scholarly journals KISS1R Intracellular Trafficking and Degradation: Effect of the Arg386Pro Disease-Associated Mutation

Endocrinology ◽  
2011 ◽  
Vol 152 (4) ◽  
pp. 1616-1626 ◽  
Author(s):  
Suzy D. C. Bianco ◽  
Lauren Vandepas ◽  
Mayrin Correa-Medina ◽  
Balázs Gereben ◽  
Abir Mukherjee ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Intracellular Trafficking ◽  
Proteasome Inhibitor ◽  
Central Precocious Puberty ◽  
Confocal Imaging ◽  
Time Dependent ◽  
Relative Distribution ◽  
Wild Type ◽  
Protein Levels ◽  
Degradation Effect

Abstract The goal of this study was to investigate how the Arg386Pro mutation prolongs KiSS-1 receptor (KISS1R) responsiveness to kisspeptin, contributing to human central precocious puberty. Confocal imaging showed colocalization of wild-type (WT) KISS1R with a membrane marker, which persisted for up to 5 h of stimulation. Conversely, no colocalization with a lysosome marker was detected. Also, overnight treatment with a lysosome inhibitor did not affect WT KISS1R protein, whereas overnight treatment with a proteasome inhibitor increased protein levels by 24-fold. WT and Arg386Pro KISS1R showed time-dependent internalization upon stimulation. However, both receptors were recycled back to the membrane. The Arg386Pro mutation did not affect the relative distribution of KISS1R in membrane and internalized fractions when compared to WT KISS1R for up to 120 min of stimulation, demonstrating that this mutation does not affect KISS1R trafficking rate. Nonetheless, total Arg386Pro KISS1R was substantially increased compared with WT after 120 min of kisspeptin stimulation. This net increase was eliminated by blockade of detection of recycled receptors, demonstrating that recycled receptors account for the increased responsiveness of this mutant to kisspeptin. We therefore conclude the following: 1) WT KISS1R is degraded by proteasomes rather than lysosomes; 2) WT and Arg386Pro KISS1R are internalized upon stimulation, but most of the internalized receptors are recycled back to the membrane rather than degraded; 3) the Arg386Pro mutation does not affect the rate of KISS1R trafficking—instead, it prolongs responsiveness to kisspeptin by decreasing KISS1R degradation, resulting in the net increase on mutant receptor recycled back to the plasma membrane.

scholarly journals The two zinc fingers in the nucleocapsid domain of the HIV-1 Gag precursor are equivalent for the interaction with the genomic RNA in the cytoplasm, but not for the recruitment of the complexes at the plasma membrane

2020 ◽  
Author(s):  
E. Boutant ◽  
J. Bonzi ◽  
H. Anton ◽  
M. B. Nasim ◽  
R. Cathagne ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Intracellular Trafficking ◽  
Zinc Fingers ◽  
Late Phase ◽  
Genomic Rna ◽  
Wild Type ◽  
Ribonucleoprotein Complexes ◽  
Viral Genomic Rna ◽  
Hiv 1

ABSTRACTThe HIV-1 Gag precursor specifically selects the unspliced viral genomic RNA (gRNA) from the bulk of cellular and spliced viral RNAs via its nucleocapsid (NC) domain and drives gRNA encapsidation at the plasma membrane (PM). To further identify the determinants governing the intracellular trafficking of Gag-gRNA complexes and their accumulation at the PM, we compared, in living and fixed cells, the interactions between gRNA and wild-type (WT) Gag or Gag mutants carrying deletions in NC zinc fingers (ZFs), or a non-myristoylated version of Gag. Our data showed that the deletion of both ZFs simultaneously or the complete NC domain completely abolished intracytoplasmic Gag-gRNA interactions. Deletion of either ZF delayed the delivery of gRNA to the PM but did not prevent Gag-gRNA interactions in the cytoplasm, indicating that the two ZFs display redundant roles in this respect. However, ZF2 played a more prominent role than ZF1 in the accumulation of the ribonucleoprotein complexes at the PM. Finally, the myristate group which is mandatory for anchoring the complexes at the MP, was found to be dispensable for the association of Gag with the gRNA in the cytosol.STATEMENT of SIGNIFICANCEFormation of HIV-1 retroviral particles relies on specific interactions between the retroviral Gag precursor and the unspliced genomic RNA (gRNA). During the late phase of replication, Gag orchestrates the assembly of newly formed viruses at the plasma membrane (PM). It has been shown that the intracellular HIV-1 gRNA recognition is governed by the two-zinc finger (ZF) motifs of the nucleocapsid (NC) domain in Gag. Here we provided a clear picture of the role of ZFs in the cellular trafficking of Gag-gRNA complexes to the PM by showing that either ZF was sufficient to efficiently promote these interactions in the cytoplasm, while interestingly, ZF2 played a more prominent role in the relocation of these ribonucleoprotein complexes at the PM assembly sites.

