 0.45 mg/liter, respectively, while concentrations in plasma were 1.23, 0.79, 1.31, 0.83, and 0.18 mg/liter, respectively. Dapsone concentrations in ELF were 76, 79, 115, 65, and 291% of those observed in plasma at the same times, respectively. These data show that dapsone is well distributed into ELF and that a twice-weekly 100-mg prophylactic regimen results in sustained concentrations in this compartment.

scholarly journals Depressed Phagocytosis and Oxidative Burst in Polymorphonuclear Leukocytes from Individuals with Pulmonary Tuberculosis with or without Human Immunodeficiency Virus Type 1 Infection

1998 ◽  
Vol 5 (1) ◽  
pp. 41-44 ◽  
Author(s):  
Sharon Shalekoff ◽  
Caroline T. Tiemessen ◽  
Clive M. Gray ◽  
Desmond J. Martin
Keyword(s):  
Human Immunodeficiency Virus ◽  
Pulmonary Tuberculosis ◽  
Oxidative Burst ◽  
Whole Blood ◽  
Opportunistic Infections ◽  
Oxygen Intermediates ◽  
E Coli ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Phagocytosis and oxidative burst in whole-blood granulocytes were assessed by flow cytometry with Phagotest and Bursttest kits, respectively. Seventy individuals were included in this study: 15 healthy, normal donors, 18 human immunodeficiency virus (HIV) type 1 (HIV-1)-seropositive patients, 19 patients with pulmonary tuberculosis (TB), and 18 patients co-infected with Mycobacterium tuberculosis and HIV-1 (TB-HIV). Granulocyte phagocytosis was assessed by incubating whole blood with fluorescence-labelledEscherichia coli and measuring the proportion of granulocytes with ingested bacteria and the capacity (fluorescence intensity) of each cell to phagocytose E. coli. The percentage of granulocytes converting nonfluorescent dihydrorhodamine to fluorescent rhodamine 123 on production of reactive oxygen intermediates (ROIs) and the mean channel shift were assessed as a measure of oxidative burst. No differences in the proportion of granulocytes that were capable of phagocytosing or producing ROIs in response to E. coli were observed between any of the study groups. Phagocytosis was significantly enhanced in granulocytes from HIV-1-infected individuals. On the other hand, granulocytes from individuals infected with M. tuberculosis alone or in combination with HIV-1 had a significantly reduced capacity to phagocytose E. coli and to produce ROIs in response toE. coli as an agonist. These results provide evidence that granulocytes from individuals with pulmonary TB with or without concomitant infection with HIV-1 have an impaired ability to phagocytose and to undergo oxidative burst, possibly contributing to the enhanced susceptibility to opportunistic infections in these patients.

scholarly journals The Role of GPR15 Function in Blood and Vasculature

2021 ◽  
Vol 22 (19) ◽  
pp. 10824
Author(s):  
Mario Bauer
Keyword(s):  
Vascular Endothelial Cells ◽  
Cell Types ◽  
Vascular Endothelial ◽  
Cytoprotective Effect ◽  
New Findings ◽  
Immunodeficiency Virus ◽  
Hiv Type 1 ◽  
G Protein Coupled

Since the first prominent description of the orphan G protein-coupled receptor 15 (GPR15) on lymphocytes as a co-receptor for the human immunodeficiency virus (HIV) type 1 and 2 and the first report about the GPR15-triggered cytoprotective effect on vascular endothelial cells by recombinant human thrombomodulin, several decades passed before the GPR15 has been recently deorphanized. Because of new findings on GPR15, this review will summarize the consequences of GPR15 signaling considering the variety of GPR15-expressing cell types and of GPR15 ligands, with a focus on blood and vasculature.

scholarly journals Allosteric Modulation of Cannabinoid Receptor 1—Current Challenges and Future Opportunities

