scholarly journals The bioactivity of soluble Fas ligand is modulated by key amino acids of its stalk region

PLoS ONE ◽  
2021 ◽  
Vol 16 (6) ◽  
pp. e0253260
Author(s):  
Osamu Kajikawa ◽  
Raquel Herrero ◽  
Yu-Hua Chow ◽  
Chi F. Hung ◽  
Gustavo Matute-Bello
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
T Cell ◽  
Epithelial Cells ◽  
Cell Line ◽  
Fas Ligand ◽  
Wild Type ◽  
Soluble Fas ◽  
Charged Amino Acids ◽  
Soluble Fas Ligand

We have previously reported that the 26-amino acid N-terminus stalk region of soluble Fas ligand (sFasL), which is separate from its binding site, is required for its biological function. Here we investigate the mechanisms that link the structure of the sFasL stalk region with its function. Using site-directed mutagenesis we cloned a mutant form of sFasL in which all the charged amino acids of the stalk region were changed to neutral alanines (mut-sFasL). We used the Fas-sensitive Jurkat T-cell line and mouse and human alveolar epithelial cells to test the bioactivity of sFasL complexes, using caspase-3 activity and Annexin-V externalization as readouts. Finally, we tested the effects of mut-sFasL on lipopolysaccharide-induced lung injury in mice. We found that mutation of all the 8 charged amino acids of the stalk region into the non-charged amino acid alanine (mut-sFasL) resulted in reduced apoptotic activity compared to wild type sFasL (WT-sFasL). The mut-sFasL attenuated WT-sFasL function on the Fas-sensitive human T-cell line Jurkat and on primary human small airway epithelial cells. The inhibitory mechanism was associated with the formation of complexes of mut-sFasL with the WT protein. Intratracheal administration of the mut-sFasL to mice 24 hours after intratracheal Escherichia coli lipopolysaccharide resulted in attenuation of the inflammatory response 24 hours later. Therefore, the stalk region of sFasL has a critical role on bioactivity, and changes in the structure of the stalk region can result in mutant variants that interfere with the wild type protein function in vitro and in vivo.

scholarly journals Two site-directed mutations abrogate enzyme activity but have different effects on the conformation and cellular content of the N-acetylgalactosamine 4-sulphatase protein

1995 ◽  
Vol 307 (2) ◽  
pp. 457-463 ◽  
Author(s):  
D A Brooks ◽  
D A Robertson ◽  
C Bindloss ◽  
T Litjens ◽  
D S Anson ◽  
...  
Keyword(s):  
Amino Acids ◽  
Enzyme Activity ◽  
Amino Acid ◽  
Cell Line ◽  
Protein Level ◽  
Structural Modification ◽  
Wild Type ◽  
Cellular Content ◽  
Control Protein ◽  
Wild Type Control

The sulphatase family of enzymes have regions of sequence similarity, but relatively little is known about either the structure-function relationships of sulphatases, or the role of highly conserved amino acids. The sequence of amino acids CTPSR at position 91-95 of 4-sulphatase has been shown to be highly conserved in all of the sequenced sulphatase enzymes. The cysteine at amino acid 91 of 4-sulphatase was selected for mutation analysis due to its potential role in either the active site, substrate-binding site or part of a key structural domain of 4-sulphatase and due to the absence of naturally occurring mutations in this residue in mucopolysaccharidosis type VI (MPS VI) patients. Two mutations, C91S and C91T, altering amino acid 91 of 4-sulphatase were generated and expressed in Chinese hamster ovary cells. Biochemical analysis of protein from a C91S cell line demonstrated no detectable 4-sulphatase enzyme activity but a relatively normal level of 4-sulphatase polypeptide (180% of the wild-type control protein level). Epitope detection, using a panel of ten monoclonal antibodies, demonstrated that the C91S polypeptide had a similar immunoreactivity to wild-type 4-sulphatase, suggesting that the C91S substitution does not induce a major structural change in the protein. Reduced catalytic activity associated with normal levels of 4-sulphatase protein have not been observed in any of the MPS VI patients tested and all show evidence of structural modification of 4-sulphatase protein with the same panel of antibodies [Brooks, McCourt, Gibson, Ashton, Shutter and Hopwood (1991) Am. J. Hum. Genet. 48, 710-719]. The loss of enzyme activity without a detectable protein conformation change suggests that Cys-91 may be a critical residue in the catalytic process. In contrast, analysis of protein from a C91T cell line revealed low levels of catalytically inactive 4-sulphatase polypeptide (0.37% of the wild-type control protein level) which had missing or masked epitopes, suggesting an altered protein structure or conformation. Subcellular fractionation studies of the C91T cell line demonstrated a high proportion of 4-sulphatase polypeptide content in organelles characteristic of microsomes. The aberrant intracellular localization and the reduced cellular content of 4-sulphatase polypeptide was consistent with the observed structural modification leading to retention and degradation of the protein within an early vacuolar compartment.