scholarly journals An Endoplasmic Reticulum Retrieval Signal Partitions Human Foamy Virus Maturation to Intracytoplasmic Membranes

1999 ◽  
Vol 73 (9) ◽  
pp. 7210-7217 ◽  
Author(s):  
Paul A. Goepfert ◽  
Kit Shaw ◽  
George Wang ◽  
Anju Bansal ◽  
Bradley H. Edwards ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Culture Media ◽  
Foamy Virus ◽  
Relative Increase ◽  
Wild Type ◽  
Virus Maturation ◽  
Protein Levels ◽  
Intracytoplasmic Membranes ◽  
Viral Maturation

ABSTRACT Among all retroviruses, foamy viruses (FVs) are unique in that they regularly mature at intracytoplasmic membranes. The envelope glycoprotein of FV encodes an endoplasmic reticulum (ER) retrieval signal, the dilysine motif (KKXX), that functions to localize the human FV (HFV) glycoprotein to the ER. This study analyzed the function of the dilysine motif in the context of infectious molecular clones of HFV that encoded mutations in the dilysine motif. Electron microscopy (EM) demonstrated virion budding both intracytoplasmically and at the plasma membrane for the wild-type and mutant viruses. Additionally, mutant viruses retained their infectivity, but viruses lacking the dilysine signal budded at the plasma membrane to a greater extent than did wild-type viruses. Interestingly, this relative increase in budding across the plasma membrane did not increase the overall release of viral particles into cell culture media as measured by protein levels in viral pellets or infectious virus titers. We conclude that the dilysine motif of HFV imposes a partial restriction on the site of viral maturation but is not necessary for viral infectivity.

Intracellular trafficking/membrane targeting of human reduced folate carrier expressed in Xenopus oocytes

2001 ◽  
Vol 281 (6) ◽  
pp. G1477-G1486 ◽  
Author(s):  
Veedamali S. Subramanian ◽  
Jonathan S. Marchant ◽  
Ian Parker ◽  
Hamid M. Said
Keyword(s):  
Plasma Membrane ◽  
Intracellular Trafficking ◽  
Fluorescent Protein ◽  
Cytoplasmic Tail ◽  
Confocal Imaging ◽  
Membrane Targeting ◽  
Reduced Folate Carrier ◽  
Animal Pole ◽  
Molecular Determinant ◽  
Green Fluorescent

The major cellular pathway for uptake of the vitamin folic acid, including its absorption in the intestine, is via a plasma membrane carrier system, the reduced folate carrier (RFC). Very little is known about the mechanisms that control intracellular trafficking and plasma membrane targeting of RFC. To begin addressing these issues, we used Xenopus oocyte as a model system and examined whether the signal that targets the protein to the plasma membrane is located in the COOH-terminal cytoplasmic tail or in the backbone of the polypeptide. We also examined the role of microtubules and microfilaments in intracellular trafficking of the protein. Confocal imaging of human RFC (hRFC) fused to the enhanced green fluorescent protein (hRFC-EGFP) showed that the protein was expressed at the plasma membrane, with expression confined almost entirely to the animal pole of the oocyte. Localization of hRFC at the plasma membrane was not affected by partial or total truncation of the COOH-terminal tail of the polypeptide, whereas a construct of the cytoplasmic tail fused to EGFP was not found at the plasma membrane. Disruption of microtubules, but not microfilaments, prevented hRFC expression at the plasma membrane. These results demonstrate that the molecular determinant(s) that directs plasma membrane targeting of hRFC is located within the backbone of the polypeptide and that intact microtubules, but not microfilaments, are essential for intracellular trafficking of the protein.