2019 ◽  
Vol 20 (23) ◽  
pp. 5874 ◽  
Author(s):  
Hryhorowicz ◽  
Kaczmarek-Ryś ◽  
Andrzejewska ◽  
Staszak ◽  
Hryhorowicz ◽  
...  
Keyword(s):  
Cannabinoid Receptors ◽  
Cannabinoid Receptor ◽  
Memory Loss ◽  
Allosteric Modulation ◽  
Site Directed Mutagenesis ◽  
New Horizons ◽  
X Ray Crystallography ◽  
Physiological Processes ◽  
G Protein Coupled

The cannabinoid receptor type 1 (CB1R), a G protein-coupled receptor (GPCR), plays an essential role in the control of many physiological processes such as hunger, memory loss, gastrointestinal activity, catalepsy, fear, depression, and chronic pain. Therefore, it is an attractive target for drug discovery to manage pain, neurodegenerative disorders, obesity, and substance abuse. However, the psychoactive adverse effects, generated by CB1R activation in the brain, limit the use of the orthosteric CB1R ligands as drugs. The discovery of CB1R allosteric modulators during the last decade provided new tools to target the CB1R. Moreover, application of the site-directed mutagenesis in combination with advanced physical methods, especially X-ray crystallography and computational modeling, has opened new horizons for understanding the complexity of the structure, function, and activity of cannabinoid receptors. In this paper, we present the latest advances in research on the CB1R, its allosteric modulation and allosteric ligands, and their translational potential. We focused on structural essentials of the cannabinoid 1 receptor- ligand (drug) interactions, as well as modes of CB1R signaling regulation.

A Synthetic Peptide Derived From Human Immunodeficiency Virus Type 1 gp120 Downregulates the Expression and Function of Chemokine Receptors CCR5 and CXCR4 in Monocytes by Activating the 7-Transmembrane G-Protein–Coupled Receptor FPRL1/LXA4R

Blood ◽  
1999 ◽  
Vol 94 (4) ◽  
pp. 1165-1173 ◽  
Author(s):  
Xiyun Deng ◽  
Hirotsugu Ueda ◽  
Shao Bo Su ◽  
Wanghua Gong ◽  
Nancy M. Dunlop ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
G Protein ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Synthetic Peptide ◽  
Immunodeficiency Virus ◽  
And Function ◽  
G Protein Coupled ◽  
Hiv 1

Abstract Because envelope gp120 of various strains of human immunodeficiency virus type 1 (HIV-1) downregulates the expression and function of a variety of chemoattractant receptors through a process of heterologous desensitization, we investigated whether epitopes derived from gp120 could mimic the effect. A synthetic peptide domain, designated F peptide, corresponding to amino acid residues 414-434 in the V4-C4 region of gp120 of the HIV-1 Bru strain, potently reduced monocyte binding and chemotaxis response to macrophage inflammatory protein 1β (MIP-1β) and stromal cell-derived factor 1 (SDF-1), chemokines that use the receptors CCR5 and CXCR4, respectively. Further study showed that F peptide by itself is an inducer of chemotaxis and calcium mobilization in human monocytes and neutrophils. In cross-desensitization experiments, among the numerous chemoattractants tested, only the bacterial chemotactic peptide fMLF, when used at high concentrations, partially attenuated calcium mobilization induced by F peptide in phagocytes, suggesting that this peptide domain might share a 7-transmembrane, G-protein–coupled receptor with fMLF. By using cells transfected with cDNAs encoding receptors that interact with fMLF, we found that F peptide uses an fMLF receptor variant, FPRL1, as a functional receptor. The activation of monocytes by F peptide resulted in downregulation of the cell surface expression of CCR5 and CXCR4 in a protein kinase C-dependent manner. These results demonstrate that activation of FPRL1 on human moncytes by a peptide domain derived from HIV-1 gp120 could lead to desensitization of cell response to other chemoattractants. This may explain, at least in part, the initial activation of innate immune responses in HIV-1–infected patients followed by immune suppression.

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