Amino Acid Uptake by Amino Acid Analog Resistant Tobacco Cell Lines

1978 ◽  
Vol 33 (9-10) ◽  
pp. 634-640 ◽  
Author(s):  
Jochen Berlin ◽  
Jade M. Widholm
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Cell Line ◽  
Transport System ◽  
Amino Acid Uptake ◽  
Resistant Cell ◽  
Resistant Cell Line ◽  
Acid Uptake ◽  
Wild Type ◽  
Phenylalanine Transport

Abstract Two tobacco cell lines resistant to p-fiuorophenylalanine (PFP) and one resistant to 5-methyltryptophan (5-MT) are compared with wild type cells in their ability to absorb amino acids from the medium. One p-fluorophenylalanine-resistant cell line shows greatly reduced uptake of all amino acids so is resistant to growth inhibition by other amino acid analogs. The impaired absorption is noted with amino acids, amino acid analogs and shikimate, but not with cinnamate, salicylate, nicotine, glucose, 3-O-methylglucose and palmitate. The phenylalanine transport system of the PFP-resistant cell line and the wild type both have Km values of 90 µᴍ, but have different V max values. Several analogs of phenylalanine and several neutral L-amino acids inhibit the phenylalanine transport system, while ʟ-aspartic acid, ʟ-arginine, ᴅ-phenylalanine or chlorogenic acid do not interfere with the ʟ-phenylalanine uptake. The results indicate the presence of more than one transport system for amino acid uptake. The lessened uptake of all amino acids, the specificity of the uptake systems and the unchanged binding let us conclude that a pleiotropic mutation or that some inhibitor causes the reduced uptake of all amino acids by the PFP-resistant cell line.

scholarly journals Molecular and Cellular Analysis of Human Immunodeficiency Virus-Induced Apoptosis in Lymphoblastoid T-Cell-Line-Expressing Wild-Type and Mutated CD4 Receptors

1998 ◽  
Vol 72 (10) ◽  
pp. 8061-8072 ◽  
Author(s):  
Laure Moutouh ◽  
Jérôme Estaquier ◽  
Douglas D. Richman ◽  
Jacques Corbeil
Keyword(s):  
Human Immunodeficiency Virus ◽  
T Cell ◽  
Cell Line ◽  
Fas Ligand ◽  
Cytoplasmic Tail ◽  
Wild Type ◽  
Ligand Interaction ◽  
Immunodeficiency Virus ◽  
Infected Cells ◽  
Induced Apoptosis

ABSTRACT We have previously shown that the presence of the CD4 cytoplasmic tail is critical for human immunodeficiency virus (HIV)-induced apoptosis (J. Corbeil, M. Tremblay, and D. D. Richman, J. Exp. Med. 183:39–48, 1996). We have pursued our investigation of the role of the CD4 transduction pathway in HIV-induced apoptosis. To do this, wild-type and mutant forms of the CD4 cytoplasmic tail were stably expressed in the lymphoblastoid T-cell line A2.01. Apoptosis was prevented when CD4 truncated at residue 402 was expressed; however, cells expressing mutated receptors that do not associate with p56 lck (mutated at the dicysteine motif and truncated at residue 418) but which conserved proximal domains of the cytoplasmic tail underwent apoptosis like wild-type CD4. The differences between wild-type and mutated receptors in the induction of apoptosis were not related to levels of p56 lck or NF-κB activation. Initial signaling through the CD4 receptor played a major role in the sensitization of HIV-infected T cells to undergo apoptosis. Incubation of HIV-infected cells with monoclonal antibody (MAb) 13B8-2, which binds to CD4 in a region critical for dimerization of the receptor, prevented apoptosis without inhibiting HIV replication. Moreover, the apoptotic process was not related to Fas-Fas ligand interaction; however, an antagonistic anti-Fas MAb (ZB-4) enhanced apoptosis in HIV-infected cells without inducing apoptosis in uninfected cells. These observations demonstrate that CD4 signaling mediates HIV-induced apoptosis by a mechanism independent of Fas-Fas ligand interaction, does not require p56 lck signaling, and may involve a critical region for CD4 dimerization.

scholarly journals Increased Levels of Soluble Fas Ligand in Serum inPlasmodium falciparum Malaria