The Natural Flavonoid Naringenin Inhibits the Cell Growth of WilmsTumorinChildren by Suppressing TLR4/NF-κB Signaling

2020 ◽  
Vol 20 ◽  
Author(s):  
Hongtao Li ◽  
Peng Chen ◽  
Lei Chen ◽  
Xinning Wang
Keyword(s):  
Cell Growth ◽  
Western Blot ◽  
Wilms Tumor ◽  
Critical Role ◽  
Time Dependent ◽  
Luciferase Assay ◽  
Dependent Manner ◽  
Tlr4 Expression ◽  
Protein Levels

Background: Nuclear factor kappa B (NF-κB) is usually activated in Wilms tumor (WT) cells and plays a critical role in WT development. Objective: The study purpose was to screen a NF-κB inhibitor from natural product library and explore its effects on WT development. Methods: Luciferase assay was employed to assess the effects of natural chemical son NF-κB activity. CCK-8 assay was conducted to assess cell growth in response to naringenin. WT xenograft model was established to analyze the effect of naringenin in vivo. Quantitative real-time PCR and Western blot were performed to examine the mRNA and protein levels of relative genes, respectively. Results: Naringenin displayed significant inhibitory effect on NF-κB activation in SK-NEP-1 cells. In SK-NEP-1 and G-401 cells, naringenin inhibited p65 phosphorylation. Moreover, naringenin suppressed TNF-α-induced p65 phosphorylation in WT cells. Naringenin inhibited TLR4 expression at both mRNA and protein levels in WT cells. CCK-8 staining showed that naringenin inhibited cell growth of the two above WT cells in dose-and time-dependent manner, whereas Toll-like receptor 4 (TLR4) over expression partially reversed the above phenomena. Besides, naringenin suppressed WT tumor growth in dose-and time-dependent manner in vivo. Western blot found that naringenin inhibited TLR4 expression and p65 phosphorylation in WT xenograft tumors. Conclusion: Naringenin inhibits WT development viasuppressing TLR4/NF-κB signaling

Intracellular trafficking and cytoplasmic streaming under abiotic stress conditions

2019 ◽  
Vol 6 (04) ◽  
Author(s):  
JESHIMA KHAN YASIN ◽  
ANIL KUMAR SINGH
Keyword(s):  
Plasma Membrane ◽  
Abiotic Stress ◽  
Confocal Microscopy ◽  
Fusion Protein ◽  
Intracellular Trafficking ◽  
Cytoplasmic Streaming ◽  
Living Cells ◽  
Stress Conditions ◽  
Vesicle Formation ◽  
External Stimuli

Cytoplasmic streaming is one among the vital activities of the living cells. In plants cytolplasmic streaming could clearly be seen in hypocotyls of growing seedlings. To observe cytoplsmic streaming and its correlated intracellular trafficking an investigation was conducted in legumes in comparison with GFP-AtRab75 and 35S::GFP:δTIP tonoplast fusion protein expressing arabidopsis lines. These seedlings were observed under confocal microscopy with different buffer incubation treatments and under different stress conditions. GFP expressing 35S::GFP:δTIP tonoplast lines were looking similar to the control lines and differ under stress conditions. Movement of cytoplasmic invaginations within the tonoplast and cytoplasmic sub vesicle or bulb budding during cytoplasmic streaming was observed in hypocotyls of At-GFP tonoplast plants. We found the cytoplasmic bulbs/ vesicles or sub vesicle formation from the plasma membrane. The streaming speed also depends on the incubation medium in which the specimen was incubated, indicating that the external stimuli as well as internal stimuli can alter the speed of streaming

scholarly journals Surfactant protein A reduces TLR4 and inflammatory cytokine mRNA levels in neonatal mouse ileum

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Lidan Liu ◽  
Chaim Z. Aron ◽  
Cullen M. Grable ◽  
Adrian Robles ◽  
Xiangli Liu ◽  
...  
Keyword(s):  
Proinflammatory Cytokine ◽  
Inflammatory Cytokine ◽  
Protein A ◽  
Surfactant Protein ◽  
Surfactant Protein A ◽  
Mrna Levels ◽  
Wild Type ◽  
Cytokine Mrna ◽  
Protein Levels ◽  
Cytokine Levels

AbstractLevels of intestinal toll-like receptor 4 (TLR4) impact inflammation in the neonatal gastrointestinal tract. While surfactant protein A (SP-A) is known to regulate TLR4 in the lung, it also reduces intestinal damage, TLR4 and inflammation in an experimental model of necrotizing enterocolitis (NEC) in neonatal rats. We hypothesized that SP-A-deficient (SP-A−/−) mice have increased ileal TLR4 and inflammatory cytokine levels compared to wild type mice, impacting intestinal physiology. We found that ileal TLR4 and proinflammatory cytokine levels were significantly higher in infant SP-A−/− mice compared to wild type mice. Gavage of neonatal SP-A−/− mice with purified SP-A reduced ileal TLR4 protein levels. SP-A reduced expression of TLR4 and proinflammatory cytokines in normal human intestinal epithelial cells (FHs74int), suggesting a direct effect. However, incubation of gastrointestinal cell lines with proteasome inhibitors did not abrogate the effect of SP-A on TLR4 protein levels, suggesting that proteasomal degradation is not involved. In a mouse model of experimental NEC, SP-A−/− mice were more susceptible to intestinal stress resembling NEC, while gavage with SP-A significantly decreased ileal damage, TLR4 and proinflammatory cytokine mRNA levels. Our data suggests that SP-A has an extrapulmonary role in the intestinal health of neonatal mice by modulating TLR4 and proinflammatory cytokines mRNA expression in intestinal epithelium.

scholarly journals Interplay between Pds5 and Rec8 in regulating chromosome axis length and crossover frequency

2021 ◽  
Vol 7 (11) ◽  
pp. eabe7920
Author(s):  
Meihui Song ◽  
Binyuan Zhai ◽  
Xiao Yang ◽  
Taicong Tan ◽  
Ying Wang ◽  
...  
Keyword(s):  
Recombination Frequency ◽  
Crossover Frequency ◽  
Wild Type ◽  
Protein Levels ◽  
Homolog Pairing ◽  
Meiotic Chromosomes ◽  
Homologous Chromosome Pairing ◽  
Axis Length ◽  
Chromosome Axis

Meiotic chromosomes have a loop/axis architecture, with axis length determining crossover frequency. Meiosis-specific Pds5 depletion mutants have shorter chromosome axes and lower homologous chromosome pairing and recombination frequency. However, it is poorly understood how Pds5 coordinately regulates these processes. In this study, we show that only ~20% of wild-type level of Pds5 is required for homolog pairing and that higher levels of Pds5 dosage-dependently regulate axis length and crossover frequency. Moderate changes in Pds5 protein levels do not explicitly impair the basic recombination process. Further investigations show that Pds5 does not regulate chromosome axes by altering Rec8 abundance. Conversely, Rec8 regulates chromosome axis length by modulating Pds5. These findings highlight the important role of Pds5 in regulating meiosis and its relationship with Rec8 to regulate chromosome axis length and crossover frequency with implications for evolutionary adaptation.

scholarly journals Low ABA concentration promotes root growth and hydrotropism through relief of ABA INSENSITIVE 1-mediated inhibition of plasma membrane H+-ATPase 2

2021 ◽  
Vol 7 (12) ◽  
pp. eabd4113
Author(s):  
Rui Miao ◽  
Wei Yuan ◽  
Yue Wang ◽  
Irene Garcia-Maquilon ◽  
Xiaolin Dang ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Root Growth ◽  
Experimental System ◽  
Aba Signaling ◽  
Wild Type ◽  
Normal Medium ◽  
C Terminus ◽  
Quadruple Mutant ◽  
Aba Insensitive ◽  
Aba Receptors

The hab1-1abi1-2abi2-2pp2ca-1 quadruple mutant (Qabi2-2) seedlings lacking key negative regulators of ABA signaling, namely, clade A protein phosphatases type 2C (PP2Cs), show more apoplastic H+ efflux in roots and display an enhanced root growth under normal medium or water stress medium compared to the wild type. The presence of low ABA concentration (0.1 micromolar), inhibiting PP2C activity via monomeric ABA receptors, enhances root apoplastic H+ efflux and growth of the wild type, resembling the Qabi2-2 phenotype in normal medium. Qabi2-2 seedlings also demonstrate increased hydrotropism compared to the wild type in obliquely-oriented hydrotropic experimental system, and asymmetric H+ efflux in root elongation zone is crucial for root hydrotropism. Moreover, we reveal that Arabidopsis ABA-insensitive 1, a key PP2C in ABA signaling, interacts directly with the C terminus of Arabidopsis plasma membrane H+-dependent adenosine triphosphatase 2 (AHA2) and dephosphorylates its penultimate threonine residue (Thr947), whose dephosphorylation negatively regulates AHA2.