2000 ◽  
Vol 68 (5) ◽  
pp. 3061-3063 ◽  
Author(s):  
Peter Kern ◽  
Manfred Dietrich ◽  
Christoph Hemmer ◽  
Nele Wellinghausen
Keyword(s):  
Plasmodium Falciparum ◽  
T Cell ◽  
Fas Ligand ◽  
Significant Decline ◽  
Disease Course ◽  
Antimalarial Treatment ◽  
Soluble Fas ◽  
Cell Counts ◽  
Soluble Fas Ligand ◽  
Induced Apoptosis

ABSTRACT Levels of soluble Fas ligand (sFasL) in serum were elevated in patients with Plasmodium falciparum malaria and showed a significant decline during disease course. sFasL levels that were elevated before antimalarial treatment began correlated significantly with depressed total lymphocyte and T-cell counts. These data suggest that Fas-induced apoptosis might play a role in malaria-associated lymphopenia.

Identification of a novel signal in the cytoplasmic tail of the Na+:HCO3− cotransporter NBC1 that mediates basolateral targeting

2007 ◽  
Vol 292 (4) ◽  
pp. F1245-F1255 ◽  
Author(s):  
Hong C. Li ◽  
Emily Y. Li ◽  
Lisa Neumeier ◽  
Laura Conforti ◽  
Manoocher Soleimani
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Epithelial Cells ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Cytoplasmic Tail ◽  
Membrane Targeting ◽  
Wild Type ◽  
Functional Studies ◽  
Basolateral Targeting

The Na+:HCO3− cotransporter NBC1 (SLC4A4, variant A, kidney specific) is located exclusively on the basolateral membrane of epithelial cells, implying that this molecule has acquired specific signals for targeting to the basolateral membrane. A motif with the sequence QQPFLS (positions 1010–1015) in the cytoplasmic tail of NBC1 was recently demonstrated to mediate targeting of NBC1 to the basolateral membrane. Here, we demonstrate that mutating the amino acid F (phenylalanine) or L (leucine) at positions 1013 or 1014 to alanine, respectively, resulted in the retargeting of NBC1 to the apical membrane. Furthermore, mutation of the FL motif to FF showed similar properties as the wild-type; however, mutation of the FL motif to LL showed significant intracellular retention of NBC1. Mutating the amino acids Q-Q-P and S (positions 1010-1011-1012 and 1015) to A-A-A and A, respectively, did not affect the membrane targeting of NBC1. Functional studies in oocytes with microelectrode demonstrated that the apically targeted mutants, as well as basolaterally targeted mutants, are all functional. We propose that the FL motif in the COOH-terminal tail of NBC1 is essential for the targeting of NBC1 to the basolateral membrane but is distinct from the membrane-targeting di-leucine motif identified in other membrane proteins.

Abstract WP104: Neuron- microglia Crosstalk Mediated by Soluble Fas Ligand after Ischemic Stroke

Stroke ◽  
2016 ◽  
Vol 47 (suppl_1) ◽  
Author(s):  
Hailan Meng ◽  
Xiaoxi Li ◽  
Dan Ye ◽  
Leihua Weng ◽  
Yanting Chen ◽  
...  
Keyword(s):  
Conditioned Medium ◽  
Neuronal Death ◽  
Fas Ligand ◽  
Neutralizing Antibody ◽  
Wild Type ◽  
Ischemic Brain ◽  
Microglial Polarization ◽  
Soluble Fas ◽  
Microglia Polarization ◽  
Soluble Fas Ligand

Objectives: Accumulating data indicate that soluble Fas Ligand (sFasL) is involved in inflammatory cascades after cerebral ischemia. However, the potential role of sFasL in modulating the neuron-microglia crosstalk is barely understood. We hypothesized that sFasL triggers neuronal death and M1-microglia polarization after ischemic injury. Methods: Middle cerebral artery occlusion (MCAO) was induced in both FasL mutant gld mice and wild-type C57BL/6J mice. Microglial polarization was determined by immunohistochemical staining and real-time polymerase chain reaction. Expression of sFasL was detected by western blot. The expression of sFasL in the medium after OGD was detected by ELISA assay. Activity of sFasL was blocked by using its neutralizing antibody. Neuronal survival and apoptosis were quantified. Results: Expression of sFasL at protein level in the ischemic cortex was increased by 451.67% in wild-type stroke mice over the sham-operated brain. The gld mice demonstrated attenuated neuronal death and M1-microglial phenotype markers (iNOS and CD86) compared to wild-type mice after MCAO. Protein expression of sFasL was increased both in the neuronal and microglial culture medium after OGD. In addition, conditioned medium obtained from ischemic neuron drove microglial polarization toward the M1 phenotype producing more pro-inflammatory cytokines (IL-1β, TNF-α and MCP-1). Meanwhile, conditioned medium from ischemic microglia exacerbated neuronal death dramatically. sFasL neutralizing antibody, however, significantly decreased these changes. Furthermore, phosphorylated JAK2/STAT3(Y705 and S727) was increased in microglia after neuronal-conditioned medium treatment, which was decreased following inhibition of sFasL activity. Conclusions: The gld mice demonstrated attenuated ischemic brain damage. sFasL is involved in modulating the crosstalk between these two cells in a contact-independent way. Microglia-released sFasL exacerbated neuronal death while neuron-released sFasL induced M1-microglia polarization after ischemic stroke. Neutralization of sFasL, thereby, may protect against ischemic brain damage and provide novel views on post-stroke neuroimmune regulation.