scholarly journals Alopecia in Harlequin mutant mice is associated with reduced AIF protein levels and expression of retroviral elements

2020 ◽  
Author(s):  
Maik Hintze ◽  
Sebastian Griesing ◽  
Marion Michels ◽  
Birgit Blanck ◽  
Lena Wischhof ◽  
...  
Keyword(s):  
Hair Growth ◽  
Transcriptional Regulators ◽  
Mutant Mice ◽  
Wild Type ◽  
Protein Levels ◽  
Expression Of Genes ◽  
Genes Encoding ◽  
Keratinocyte Cell ◽  
Apoptosis Inducing Factor ◽  
Hair Cortex

AbstractWe investigated the contribution of apoptosis-inducing factor (AIF), a key regulator of mitochondrial biogenesis, in supporting hair growth. We report that pelage abnormalities developed during hair follicle (HF) morphogenesis in Harlequin (Hq) mutant mice. Fragility of the hair cortex was associated with decreased expression of genes encoding structural hair proteins, though key transcriptional regulators of HF development were expressed at normal levels. Notably, Aifm1 (R200 del) knockin males and Aifm1(R200 del)/Hq females showed minor hair defects, despite substantially reduced AIF levels. Furthermore, we cloned the integrated ecotropic provirus of the Aifm1Hq allele. We found that its overexpression in wild-type keratinocyte cell lines led to down-regulation of HF-specific Krt84 and Krtap3-3 genes without altering Aifm1 or epidermal Krt5 expression. Together, our findings imply that pelage paucity in Hq mutant mice is mechanistically linked to severe AIF deficiency and is associated with the expression of retroviral elements that might potentially influence the transcriptional regulation of structural hair proteins.

scholarly journals Aggregated Tau-PHF6 (VQIVYK) Potentiates NLRP3 Inflammasome Expression and Autophagy in Human Microglial Cells

Cells ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1652
Author(s):  
Chinmaya Panda ◽  
Clara Voelz ◽  
Pardes Habib ◽  
Christian Mevissen ◽  
Thomas Pufe ◽  
...  
Keyword(s):  
Tau Protein ◽  
Nlrp3 Inflammasome ◽  
Time Dependent ◽  
Microglial Cells ◽  
Inflammasome Activation ◽  
Dependent Manner ◽  
Progressive Dementia ◽  
Microglial Polarization ◽  
Protein Levels ◽  
Pyrin Domain

Intra-neuronal misfolding of monomeric tau protein to toxic β-sheet rich neurofibrillary tangles is a hallmark of Alzheimer’s disease (AD). Tau pathology correlates not only with progressive dementia but also with microglia-mediated inflammation in AD. Amyloid-beta (Aβ), another pathogenic peptide involved in AD, has been shown to activate NLRP3 inflammasome (NOD-like receptor family, pyrin domain containing 3), triggering the secretion of proinflammatory interleukin-1β (IL1β) and interleukin-18 (IL18). However, the effect of tau protein on microglia concerning inflammasome activation, microglial polarization, and autophagy is poorly understood. In this study, human microglial cells (HMC3) were stimulated with the unaggregated and aggregated forms of the tau-derived PHF6 peptide (VQIVYK). Modulation of NLRP3 inflammasome was examined by qRT-PCR, immunocytochemistry, and Western blot. We demonstrate that fibrillar aggregates of VQIVYK upregulated the NLRP3 expression at both mRNA and protein levels in a dose- and time-dependent manner, leading to increased expression of IL1β and IL18 in HMC3 cells. Aggregated PHF6-peptide also activated other related inflammation and microglial polarization markers. Furthermore, we also report a time-dependent effect of the aggregated PHF6 on BECN1 (Beclin-1) expression and autophagy. Overall, the PHF6 model system-based study may help to better understand the complex interconnections between Alzheimer’s PHF6 peptide aggregation and microglial inflammation, polarization, and autophagy.

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