scholarly journals Soluble FAS Ligand directs human memory B cells towards antibody-secreting cells

2020 ◽  
Author(s):  
Saskia D. van Asten ◽  
Peter-Paul Unger ◽  
Casper Marsman ◽  
Sophie Bliss ◽  
Tineke Jorritsma ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Fas Ligand ◽  
Memory B Cells ◽  
B Cell Differentiation ◽  
Soluble Fas ◽  
Soluble Fas Ligand ◽  
Antibody Secreting Cells

AbstractDifferentiation of antigen-specific B cells into class-switched, high affinity antibody-secreting cells provides protection against invading pathogens but is undesired when antibodies target self-tissues in autoimmunity, beneficial non-self blood transfusion products or therapeutic proteins. Essential T cell factors have been uncovered that regulate T cell-dependent B cell differentiation. We performed a screen using a secreted protein library to identify novel factors that promote this process and may be used to combat undesired antibody formation. We tested the differentiating capacity of 756 secreted proteins on human naive or memory B cell differentiation in a setting with suboptimal T cell help in vitro (suboptimal CD40L and IL-21). High-throughput flow cytometry screening and validation revealed that type I IFNs and soluble FAS ligand (sFASL) induce plasmablast differentiation in memory B cells. Furthermore, sFASL induces robust secretion of IgG1 and IgG4 antibodies, indicative of functional plasma cell differentiation. Our data suggest a mechanistic connection between elevated sFASL levels and the induction of autoreactive antibodies, providing a potential therapeutic target in autoimmunity. Indeed, the modulators identified in this secretome screen are associated with systemic lupus erythematosus and may also be relevant in other autoimmune diseases and allergy.

scholarly journals Primary structure requirements for correct sorting of the yeast mitochondrial protein ADH III to the yeast mitochondrial matrix space.

1987 ◽  
Vol 7 (1) ◽  
pp. 294-304 ◽  
Author(s):  
D Pilgrim ◽  
E T Young
Keyword(s):  
Amino Acids ◽  
Enzyme Activity ◽  
Amino Acid ◽  
Proteolytic Processing ◽  
Mitochondrial Matrix ◽  
Wild Type ◽  
Mature Form ◽  
Amino Terminal ◽  
The Matrix

Alcohol dehydrogenase isoenzyme III (ADH III) in Saccharomyces cerevisiae, the product of the ADH3 gene, is located in the mitochondrial matrix. The ADH III protein was synthesized as a larger precursor in vitro when the gene was transcribed with the SP6 promoter and translated with a reticulocyte lysate. A precursor of the same size was detected when radioactively pulse-labeled proteins were immunoprecipitated with anti-ADH antibody. This precursor was rapidly processed to the mature form in vivo with a half-time of less than 3 min. The processing was blocked if the mitochondria were uncoupled with carbonyl cyanide m-chlorophenylhydrazone. Mutant enzymes in which only the amino-terminal 14 or 16 amino acids of the presequence were retained were correctly targeted and imported into the matrix. A mutant enzyme that was missing the amino-terminal 17 amino acids of the presequence produced an active enzyme, but the majority of the enzyme activity remained in the cytoplasmic compartment on cellular fractionation. Random amino acid changes were produced in the wild-type presequence by bisulfite mutagenesis of the ADH3 gene. The resulting ADH III protein was targeted to the mitochondria and imported into the matrix in all of the mutants tested, as judged by enzyme activity. Mutants containing amino acid changes in the carboxyl-proximal half of the ADH3 presequence were imported and processed to the mature form at a slower rate than the wild type, as judged by pulse-chase studies in vivo. The unprocessed precursor appeared to be unstable in vivo. It was concluded that only a small portion of the presequence contains the necessary information for correct targeting and import. Furthermore, the information for correct proteolytic processing of the presequence appears to be distinct from the targeting information and may involve secondary structure information in the presequence.

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